Mitchell Meritorious Research Travel Awardees




Name:  Estefany Reyes
Lab:  Mari Shinohara
Conference: Keystone Symposia - Fungal Pathogens :Emerging  Threats and Future Challenges
Date:  February 18-21, 2024
Location:  Banff, Alberta, Canada 
Purpose:  To present a talk





Name:  Lupeng Li 
Lab:  Miao
Conference: Gordon Research Conference on Cell Death
Date:  July 27-August 2, 2024 
Location:  Newry, ME, US 
Purpose:  To present a poster and potentially give a talk




Name:  Katherine Mueller 
Lab:  Valdivia 
Conference: The Human Microbiome
Date:  September 20-23, 2023 
Location:  Heidelberg, Germany 
Purpose:  Poster




Name: Jake Nash 
Lab:  Vilgalys
Conference: Ecological Society of America Annual Meeting
Date:  August 6-11, 2023 
Location:  Portland, OR
Purpose:  Talk


Ben Neubert


Name: Ben Neubert
Lab:  David 
Conference: International Conference on Diet and Activity Methods 2023 
Date:  June 26-29, 2023
Location:  Limerick, Ireland
Purpose:  Poster 




Name:  Corinna Probst
Lab:  Alspaugh 
Conference: Molecular Mycology: Current Approaches to Fungal Pathogenesis 
Date:  July 29 - August 14, 2023 
Location:  Woods Hole, MA 
Purpose:  Course 


Agastya Sharma


Name:  Agastya Sharma
Lab:  Valdivia 
Conference: Mechanisms of Microbiota-Immune Interactions Towards the Next Decade
Date:  October 8-11, 2023 
Location:  Snowbird, Utah 
Purpose:  Poster 




Name:  Angela Rivera 
Lab:  Heitman
Conference: 19th World Congress of Basic and Clinical Pharmacology
Date:  July 2-7, 2023 
Location:  Glasgow, Scotland
Purpose:  Poster 




Name:  Anna Truong 
Lab:  Derbyshire
Conference: Biology and Pathology of the Malaria Parasite (BioMalPar) XIX 
Date:  May 23-25, 2023 
Location:  Heidelberg, Germany 
Purpose:  Talk




Name:  Raul Zavaliev
Lab:  Xinnian Dong
Conference: IS-MPMI Congress 2023 
Date:  July 16-20, 2023
Location:  Providence, Rhode Island, USA 
Purpose:  Poster Presentation 



Name:  Mary Dickinson 
Lab:  Coers
Conference: ASM Microbe
Date:  June 9-13, 2022 
Location:  Washington DC 
Purpose:  Talk

GBP2 aggregates LPS and promotes caspase-4-dependent pyroptosis 

The guanylate binding proteins (GBPs) are a family of gamma interferon (IFNγ) inducible proteins that are important for immunity to cytosolic pathogens such as gram-negative bacteria Shigella flexneri. GBPs are required for activation of the caspase-4 non-canonical inflammasome, triggering host cell pyroptosis and killing of Shigella. GBP1, 2, 3, and 4 are recruited to the Shigella surface, but it is unclear how this recruitment affects inflammasome activation and how each GBP contributes to pyroptosis or bacterial killing. Here we show using knockout cells that GBP1, but not GBP2-5, is required for pyroptosis and bacterial restriction. Additionally, a mutant of GBP1 that is unable to bind the bacterial outer membrane (R584-586A) can mediate pyroptosis and bacterial killing, indicating GBP1 activates pyroptosis without docking on the bacterial surface. Other GBP family members are structurally similar to GBP1, so we tested if overexpression of GBP2, 3, 4, or 5 could promote pyroptosis. Both GBP1 and GBP2 rescued pyroptosis and bacterial restriction in GBP1 knockout cells. Transfection of LPS into cells overexpressing GBP1, GBP1R584-586A, or GBP2 induced cell death in GBP1 knockout cells. Pyroptosis occurred even in the absence of IFNγ, suggesting expression of GBP1 or GBP2 in the absence of other GBPs is sufficient to activate caspase-4 in response to cytosolic LPS. It has been shown that caspase-4 preferentially binds to aggregates of LPS rather than monomers. In vitro, we found that purified GBP1 and GBP2 clustered free LPS into larger structures, suggesting they may promote caspase-4 activation through aggregation of LPS. These findings highlight the importance of GBP1 and GBP2, and not other GBPs, in the activation of caspase-4, and show that GBP1/2 binding to the bacterial surface is dispensable for their role in pyroptosis and bacterial killing.  

Name:  Carissa Harvest 
Lab:  Miao
Conference: EMBO/EMBL Symposium
Date:  July 17 - 20, 2022 
Location:  Heidelberg, Germany 
Purpose:  Poster presentation

A novel mouse granuloma model pathogen is cleared by nitric oxide

Granulomas are an organized collection of immune cells characterized histologically by a specific organizational ring of activated macrophages that surround a persistent stimulus. Clinically, some granulomas can become chronic and are unable to clear a pathogen. These chronic granulomas are thought of as the immune system failing and the end result is to wall off the pathogen without every clearing the infection. Currently there few animal models that recapitulate necrotic granuloma formation that is characteristic of the granuloma response during Mycobacterium tuberculosis infection. We discovered a model infection wherein necrotic granulomas form, and then readily clear the infectious agent – Chromobacterium violaceum. Remarkably, a single bacterium gives rise to the infected abscess at one day post infection, however, neutrophils fail to clear the infection. This failure triggers a granuloma response, and forms a necrotic core that is typically absent in other murine granuloma models. Using immunohistochemistry we can show the remarkable multilayered architecture of the granuloma, as well as the location of the different immune cell types within the granuloma. We used 10x Genomics’ Visium Spatial Gene Expression to analyze gene expression during the granuloma response, and to localize each expressed gene to a specific granuloma layer. This identified nitric oxide synthase 2 (Nos2) as highly upregulated in the macrophage layer. Mice deficient in Nos2 succumb to the infection and have worse granuloma pathology marked by visible breaks in the protective macrophage ring. Because this granuloma successfully clears the infection, our model has significant impact in revealing that granulomas are not the last line of the immune system failing and walling off an infection that it cannot clear, but rather that granulomas represent an organized immune response that successfully clears a pathogen.

Name:  Rachel Keener 
Lab:  Ko
Conference: American Society of Human Genetics
Date:  October 25 - 29, 2022
Location:  Los Angeles, California
Purpose:  Presentation

Human genetic variation reveals regulators of Yersinia pestis cellular Infection.

Yersinia (Y.) pestis is the gram-negative pathogenic bacterium responsible for the deadliest pandemic in recorded human history, the Black Death. This pathogen causes such catastrophic pandemics because of its variable 30–100% mortality rate in infected humans when antibiotic intervention is unavailable. Factors like time to treatment and nutrition can cause some of this variation but, like other infectious diseases, we hypothesize host genetic variation also plays a role. These genetic differences can normally be identified by human challenge studies or genome-wide association studies (GWAS) of natural infection. Both quantify disease symptoms, physiology, and outcomes then compare these phenotypes to the genetics of the individual. Unfortunately, using these techniques for Y. pestis is difficult: high lethality makes human challenges unethical, and the infrequency of present-day Y. pestis outbreaks coupled with non-random population exposure make GWAS too underpowered and confounded. To overcome these hurdles, we developed a genetically diverse cellular GWAS approach. This method, called Hi-HOST (high-throughput human in vitro susceptibility testing), is unhindered by pre-existing conditions, medical care access, diet, or other environmental and life history confounders found in traditional human disease related GWAS. Additionally, Hi-HOST previously identified variation in VAC14 and ARHGEF26 loci that modulate Salmonella infection. For Y. pestis Hi-HOST, we infected LCLs (lymphoblastoid cell lines; immortalized B cells) from 978 genetically distinct individuals in eight global populations with GFP-tagged Y. pestis. Using flow cytometry, we measured percent infection (proportion of cells that are GFP+) as well as bacterial burden (median fluorescence of GFP+ cells). Each phenotype was tested for association with approximately 15 million single nucleotide polymorphisms (SNPs), using family-based association to control for population stratification while also allowing for heritability estimates. This uncovered a genome-wide significant hit (p = 3×10−9) in the intracellular survival phenotype. We hypothesize the gene containing this SNP helps modulate the intracellular niche of Y. pestis. Additionally, stratifying for likely functional variants revealed a nonsynonymous SNP associated with 24-hour percent infection with greater significance (p=3.5x10-07) than expected based on the neutral distribution. Experimental follow-up studies suggest the protein product of this gene acts as a previously unrecognized receptor for Y. pestis in lymphoid lineage cells, regulating Y. pestis attachment and uptake.

Name:  Aruna Menon
Lab:  Tobin 



Name:  Hannah Schmidt
Lab:  Horner



Name:  Chin Yee Tan
Lab:  Surana



Name:  Nicolás M. Reinoso-Vizcaíno
Lab:  Luftig 



Name:  Sebastian Wellford 
Lab:  Moseman



Name:  Vikas Yadav 
Lab:  Heitman
Conference: Gordon Research Conference on “Cellular and Molecular Fungal Biology
Date:  June 26 - July 1, 2022 
Location:  NH, USA
Purpose:  Talk and Poster presentation

Dynamics of the calcineurin interactome in Cryptococcus neoformans

Calcineurin signaling is a highly conserved signaling cascade that governs virulence of fungal pathogens in response to stress conditions that lead to calcium influx into cells. This  calcium signal is sensed by calmodulin that then binds to a series of targets including calcineurin. Ca2+-calmodulin binding to calcineurin triggers conformational changes that activate serine-threonine specific phosphatase activity of the enzyme. In the human fungal pathogen Cryptococcus neoformans calcineurin is activated by heat stress as well as in response to excess calcium.  Previous studies established an essential role for calcineurin in growth at high temperature and virulence as well as during mating via its targets in P-bodies and the transcription factor Crz1. In this study, we employed two approaches to better understand calcineurin localization and its interacting proteins. In the first, we utilized the proximity ligation approach TurboID to identify the pool of calcineurin interacting proteins and candidate substrates. In the second approach, we generated truncation alleles of calcineurin lacking one or more domains to elucidate the role of these domains and their contribution to calcineurin localization and function. The first approach revealed novel calcineurin-interacting proteins in localized in novel cellular compartments, whereas the second approach revealed functional divergence of calcineurin domains for its various roles. Combined, our studies provide detailed insights into the dynamics of calcineurin and its interactions that are crucial for its function. These studies also highlight previously unexplored sites of calcineurin that might prove key for its role in C. neoformans virulence and stress response as well as could apply to other fungal pathogens.


Name:  Heejin Yoo
Lab:  Dong
Conference: The Annual Plant Biology Meeting 2019 (PB19)
Date:  August 3-7, 2019
Location:  San Jose, CA
Purpose:  Poster presentation

Global translational regulation during plant effector triggered immunity 

Plants are sessile organisms that have evolved multi-layered defense mechanisms including transcriptional and translational reprogramming to counter pathogen infection. Transcriptional reprogramming in plant immunity has been extensively studied during the last decades, but the impact of translational regulatory mechanisms are largely unknown. To understand genome-wide global translational regulatory mechanisms during the immune response, we performed global translatome analysis during effector-triggered immunity (ETI) to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 carrying the effector AvrRpt2. We used ribosome footprinting, which is the deep sequencing of ribosome protected mRNA fragments. Interestingly, translational regulation during ETI is very different from our previous translational analysis on pattern triggered immunity (PTI), with induction of genes in metabolic pathways, especially aromatic amino acid metabolism. We further identified novel immune translational regulators that are specifically involved in several metabolic pathways. Together, our study provides novel molecular mechanism for global translational reprogramming during ETI, connecting metabolic dynamics and translational regulation in plants.


Name:  Jeff Bourgeois
Lab:  Ko
Conference: Gordon Research Conference for Salmonella Biology and Pathogenesis
Date:  June 2-7, 2019
Location:  Stonehill College
Purpose:  Poster presentation

Methionine metabolism facilitates cross-kingdom signaling to suppress Salmonella virulence

Background: During infection, Salmonella are bombarded by an array of intercellular, interspecies, and cross kingdom metabolites and proteins. To survive, the bacteria need to rapidly respond to these signals in order to appropriately deploy virulence factors and evade host defenses. Previously, we have shown that the methionine derived metabolite methylthioadenosine (MTA) is not only regulated in response to Salmonella infection, but also directly suppress S. Typhimurium SPI-1 secretion and motility. Our current focus is on understanding how MTA regulates virulence, and what role this regulation has during natural models of infection.

Methods and Results: Our work examines how MTA impacts Salmonella on two fronts. First, we study how the molecule affects S. Typhimurium virulence from the perspective of the microbe. In order to study how MTA impacts virulence in murine models of infection, we generated a genetic mutant (ΔmetJ S. Typhimurium) that has constitutively high endogenous MTA production. This mutant has attenuated fitness in the murine gut, in line with our observation that MTA results in reduced SPI-1 secretion, motility, and host cell invasion in vitro. Surprisingly, however, we find that this reduction in fitness
is not dependent on SPI-1 or flagella, as loss of either pathway does not result in reduced sensitivity to MTA. Further, we find that the ΔmetJ mutant also has reduced fitness in intraperitoneal models of infection, where SPI-1 and flagella are thought to be largely off. Therefore, additional virulence machinery must be regulated by MTA. To identify additional pathways regulated by MTA, we have performed RNA-seq on WT and ΔmetJ S. Typhimurium grown under SPI-1 and SPI- 2 inducing conditions. Our future work will examine how MTA is directly sensed by the bacterial cell, and how this sensing leads to changes in gene expression.
The second focus of our work is to study how the host environment regulates MTA during Salmonella infection, and how this environment can be manipulated to improve infection outcomes. We have previously shown that MTA is released from lymphatic organs into plasma during infection, but were interested in examining MTA concentrations at the original site of infection, the gut. Our data show that MTA is found in high abundance in the gut, supporting our hypothesis that MTA is able to serve as a cross-kingdom signal during infection. Further, we find that MTA abundance is regulated by microbiota,
as germ-free animals have significantly elevated concentrations compared to conventional mice. This demonstrates a role for microbiota in regulating bioavailable MTA and suggests that microbial-regulation of MTA concentrations may help to either 1) target Salmonella virulence factor deployment or 2) contribute to the differential capacity for microbiota to provide colonization resistance against Salmonella infection. Our current work focuses on studying how MTA concentrations change along the gut, as well as how concentrations change during S. Typhimurium infection.

Conclusions: These data greatly expand our previous finding that MTA suppresses S. Typhimurium virulence. We find that MTA is dynamically regulated in the murine gut by microbiota, and provide evidence that concentrations likely reach levels that enable the molecule to serve as a cross-kingdom signal in the mouse ileum. Our future studies will focus on expanding our understanding of this novel example host-microbe communication in the gut, and will provide mechanistic insight into how MTA is able to suppress virulence gene expression in S. Typhimurium.


Name:  Jia Wen
Lab:  Rawls
Conference: FASEB “The Gastrointestinal Tract XVII Conference: Integrated Biology of the GI Super-Organ”
Date:  July 28- August 2, 2019
Location:  Steamboats Springs, Colorado
Purpose:  Poster presentation

Genetic analysis of ileal identity in the zebrafish intestine

The intestinal tract is regionalized along the cephalocaudal axis to coordinate region specific functions for efficient nutrient assimilation. The ileum is the only region of the small intestine that actively absorbs bile salts, amphipathic molecules that serve as lipid emulsifiers and signaling molecules regulating metabolism. Disruption of ileal function can cause malnutrition and intestinal failure. The molecular mechanisms determining ileal regional functions, however, remain largely unknown. We recently found that the cephalocaudal regionality of the intestine is conserved between fishes and mammals and
identified a region of zebrafish intestine with orthologous gene expression to the mouse ileum. Many of the conserved markers found in the zebrafish ileum-like region are targets of the bile salt activated transcription factor Farnesoid X receptor (Fxr/NR1H4). To test if Fxr contributes to ileal identity, we generated fxr mutant zebrafish that exhibited reduced expression of conserved ileal markers including fabp6. Using a ileal transgenic reporter zebrafish fabp6:GFP, we observed a complete loss of the GFP-labelled ileal region upon fxr disruption, suggesting that Fxr is required to maintain normal ileal identity. To evaluate if the Fxr ligand bile salt is necessary for the regulatory impacts of Fxr, we utilized
zebrafish cyp7a1 and slc10a2 mutants that have defective bile salt synthesis or absorption respectively. The GFP-labelled ileal region was attenuated in both mutants, suggesting that the establishment of ileal identity requires activation of Fxr by bile salts. In mammals, the primary bile acids are modified by the gut microbiota into secondary bile acids with varying activities as Fxr ligands. We found that the primary bile salts in zebrafish consist of mostly bile alcohol with few bile acid species, and that bile modification by gut microbiota still occurs though the modifications are distinct from
mammals. We detected dehydrogenation, epimerization, and deconjugation of the primary bile salts by the zebrafish microbiota, but no activity of dehydroxylation, the most common modification in mammals. Together, these results establish that the ileum of the intestine and bile salt modification by gut microbiota are conserved features in fishes and mammals, and that Fxr function is critical for the establishment of ileal identity. This provides a critical foundation for using zebrafish to identify contributions of host and microbiota in governing regional identity along the length of the intestine.


Name:  Maria Toro-Moreno
Lab:  Derbyshire
Conference: EMBL: New Approaches and Concepts in Microbiology
Date:  July 10-13, 2019
Location:  Heidelberg, Germany
Purpose:  Poster presentation


To successfully complete its life cycle in the human host after being transmitted by mosquitoes, Plasmodium parasites must first invade hepatocytes. Within these cells, parasites replicate extensively to form bloodinfectious forms that are released into the bloodstream. Despite clear exploitation of host cell resources during infection, little is known about the host factors that are essential for parasite development in hepatocytes. To identify these host factors, we executed a high-throughput siRNA screen of the human druggable genome. Interestingly, we discovered that the host vesicular transport pathway is critical in hepatic development of Plasmodium parasites. Moreover, chemical inhibition of the vesicular transport pathway significantly reduces Plasmodium berghei parasite load in hepatocytes. The disruption of protein trafficking modulators, including COPB2 and GGA1 decreases P. berghei parasite size and an immunofluorescence study indicates that these proteins are recruited to the Plasmodium parasitophorous vacuole in infected hepatocytes. Furthermore, phenotypic analyses upon genetic or pharmacological disruption indicate that a non-canonical trafficking pathway modulates this recruitment to the parasitophorous vacuole. Our data reveal that the host vesicle-mediated trafficking pathway, which is subverted by various intracellular pathogens, also has an important function in the development of Plasmodium parasites during their exo-erythrocytic stage in liver cells.


Name:  Blanca Rodriguez
Lab:  Kuehn
Conference: Microbial Adhesion and Signal Transduction Gordon Research Conference and Gordon Research Seminar
Date:  July 20-26, 2019
Location:  Newport, Rhode Island
Purpose:  Poster presentation

Bacterial Membrane Vesicle-associated Nucleic Acids Induce Interferon-ß Expression in Murine

Bacterial nucleic acids are well recognized as important pathogen associated molecular patterns sensed by innate immune cells during infection. Intriguingly, RNA-secretion structures analogous to protein secretion systems have not been identified in bacteria. It is therefore unclear how bacterial RNA is secreted and whether specific types of RNAs are selected for secretion during infection of host cells. Pathogenic bacteria share a common secretion mechanism that enables the packaging of soluble and insoluble macromolecules into nanosized, spherical, proteolipidic structures termed membrane vesicles (MV). Although MVs originate from the cellular membrane, reports now show that during the course of their biogenesis, bacterial MVs are loaded with a subset of nucleic acids and cytoplasmic proteins lacking export signals. Those findings position MV formation as a non-canonical secretion mechanism for cytoplasmic molecules. MVs are important mediators of pathogen-host interactions and are often internalized via receptor-mediated endocytosis, leading to the intracellular delivery of bioactive macromolecules—which can alter the function of recipient cells. The present study sought to examine whether MVs produced by the human pathogen, Staphylococcus aureus, contain RNA and DNA that can be transferred to host cells to modulate innate immune responses.
Here we report that S. aureus produce MV-associated nucleic acids that are protected from nuclease
degradation. MV-associated nucleic acids were transferred to cultured murine macrophages and induced significant Interferon-ß mRNA expression largely through endosomal Toll-like receptor (TLR) signaling. Upon exposure to nuclease-treated MVs, TLR3-/- and TLR7-/- macrophages produced very little IFN-ß mRNA. TLR9-/- macrophages also produced less IFN-ß mRNA relative to wild-type macrophages, although more IFN-ß mRNA was produced compared to both TLR3- and TLR7-deficient macrophages. TLR3 recognizes dsRNA, which points to the possibility that S. aureus MVs are packaged with immunostimulatory dsRNA molecules. TLR7 has previously been found to recognize S. aureus tRNA, as well as ssRNA molecules. Meanwhile, TLR9 is known to recognize Staphylococcal CpG DNA, which can lead to Type I Interferon production. Altogether, these data indicate that endosomal nucleic acid receptors are activated in cultured mouse macrophages upon MV exposure, likely due to immunostimulatory properties of MV-associated nucleic acids. Our findings show for the first time an MV-mediated pathway by which S. aureus-derived immunomodulatory nucleic acids are delivered to host cells. How MVassociated nucleic acids are trafficked intracellularly and recognized by endosomal TLRs will be examined in future experiments.


Name:  Michael Hoy
Lab:  Heitman
Conference: 8th FEBS Advanced Lecture Conference on Human Fungal Pathogens
Date:  May 18-24, 2019
Location:  La Colle sur Loop, France
Purpose:  Poster presentation

Increased fungal specificity of novel FK506 analogs confers both in vitro and in vivo antifungal efficacy

Invasive fungal infections caused by Aspergillus fumigatus, Candida albicans, and Cryptococcus
neoformans total more than 1.6 million cases per year with up to 95% mortality. Although many
antifungal drugs are used clinically, new antifungal drug targets are uncommon. The natural product, FK506, inhibits calcineurin which is a serine-threonine specific phosphatase and a key virulence factor in pathogenic fungi. However, FK506 is not used to treat fungal infections due to the immunosuppressive effect of inhibiting human calcineurin function. In fact, FK506 is commonly used clinically to immunosuppress patients receiving solid organ transplants. In both fungi and mammals, FK506 binds to the immunophilin, FKBP12, and then binds calcineurin and inhibits its activity by blocking binding to substrates. Although mammalian and fungal FKBP12 are highly conserved, we have identified a key differential residue in the 80s loop proximal to the FK506 binding pocket that can be exploited to design FK506 analogs with increased fungal specificity.
One FK506 analog, APX879, differs from FK506 by a single chemical substitution. APX879 retains antifungal activity in vitro, and in vivo reduces fungal burden and extends animal survival in a murine inhalation model of cryptococcosis. The structure of C. neoformans calcineurin-FK506-FKBP12 suggests that the moiety modified in APX879 will not affect its binding to FKBP12, but instead will alter how it interacts with calcineurin. Importantly, compared to FK506, APX879 exhibits a marked decrease in immunosuppressive activity assessed by IL-2 production in primary T cells. Additional FK506 analogs modified at the same site maintain antifungal activity similar to APX879 and provide a platform for probing the differences between the fungal and human ternary complexes. By utilizing a combination of structural biology and medicinal chemistry, calcineurin inhibitors can be generated with an improved therapeutic window due to decreased action on the host.



Name:  Stephen Walsh
Lab:  Coers
Conference: Chlamydia Basic Research Society (CBRS)
Date:  March 18-21, 2019
Location:  Seattle, WA
Purpose:  Poster presentation

Defining the role of interferon-stimulated genes in restricting Chlamydia growth

The bacteria Chlamydia trachomatis is a prevalent human pathogen that infects over 100 million people every year, leading to significant morbidity across the globe. Chlamydia-associated disease is not always amenable to standard antibiotic treatment; the development of an effective vaccine or novel host-directed therapeutic could alleviate the health burden of Chlamydia-infected populations and decrease the economic cost in treating its related disease. To develop these medicines, critical steps include the identification of host factors deployed against the pathogen and an understanding of why these factors frequently fail to provide a lasting immune protective effect for the host. Many host innate immune responses are executed by an array of interferon-stimulated genes (ISGs), but one of the most robustly-induced and well-studied ISGs, indole-2,3-dioxygenase (IDO), is unable to fully eradicate Chlamydia trachomatis infections due to a co-evolved bacterial counter-defense. Additionally, little is known about the function of other ISGs that may fight against Chlamydia infectionand if specific bacterial counter-defenses to these ISGs also exist. A recent functional genomics screen in our lab revealed five ISGs that exert significant anti-Chlamydia innate immune responses, two of which have novel anti-chlamydia functions by currently undefined mechanisms. We have termed these two genes “chlamydia restriction factors” (CRF) 1 and 2. Additionally, CRF1 and CRF2 both function in an IDO-independent manner and are required for the maximum level of interferon-mediated restriction of certain Chlamydia species in human epithelial cells. Future studies will clarify the cell-intrinsic effects of CRF1 and CRF2 in restricting Chlamydia infection, and will also delineate the counter-resistance mechanisms Chlamydia may utilize to block the functions of these two CRF genes. Implications of these studies will provide important platforms and lead molecules in the development of anti-Chlamydia medicines._____________________________________________________________________

Name:  Calla Telzrow
Lab:  Alspaugh
Conference: 2019 Eight FEBS Advanced Lecture Course
Date:  May 18-24, 2019
Location:  La Colle sur Loup, France
Purpose:  Poster presentation

Defining arrestin-regulated adaptive cellular responses in fungal virulence

In order to effectively cause disease, pathogens must sense and adapt to dynamic environments. Arrestin proteins are potent regulators of these adaptive cellular responses. Arrestins are a structurally
specialized and functionally diverse group of proteins that modulate intracellular responses to extracellular signals. Previous studies on fungal arrestins have demonstrated their roles in classical arrestin functions, such as G protein-coupled receptor desensitization and membrane protein endocytosis, but the mechanisms by which arrestin-regulated processes are involved in fungal virulence remained unexplored. Based on protein structure homology to human arrestins, we identified a family of four putative arrestin proteins – Ali1, Ali2, Ali3, and Ali4 – in the human fungal pathogen Cryptococcus neoformans. The arrestin loss-of-function mutants exhibited varying degrees of sensitivity to cell surface stressors and high temperature. Additionally, macrophage co-culture assays indicated that the arrestin family supported virulence. To probe these shared phenotypes, we have focused initial studies on Ali1. We observed that Ali1 localized to the septum, and that the Ali1 loss-of-function mutant displayed cytokinesis defects. These data implicate Ali1 in the regulation of cytokinesis. Based on preliminary co-immunoprecipitation experiments, Ali1 potentially interacted with
multiple proteins involved in cell surface synthesis. Collectively, we hypothesize that Ali1 acts as an adaptor protein for enzymes that synthesize and rebuild the cell membrane and cell wall during cytokinesis. Future studies will focus on mechanisms, such as ubiquitination, by which Ali1 regulates the localization and function of these enzymes throughout the cell cycle. By functionally characterizing each of the C. neoformans four arrestin proteins, this work will enhance the basic biological understanding of arrestin functions, as well as establish the active role of fungal arrestins in virulence.


Name:  Ria Goswami
Lab:  Permar
Conference: Conference on Retroviruses and Opportunistic Infections (CROI)
Date:  March 4-7, 2019
Location:  Seattle, WA
Purpose:  Poster presentation

SHIV CH505 T/F Reservoir and Rebound in Infant and Adult Rhesus Macaques 

Each year >150,000 infants get infected with HIV, ~50% infections occurring during
breastfeeding. While lifelong ART results in effective viral suppression, these infants are
predisposed to long-term metabolic consequences and development of drug-resistant viral
strains. Therefore, a functional cure is required to attain an ART-free life of sustained viral
remission. The primary barrier for a cure is the ability of HIV to establish persistent viral
reservoirs, immediately upon infection. Therefore, strategies to reduce viral reservoirs are
urgently needed to delay viral rebound, and attain prolonged viral remission. For that, it is critical
to monitor establishment of HIV reservoirs and kinetics of viral rebound.
In this pilot study, 6 SHIV.C.CH505 T/F infected infants and 6 adult rhesus macaques (RMs)
were used to characterize viral replication and establishment of reservoirs upon ART initiation at
12 wpi. After 8 wk of ART, the kinetics and anatomic distribution of viral rebound upon ART
interruption was measured using ddPCR and co-culturing mononuclear cells with Tzm-bl
reporter cells.
Plasma viral RNA (vRNA) in infants and adults peaked at 2 wpi (infant mean: 6.9X106 vRNA
copies/ml; adult mean:4.2X106 vRNA copies/ml). While both groups demonstrated similar viral
load kinetics until ART initiation at 12 wpi, 33% infants showed pre ART control. Cell-associated
viral DNA (vDNA) in PBMCs were comparable at 6 wpi in infant (Mean: 1.5X107 copies/million
CD4+ T cells) and adult (Mean: 2.1X107 copies/million CD4+ T cells) RMs. Upon initiation of
ART, plasma vRNA suppressed below levels of detection within 2-4 wk. Interestingly, ARTsuppressed
RMs showed similar frequencies of cell-associated vDNA in naïve, Tfh and memory
CD4+ T cell populations in LNs. (Range: undetectable-2.6X104 copies/million CD4+ T cells).
Upon ART interruption, 5/6 infants and 3/6 adult RMs rebounded to >150 vRNA copies/ml of
plasma. Plasma VL at ART initiation was a predictor of time to viral rebound, and infants had
more variability in time to rebound. While all the adults controlled systemic virus within 3-4 wk of
rebound, only 3/5 infants demonstrated post-rebound viral control. Oral LNs were a primary site
of vRNA and infectious virus in both adults and infant RMs.
Using a RM model of postnatal infection, we have characterized SHIV.C.CH505 reservoirs and
rebound kinetics, which can inform correlates of viral rebound, and design immune-based
interventions to reduce pediatric HIV reservoirs.


Name:  Asiya Gusa
Lab:  Jinks-Robertson
Conference: 2019 Eight FEBS Advanced Lecture Course
Date:  May 18-24, 2019
Location:  La Colle sur Loup, France
Purpose:  Poster presentation

Temperature stress, transposon-mediated mutagenesis and drug resistance by
Cryptococcus neoformans in a host model of infection 

Cryptococcus neoformans is a human fungal pathogen causing infections primarily in
immunocompromised individuals, such as those with HIV/AIDS or cancer patients undergoing
chemotherapy. Dissemination of Cryptococcus to the brain causes meningoencephalitis, a
condition with a high mortality rate and limited treatment success due to drug resistance.
Previous studies have shown temperature-induced mutagenesis and increased survival of C.
neoformans serotype D (XL280α) in the presence of antifungal drugs in culture. Compared to
growth at 30˚C, there was a 25-fold increase in mutagenesis and drug resistance when
Cryptococcus was grown at 37˚C (human host temperature), largely due to insertions by
transposable elements identified as the DNA transposon T1 and retrotransposon Tcn12. Using a
murine model of infection (37˚C host temperature), this study provides the first evidence that
transposon insertions in the XL280α cryptococcal genome in vivo result in mutations and drug
resistance to 5-fluoroorotic acid and rapamycin/FK506. Cryptococcus mutants resistant to 5-
fluoroorotic acid (insertions in the URA3 or URA5 genes) or rapamycin/FK506 (insertions in
FRR1) were recovered from the organs of mice infected intravenously at 3 and 7 days postinfection.
The number of resistant isolates was compared to the total number of colonies recovered from the lung, brain, spleen and kidney tissue to determine the mutation frequency. Significantly, nearly half of the loss of function mutations in the URA3, URA5 and FRR1 genes were due to transposon insertions. Both the T1 and Tcn12 transposable elements were present at insertion sites, supporting previous results that showed these elements to be highly mobile at 37˚C in vitro. These results suggest that transposon-mediated mutagenesis at elevated temperatures may provide a selective advantage for the survival of Cryptococcus in the presence of antifungal drugs or other hostile host conditions. To explore the effect of temperature on host survival in a cryptococcal infection, Galleria mellonella larvae were infected with XL280α then incubated at 30˚C or 37˚C. Killing of the larvae was significantly more rapid at 37˚C, with the majority of larvae dying between Day 6 and 10 post-infection. Future experiments are planned to determine if there is a difference in the frequency of transposition in vivo at 30˚C and 37˚C
and whether transposition is important for the survival and pathogenesis of Cryptococcus in the host.


Name:  Giuseppe Ianiri
Lab:  Heitman
Conference: 30th Fungal Genetics Conference
Date:  March 12-17, 2019
Location:  Pacific Grove, CA
Purpose:  Poster & Oral presentation

Horizontal gene transfer in the skin fungus Malassezia: NO resistance is mediated by a bacterial

The skin of humans and animals is colonized by numerous microganisms that comprise the skin
microbiome. They mainly include both commensal and pathogenic fungi and bacteria that share the
same ecological niche and established intense interactions. Malassezia species are the most abundant
fungal skin inhabitant of all warm-blooded animals. Previous genomics studies revealed the presence
of genes of bacterial origins in the Malassezia genus likely acquired through horizontal gene transfer,
including the flavohemoglobin-encoding gene YHB1. The aims of the present study are to 1) use
functional genetics to characterize the role of the bacterial YHB1 gene in Malassezia sympodialis, and
2) identify additional Malassezia species-specific horizontal gene transfer events.


Name:  Nichole Orench-Rivera
Lab:  Kuehn
Conference: ASBMB 2019 Annual Meeting
Date:  April 6-9, 2019
Location:  Orlando, FL
Purpose:  Poster presentation

Oxidative stress-induced differential packaging of proteins in Enterotoxigenic Escherichia coli outer membrane vesicles and its impact on host-pathogen interactions 

Enterotoxigenic Escherichia coli is the leading cause of traveler’s diarrhea worldwide and it remains
an important causal agent of diarrheal disease, especially among children of lower income countries. Because it must adjust to different ecological niches such as fruit and vegetable surfaces, water, and its animal or human host; its cell envelope must undergo remodeling. In Gram-negative bacteria, the cell envelope is the first barrier against environmental stress and studies have shown that the process of outer membrane vesiculation is essential for bacterial growth and survival during stressful conditions. Recent studies in our lab revealed that outer membrane vesicles (OMVs) can contain varying ratios of lipopolysaccharide (LPS) types during cell envelope remodeling under stress in Salmonella enterica, suggesting a role for vesicles in membrane remodeling. In this study, outer membrane vesicles and outer membrane fractions from ETEC were isolated from bacterial cultures, purified via density centrifugation, and submitted for proteomic analysis using quantitative onedimensional liquid chromatography, tandem mass spectrometry (1D-LC-MS/MS) to quantify differences in protein expression in vesicles and membranes during oxidizing and non-oxidizing conditions (plus or minus Hydrogen Peroxide). Protein packaging into vesicles was determined by calculating the log2 of the ratio of protein expression in OMVs/outer membranes for both treatment conditions. Results indicated that two distinct sets of proteins were shown to be differentially packaged into OMVs as a function of peroxide treatment. Upon further analysis implementing a Bayesian hierarchical model, lipoproteins were observed to be preferentially exported during stress, in contrast to integral proteins, which were preferentially retained in the outer membrane. These results suggest possible roles for previously uncharacterized lipoproteins in the oxidative stress pathway and allow us to speculate about potential roles for OMVs in membrane remodeling of pathogenic bacteria undergoing environmental shifts both outside and inside the human host.


Name:  Francis Fang
Lab:  Heitman
Conference:  Molecular Mycology Course
Date:  July 30-August 15, 2018
Location:  Woods Hole, MA
Purpose:  To present a poster

Investigation of the DNA repair proteins involved in RNA-induced gene
silencing in Cryptococcus neoformans
RNA-induced gene silencing (RNAi) is a ubiquitous pathway in most of the eukaryotes
that have been examined. RNAi serves central functions including regulation of gene
expression, repression of transposable element expression and movement, and
maintenance of genome stability. Previous studies have shown that tandem DNA
repeats can induce gene silencing during vegetative growth (mitotic-induced silencing,
MIS) and sexual reproduction (sex-induced silencing, SIS) in Cryptococcus neoformans,
an opportunistic human pathogen that causes lethal meningitis in almost two hundred
thousand immunocompromised patients each year. However, the mechanism regulating
gene silencing of these repetitive sequences is still unknown. Here, we tested the
hypothesis that the Rad51, Rad52 and Rad54 proteins, mediators of recognition of
homologous DNA sequences during DNA repair and recombination, may activate MIS
and SIS RNAi silencing. This is based on an earlier model proposed by Zhang et al,
Genes & Development 2013, that Rad51/52/54 are required for RNAi silencing of
repetitive sequences in the model filamentous fungus Neurospora crassa. We have
isolated independent rad51, rad52 and rad54 mutants of C. neoformans, but studies
showed that none of those genes is required for mitotic transgene silencing (MIS). To
rule out the possible functional redundancy of those proteins in C. neoformans, we
constructed double- and triple-mutants of rad51, rad52 and rad54 by genetic crosses.
Interestingly, the triple mutant abolished MIS. To further validate this observation, we are
using the recently developed transient CRISPR/Cas9 approach to generate de novo
deletions of the Rad genes for independent analysis. In parallel, we also included
another homologous recombinase, Dmc1, in our MIS study. Dmc1 is a homolog of
Rad51, but functions specifically in meiosis. Our preliminary results demonstrate that
Dmc1 is also not involved in MIS, but may work together with other Rad proteins
contributing to RNAi-mediated silencing.



Name:  Kyle Gibbs
Lab:  Ko
Conference:  ASM Microbe
Date:  June 7-11, 2018
Location:  Atlanta, GA
Purpose:  Oral presentation and present a poster

Human genetic variation identifies an endosomal cation channel that restricts
Salmonella enterica replication
Background: Salmonella enterica spp. infections manifest in a diverse array of disease
phenotypes that range from asymptomatic infection to mild gastroenteritis, bacteremia, and
septic shock. While the diversity of Salmonella serovars and variation in exposure explain some of this range, genetic variation in host response also contributes to the diverse clinical
manifestations. This presents an opportunity for new approaches that use natural genetic
diversity to gain mechanistic understanding of host-pathogen interactions.
Method and Results: We use cellular genome-wide association studies (GWAS) on infected
lymphoblastoid cell lines (LCLs; EBV-immortalized B cells) to uncover host genetic variants
associated with natural diversity in infections. Meta-analysis of two cellular GWAS identified a locus near the mucolipin-2 (MCOLN2; TRMPL2) gene that associated with both Salmonella
enterica spp. intracellular replication and expression of the MCOLN2 gene. The directionality of these associations led to the hypothesis that MCOLN2, an endosomal cation channel, restricts Salmonella intracellular replication. Consistent with this, siRNA knockdown of MCOLN2, but not the neighboring MCOLN3, caused more S. enterica intracellular replication. Comparison of wildtype and MCOLN2-/- mice and bone marrow-derived macrophages also showed that MCOLN2 restricts Salmonella replication.
Conclusion: These results demonstrate that MCOLN2 is a host restriction factor during
Salmonella infection. In ongoing work, we are examining possible mechanisms for this
restriction including MCOLN2 modulation of recycling endosome function and host cytokine
secretion. Using the novel tool of cellular GWAS on bacterial, viral, and protozoal pathogens, we are articulating how natural human genetic diversity contributes to host-pathogen interactions.



Name: Mike McFadden
Lab:  Horner
Conference:  6th Annual International Cytokine and Interferon Society
Date:  October 27-30, 2018
Location:  Boston, MA
Purpose: Oral Presentation

RNA Modifications in Type I Interferon Response Regulation
Gene expression during the type I interferon response must be strictly regulated to allow for both efficient production of antiviral effectors and controlled shut-off of inflammatory factors to avoid tissue damage and autoimmunity. RNA base modifications represent an emerging paradigm of gene regulation, which occurs at the post-transcriptional level. N6-methyladenosine (m6A) is a reversible modification whose pattern on mRNA can change during cellular stress. m6A deposition is controlled by a “writer” complex including METTL3 and METTL14, and its removal is catalyzed by demethylase proteins such as FTO and ALKBH5. We have previously shown that the m6A machinery regulates hepatitis C virus infection, however the regulatory role of m6A in innate immunity warrants further investigation. To determine whether m6A may regulate innate immunity, we mapped m6A modifications during the type I interferon response and show dynamic changes on mRNAs of many interferon-stimulated genes (ISGs). Additionally, we show that perturbation of the cellular m6A-related machinery affects production of a subset of interferon-stimulated genes, including the antiviral IFITM family proteins. Depletion of the demethylase FTO increases IFITM1 production. Conversely, ectopic expression of FTO decreases IFITM1 production and is dependent on the catalytic activity of FTO. Additionally, depletion of the methyltransferase components METTL3/14 decreases the abundance of IFITM1, whereas overexpression strongly increases IFITM1 abundance. Taken together, these data suggest that m6A contributes to the production of IFITM1 and other antiviral ISGs. Interestingly, many ISGs are unaffected by perturbation of the m6A machinery, suggesting a direct effect of m6A methylation on the mRNA transcripts of individual ISGs. Future studies will focus on the mechanisms by which m6A regulates the production of antiviral ISGs and the overall contribution of m6A to gene expression regulation during the type I interferon response.



Name:  Adam Mefferd
Lab:  Cullen
Conference:  American Society for Gene and Cell Therapy Annual Conference
Date:  May 16-19, 2018
Location:  Chicago, IL
Purpose:  To present a poster

Expression of CRISPR-Cas9 Single Guide RNA’s Using Small tRNA Promoters
The CRISPR-Cas9 DNA editing system has emerged as a powerful tool for the targeting and mutagenesis of specific DNA sequences. The in vivo applications of CRISPR-Cas9 will likely require the use of adenoassociated virus (AAV)-based viral vectors due to their lack of pathogenicity in humans and their ability to be produced at very high viral titers. Unfortunately, AAV vectors are capable of packaging only a modest ~4.7 kb
of DNA sequence which is problematic given the large size of the Cas9 protein and the pol III based cassette necessary for single guide RNA (sgRNA) expression. Much work has been done in order to identify and characterize highly active Cas9 proteins that are significantly smaller than the prototypical Streptococcus pyogenes Cas9 protein. However, little innovation has been described with regard to novel sgRNA expression
methods that are more compact that the typical ~250 bp U6 promoter expression cassette. Here, we report that small, ~70 bp tRNA promoters of human and viral origin can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently cleaved by endogenous tRNase Z to release fully functional sgRNAs. This work has the potential to greatly facilitate the construction of highly effective CRISPR-Cas9
based AAV vectors for in vivo studies and medical interventions.



Name:  Caitlin Murdoch
Lab:  Rawls
Conference:  Beneficial Microbes
Date:  July 8-11, 2018
Location:  Madison, WI
Purpose:  Oral Presentation and to present a poster

Microbiota-induced Serum Amyloid A in the intestine directs systemic neutrophil function
The intestinal microbiota influences diverse aspects of intestinal and extra-intestinal physiology, including the development and function of myeloid lineages. Numerous host factors are known to poise neutrophils and other granulocytes for response to pathogens and danger signals through a process known as priming. However, mechanisms by which intestinal microbiota regulate neutrophil priming and innate immunity have remained elusive. Using gnotobiotic zebrafish, we identified the immune effector Serum Amyloid A (Saa) as the most highly induced transcript in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however Saa’s functions in vivo remain poorly understood. Using CRISPR/Cas9 gene editing in zebrafish, we generated the first entirely Saa-deficient vertebrate, providing novel in vivo insights into Saa’s effects on neutrophil priming and immunity. Zebrafish saa mutants displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa deficient zebrafish exhibited moderate neutrophilia and altered neutrophil tissue distribution. RNA profiling of flow-sorted neutrophils from wild-type and mutant zebrafish larvae revealed Saa suppresses pro-inflammatory transcripts. Further, ex vivo co-culture assays showed that Saa attenuates the bactericidal activity of neutrophils. We observed that Saa’s effect on neutrophils depends on microbiota colonization, implicating this protein in mediating the microbiota’s influence on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we engineered transgenic zebrafish which over-express saa in the intestine. Transgenic expression of saa in the intestine of saa mutants was sufficient to partially complement the observed neutrophil phenotypes. These results indicate that Saa produced by the intestine in response to microbiota, signals to extra-intestinal neutrophils to restrict aberrant priming, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to sites of injury.Genetic analysis of colonization fitness for a zebrafish gut commensal
A plethora of high-throughput sequencing studies have illustrated that distinct microbial communities colonize diverse vertebrate mucosal surfaces including the intestinal tract. These microbiota impact many aspects of host physiology including metabolism, behavior, and immune development. However, a majority of gut microbes are genetically intractable and thus the mechanisms that mediate their colonization and influence on host remain undefined. Previous studies from our lab have identified a Firmicutes species, Exiguobacterium acetylicum, that promotes lipid absorption in larval zebrafish. Lacking genetic tools, we constructed a draft genome sequence and generated a library of isogenic mutants using EMS chemical mutagenesis. By integrating in vitro motility assays and high-throughput sequencing, we identified novel genes implicated in bacterial motility. Currently, we are defining the essential gene set for E. acetylicum both in rich-media culture and in zebrafish by performing parallel in vitro and in vivo competitions of large pools of E. acetylicum mutants. These experiments have identified several strains that are differentially enriched specifically in vivo. We are elucidating the ability of different strains to colonize the intestine in subsequent studies with gnotobiotic zebrafish utilizing a combination of pairwise in vivo competitions and high resolution live imaging of labeled strains. Interestingly, we have isolated a non-motile mutant strain that hyper-colonizes in vivo, and preliminary evidence suggests that a motile isogenic suppressor strain is depleted. Our data indicate that certain phenotypic traits traditionally thought to be positively associated with successful colonization, such as motility, may not be the primary drivers of successful colonization. Collectively, our data demonstrate in vivo screening of mutant libraries can be used to identify novel genes that promote colonization. More broadly, this experimental platform could be applied to other genetically intractable gut isolates.__________________________________________________________________________


Name: Victor Ocasio
Lab:  Valdivia
Conference:  ASM Microbe
Date:  June 7-11, 2018
Location:  Atlanta, GA
Purpose:  To present a poster

pH Mediated Regulation of Mcsc-Dependent Effector Secretion in Chlamydia
The obligate intracellular pathogen Chlamydia trachomatis, causes sexually
transmitted infections; which, if left untreated, can result in infertility, ectopic pregnancy
and pelvic inflammatory disease. Chlamydia delivers type 3 secretion (T3S) effector
proteins to target different host cellular processes to enable invasion, intracellular
residence and evasion of innate immune responses. Secretion of T3S effectors is
facilitated by specialized chaperones. Mcsc, for instance, interacts with several
Chlamydia effectors secreted early during the first few hours post invasion. We
hypothesize that Mcsc plays a critical role in establishing a hierarchy to the secretion of
these early effectors. Biochemical and structural work suggests that the binding of Mcsc
to its cognate early effector cargo is regulated by pH. Isothermal calorimetry
experiments show that under mild acidic conditions, the binding affinity of chaperone to
effector is weakened. Additionally, structural analysis of Mcsc in complex with the
effector Ct618 or Cap1, revealed that a tyrosine and aspartate residue of Mcsc engages
in hydrogen bonding with a conserved histidine residue found in both, Ct618 and Cap1,
potentially stabilizing this interaction. Based on these observations, we hypothesize that
subtle variations in pH at the base of an active T3S apparatus influences protein-protein
interaction, thus differentially affecting the affinity of each effector for Mcsc and
establishing a hierarchy to their secretion. To determine how pH influences secretion of
effectors into the host cytosol during Chlamydia infection, we generated genetic
reporters of Cap1 translocation consisting of the glycogen synthase kinase (GSK),
which is phosphorylated only in the host cytosol. Preliminary data shows that changing
the media to a lower pH enhanced the secretion of Cap1 without altering the steady
state levels of total recombinant protein produced. Furthermore, mutations of the
conserved histidine destabilized Cap1, presumably due to poor binding to the
chaperone. These findings support a model wherein changes in pH affect the nature of
interaction between Cap1 and Mcsc, weakening protein binding and facilitating the
process of translocation of Ct618 and Cap1.



Name:  Kaila Pianalto
Lab:  Alspaugh
Conference:  Gordon Research Conference: Cellular & Molecular Fungal Biology
Date:  June 16-22, 2018
Location:  Holderness, NG
Purpose:  To present a poster

Multifunctional Nap1 Promotes Rim Alkaline pH Pathway Signaling in Cryptococcus neoformans through Protein Stability Maintenance
A crucial adaptation for many pathogenic organisms is the transition from the environment to the host. In the case of the fungal pathogen Cryptococcus neoformans, one of many changes during this transition is the shift from the acidic external environment to the relatively alkaline pH of the human host in order to establish infection. The Rim alkaline pH signaling pathway is crucial for adjusting to this elevated pH in this organism. Our lab has identified downstream components of this signaling pathway through homology with proteins from other fungi, but many of the upstream components lack homologous genes in C. neoformans. Through an immunoprecipitation experiment using the most upstream known component of this pathway, the putative pH sensor Rra1, we have identified a novel interactor of the Rim pathway, Nucleosome Assembly Protein 1 (Nap1). Nap1 is required for Rim pathway activation based on several hallmark phenotypes of Rim pathway activators. Through epistasis experiments, this protein acts to activate the Rim101 transcription factor upstream of other known pathway components, aligning with its specific interaction with the Rra1 protein. Nap1 is likely playing a role in the maintenance of Rra1 protein levels within the cell, stemming from its interaction with the unstructured C-terminus of the Rra1 protein. Additionally, preliminary proteomics and localization results suggest a novel mechanism for Rra1 regulation via internalization of the protein under activating conditions. Future work will explore Rra1 protein regulation through protein trafficking pathways within the cell. Additionally, current experiments are probing the mechanism behind Rra1-Nap1 interactions and the impact that these interactions have on Rra1 protein function within the Rim pathway. Given that the role of Nap1 as a Rim pathway activator has never before been described, current studies are also determining whether this role of Nap1 is Cryptococcus-specific, or if this role occurs more broadly among basidiomycete fungi, including the skin commensal and pathogen Malassezia furfur. This work is making advances in the understanding of how this important fungal pathogen senses and responds to changes in its environment, giving insight into mechanisms of pathogenesis in this organism.



Name: Dia Beachboard
Lab: Horner
Conference: American Society for Virology Conference
Date: June 24-28, 2017
Location: Madison, WI
Purpose: Talk

RAB1B positively regulates antiviral innate immunity by activating IKK-epsilon
The antiviral innate immune response is activated when pattern recognition receptors, such as
RIG-I, sense viral RNA. RIG-I signals through the adaptor protein, MAVS, to activate kinases,
including IKKε, for the transcriptional induction of type I interferons (IFN). We have previously
identified RAB1B, a GTPase required for ER-Golgi vesicle trafficking, as interacting with MAVS,
suggesting that it regulates antiviral innate immune signaling. To test this, we overexpressed
RAB1B and found that RAB1B enhances RIG-I signaling to IFN-β. Next, we generated RAB1B
knockout cells (KO) using CRISPR/Cas9 genome editing. Using these cells, we found that
following RIG-I activation, loss of RAB1B results in less phosphorylation of IRF3, decreased
signaling to IFN-β, and increased RNA virus replication. To define the step of the IFN-induction
signaling cascade where RAB1B functions, we overexpressed constitutively active innate
immune signaling molecules in RAB1B KO cells and tested for restoration of RIG-I signaling to
IFN-β. We found that overexpression of IKKε restored signaling to IFN-β in the RAB1B KO cells,
demonstrating that RAB1B likely modulates the function of IKKε. IKKε function is known to be
activated by unanchored K48-linked ubiquitination. Therefore, to test if RAB1B is required for
IKKε ubiquitination, we performed IKKε ubiquitination immunoprecipitations, and found that
RAB1B is required for IKKε ubiquitination. In addition to acting in the IFN-induction pathway,
IKKε also acts in the IFN-response pathway by phosphorylating a specific residue in STAT1
resulting in induction of a subset of IKKε-dependent IFN-stimulated genes (ISGs). Importantly,
we found that loss of RAB1B specifically prevents induction IKKε-dependent ISGs in response
to type I IFN. Taken together, these data reveal that RAB1B is a novel regulator of IKKε
activation, which promotes IFN-induction and restriction of RNA virus infection.


Name:  Xiou Cao
Lab:  Aballay
Conference:  47th Annual Meeting, Neuroscience 2017
Date:  November 11-15, 2017
Location:  Washington, DC
Purpose:  To present a poster

Neuronal basis of the adrenergic receptor OCTR-1 in regulating the innate immune response of Caenorhabditis elegans
Increasing evidence from studies in neurobiology and immunology implies an extensive and universal interaction between the nervous and immune systems, which is responsible for organismal control of immune homeostasis. In contrast to the complexity of mammalian systems, the simple model organism C. elegans
has been validated as a powerful tool to study host-pathogen interactions and investigate the principles of neural regulation of immunity. Previous studies in our laboratory have shown that OCTR-1, a neuronal G protein-coupled receptor (GPCR) analogous to human norepinephrine receptors, functions in two sensory
neurons, ASH and ASI to control the gene expressions of both the microbial killing pathways and the unfolded protein response (UPR) in C. elegans. To examine the precise molecular and neuronal mechanism of the regulation of pathogen defenses, we performed the targeted ablation of each neurons and found that OCTR-1-expressing neurons, ASH, are involved in controlling the resistance to pathogen infections. In contrast, another group of OCTR-1- expressing neurons, ASI, were shown to promote pathogen avoidance behavior.
Through the analysis of the previous microarray data, we were able to identify a neuropeptide gene, nlp-20 that is upregulated in octr-1(ok371) mutant and functions downstream of OCTR-1 to control the innate immune response in C. elegans. In addition, interneurons AIA were found to modulate immune response
and function as a putative linker between OCTR-1-expressing and NLP-20- expressing neurons. Taken together, these data reveal the downstream molecules that are responsible for OCTR-1-mediated immune regulation and provide new insights into the neuronal network involved in regulating the pathogen defense response in C. elegans.


Name:  Zanetta Chang
Lab:  Heitman
Conference:  7th FEBS Advanced Lecture Course on Human Fungal Pathogens
Date:  May 13-19, 2017
Location:  La Colle sur Loup, France
Purpose:  To present a poster and an elevator talk

Drug resistance via RNAi-dependent epimutations in Mucor circinelloides 
The opportunistic fungal infection mucormycosis is notable for high mortality as well as increasing incidence. Treatment is complicated by the fact that Mucor circinelloides, a major cause of mucormycosis, demonstrates high intrinsic resistance to most antifungal agents. However, the mechanisms driving this extensive resistance remain poorly understood. Previous work demonstrated that Mucor is capable of developing transient resistance to the antifungal FK506 through a novel, RNA interference-dependent mechanism known as epimutation. Epimutants silence the drug target gene and can be selected by exposure to FK506; the target gene is re-expressed in these strains following passage without selective pressure. This silencing process involves the generation of small RNAs (sRNA) against the target gene via core RNAi pathway proteins. To further investigate the role of epimutation in Mucor’s intrinsic antifungal resistance, we studied the development of resistance to a second drug, 5-fluoroorotic acid (5-FOA). We have identified epimutants that exhibit resistance to 5-FOA without mutations in either of the target genes, pyrF or pyrG. We conducted sRNA hybridization analysis to document the presence of sRNA against pyrF or pyrG in these epimutants, and that this sRNA is lost after reversion to drug sensitivity. Analysis of sRNA libraries generated from these epimutants demonstrated expression of sRNA against the pyrF and pyrG loci, respectively. From this, we conclude that epimutation is a general mechanism through which Mucor can develop resistance to multiple antifungal agents.


Name:  Sarela Garcia-Santamarina
Lab:  Thiele
Conference:  10th International conference on Cryptococcus and Cryptococcosis
Date:  March 26-30, 2017
Location:  Foz do Iguacu, Brazil
Purpose:  To present a poster

At the cross-roads of copper and iron-sulfur clusters as virulence factors in the human
fungal pathogen Cryptococcus neoformans
The copper (Cu) homeostasis machinery is important for virulence in the fungal pathogen Cryptococcus neoformans. Using mRNA-seq and ChIP-seq, our laboratory has demonstrated a unique rich biology of Cu homeostasis mechanisms in C. neoformans, highlighting the importance of having a fine-tuning control of Cu-related pathways in this fungus. In C. neoformans transcriptional responses to changes in Cu concentrations are mediated by the transcription factor Cuf1, which activates, amongst many others, the Cu detoxifying Cmt1/Cmt2 metallothioneins in response to high Cu concentrations or the Ctr1/Ctr4 Cu importers under Cu deficiency. This discovery is of great relevance to pathogenesis, as during the course of an infection, C. neoformans needs to adapt to extreme and variable host Cu environments. In the initial stages of an infection, lung innate immune cells use Cu as part of their anti-pathogenicity arsenal, resulting in C. neoformans inducing the expression of the metallothionein proteins Cmt1 and Cmt2, critical to protect the fungus from Cu toxicity, as revealed by their requirement for full virulence in intranasal mouse models of infection. However, during brain colonization, C. neoformans activates expression of the Cu importers Ctr1 and Ctr4, which are required for virulence in intra-cerebral mouse models of lethal meningitis, suggesting that Cu is scarce in this environment. Here I present our studies focused on a novel Cuf1 direct target gene, induced under high Cu concentrations, which is homologous of the Saccharomyces cerevisiae mitochondrial ABC transporter Atm1, which exports an uncharacterized Fe-S cluster precursor from the mitochondria to the cytosol. Classically, due to its redox properties, Cuassociated toxicity was attributed to an increase in the intracellular levels of reactive oxygen species, which irreversibly damage DNA, lipids, and/or proteins. More recently, Cu toxicity mechanisms were revisited, and it has been suggested that iron-sulfur (Fe-S) cluster interference is a major mechanism for Cu toxicity in bacterial cells. Consistent with the latter hypothesis, we present evidence for a novel mechanism of protection against Cu toxicity in C. neoformans, where Cu activation of ATM1 expression contributes to C. neoformans Cu resistance by maintaining Fe-S cluster homeostasis, even in the presence of the highly induced and high affinity Cu-binding metallothioneines. Results from this work have revealed that ATM1 induction under Cu stress is important for survival to the high Cu environment found in the interior of a phagolysosome, and therefore is potentially required for the full virulence of this organism during host infection, highlighting the relevance of the several layers of mechanisms that this organism has uniquely adapted to survive high Cu environments.


Name:  Yi-Hong Ke
Lab:  Vilgalys
Conference:  Workshop on Molecular Evolution
Date:  July 20-30, 2017
Location:  Woods Hold, MA
Purpose:  Training and Networking

Genetic basis of mating in ectomycorrhizal bolete Suillus brevipes
Understanding genetic control of the fungal life cycle is critical for understanding fungus-host interactions. For many groups of symbiotic fungi, transitions in fungal life cycle are highly integrated with changes in gene expression and development of both the fungus and its host. For example, to complete their sexual cycle, rust fungi form as many as five distinctive life cycle stages on multiple plant hosts. Similarly, mating/dikaryotization and are strongly linked to pathogenesis/colonization for many pathogenic fungi including the corn smut fungus Ustilago maydis and the human pathogen Cryptococcus gattii. Though genetic basis of life cycle, mating system and mating types in many pathogenic fungi are well documented, these
features of other symbiotic mutualistic fungi are less understood due to difficulties in manipulations, including ectomycorrhizal fungi (EMF) that are essential for nutrient uptake by many forest trees and shrubs. The initiation of dikaryotization and mating in Fungi is controlled by MAT loci that define different mating types. In this study, we employed genomic shotgun sequencing data to recover haplotypes of the HD MAT locus from bolete mushroom Suillus brevipes. The HD MAT locus of S. brevipes only contains a pair of homeodomain encoding HD protein. De novo assembly of shotgun sequence data shows 32 distinct alleles among 25 dikaryotic isolates, where heterozygotes in both HD1 and HD2 are consistently found. Comparison of different
haplotypes shows that the same HD1 allele always coexists with the same HD2; multipartite linkages are not observed. Phylogenetic analysis of HD1 and HD2 coding regions reveals an early origin of HD MAT alleles with a long history of trans-specific polymorphism. Our results confirm the high mating type diversity under balancing selection and restricted recombination within the HD MAT locus, which indicates its functions in mating compatibility and dikaryotization. Phylogenetic incongruence between HD1 and HD2 phylogenies surprisingly implies that recombination also contributes to the generation of MAT allele diversity.


Name:  Charmaine Mutucumarana
Lab:  Permar
Conference:  IAS 2017
Date:  July 23-26, 2017
Location:  Paris, France
Purpose:  To present a poster

Maternal Humoral Immune Correlates of Mother to Child Transmission of HIV-1 in the Setting of Peripartum Antiretrovirals
Background: More than 150,000 annual pediatric HIV-1 infections occur due to mother to child transmission (MTCT) despite the availability of antiretrovirals (ARVs). In the pre-ARV era U.S. Women and Infants Transmission study (WITS), we previously reported that maternal HIV envelope-specific anti-V3 IgG, CD4 binding site antibodies, and tier 1 virus neutralization predicted reduced HIV-1 MTCT. As the majority of pediatric HIV infections occur in clade C HIV-infected populations with increased access to ARVs, we studied a Malawian HIV-infected pregnant women cohort from the Breastfeeding, Antiretrovirals, and Nutrition (BAN) study. We sought to determine if immune factors in the setting of ARVs predict reduced MTCT and help eliminate pediatric HIV-1.
Methods: Plasma from a subset of BAN clade C HIV-infected Malawian mothers (n=88, 45 transmitting and 43 non-transmitting) and their infants were studied. Women and infants received ARVs at delivery, and the majority of peripartum MTCT was during pregnancy (91%). Binding antibody multiplex assays, HIV-1 neutralization assays, and soluble CD4 blocking ELISAs measured plasma IgG against multiclade HIV Env antigens, neutralizing capacity, and CD4 binding site
antibodies, respectively. A multivariable logistic regression model analyzed the association of maternal and infant immune responses with peripartum MTCT risk.
Results: No significant association was detected between maternal anti-clade C V3 IgG (OR 0.57, p=0.42) or tier 1 neutralization (OR 1.37, p=0.70) and MTCT. Surprisingly, plasma blocking of the CD4 binding site (OR 1.06, p=0.03) and maternal anti-clade C V1V2 IgG (OR 1.62, p=0.04) were associated with increased MTCT independent of maternal viral load. Maternal anti-V1V2 IgG
transfer efficiency to infants was not associated with transmission (OR 1.00, p=0.67).
Conclusions: This study revealed an association between high maternal CD4 binding site antibodies and anti-V1V2 IgG and transmission. Distinct humoral immune correlates of MTCT in the BAN and previous studies could be due to differences between transmission mode, virus clade, or maternal antiretroviral use. The association between specific maternal antibody responses and in utero transmission, distinct from potentially protective maternal IgG in the WITS cohort, underlines the importance of investigating additional cohorts with well-defined transmission modes to understand the role of maternal antibodies during HIV-1


Name:  Eric Walton
Lab:  Tobin
Conference:  ZDM10 (Zebrafish Disease Models Society)
Date:  August 5-8, 2017
Location:  San Diego, CA
Purpose:  To present a poster

Mycobacterial lipid modifications regulate vascularization and infection outcome 
The zebrafish model of mycobacterial infection recapitulates important aspects of Mycobacterium tuberculosis pathogenesis in an optically and genetically tractable host. Vascularization of granulomas has long been observed in humans, but its role in pathogenesis has not been fully defined. In previous work, we found that granuloma-associated angiogenesis promotes mycobacterial proliferation, suggesting that this angiogenesis may be driven in part by the pathogen itself. In order to understand how pathogenic mycobacteria accelerate bacteria-beneficial angiogenic responses in their hosts, we have taken a bacterial genetic approach. We identified a set of transposon mutants that produces largely avascular infection foci during otherwise stereotypical infections of zebrafish larvae. We show that specific bacterial modifications to cell wall lipids are required for robust granuloma-associated angiogenesis. We will present data on how bacterial engagement of host signal transduction pathways mediates this host response and promotes infection. Modulation of these pathways, both in bacteria and host, may suggest potential new targets for therapeutic interventions in tuberculosis.


Name:  Guoyong Xu
Lab:  Dong
Conference:  5th International Conference on Biotic Plant Interactions (5th ICBPI)
Date:  August 17-21, 2017
Location:  Xiamen, China
Purpose:  To present a poster

In the absence of specialized immune cells, the need for plants to reprogram transcription to transition from growth-related activities to defense is well understood. However, little is known about translational changes that occur during immune induction. Using ribosome footprinting (RF), we found that during pattern-triggered immunity (PTI), translational reprogramming is a fundamental layer of immune regulation. Further investigation of these genes with altered translational efficiency (TE) showed that mRNA sequence features are major determinants of the observed TE changes. In the 5’ leader sequences of transcripts with increased TE, we found a highly enriched mRNA consensus sequence, R-motif, consisting of mostly purines. We showed that R-motif regulates translation in response to PTI induction through interaction with poly(A)- binding proteins. Besides R-motif, we also found that upstream open reading frames (uORFs) are an important RNA feature conferring stringent translational control of downstream major ORFs (mORFs). Using the two uORFs within the 5’ leader sequence of TBF1, an important transcription factor for the growth-to-defense switch upon immune induction, we demonstrate that inclusion of the uORFsTBF1-mediated translational control over the production of snc1 (an autoactivated immune receptor) in Arabidopsis (At) and
AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field, resolving the conflict of compromising plant growth with enhanced resistance. This broadly applicable new strategy may lead to reduced use of pesticides and lightening of selective pressure for resistant pathogens.


Name:  Sophia Zebell
Lab:  Xinnian Dong
Conference:  5th International Conference on Biotic Plant Interactions (5th ICBPI)
Date:  August 17-21, 2017
Location:  Xiamen, China
Purpose:  To present a poster

Non-canonical phosphorylation regulates RBR1 induction of Arabidopsis Effector-Triggered Immunity
Programmed cell death (PCD) is a highly conserved phenotype of effector-triggered immunity (ETI) to biotrophic plant pathogens. Although much is known about the receptor-mediated perception of pathogen effectors, downstream factors that coordinate a specific PCD gene expression program have yet to be identified. We have previously shown that during Arabidopsis thaliana ecotype Columbia interactions with the oomycete
pathogen Hyaloperonospora arabidopsidis EMWA1 and the bacterial pathogen Pseudomonas syringae carrying the effectors AvrRpt2 and AvrRps4, the cell-cycle checkpoint regulator RETINOBLASTOMA-RELATED protein (RBR1) is hyperphosphorylated [1]. This likely leads to an increase in active E2F-family
transcription factors, which, in addition to their role as regulators of cell-cycle progression, we have shown to be essential for robust effector-triggered PCD, in a partially redundant manner. In this study, we further characterize the hyperphosphorylation of RBR1 during plant immunity by phosphoproteomic enrichment mass spectrometry and pharmacological kinase manipulation. We find that RBR1 phosphorylation during ETI is not accomplished by the canonical cell-cycle related Cyclin/Cyclin Dependent Kinase pairs, and utilize a proximity-labeling mass spectrometry approach to identify interactors of RBR1 during ETI. Of particular interest
among these are potential novel, ETI-specific RBR1 kinases. Our finding of a cell-cycle related signaling module appropriated for immune signaling suggests interesting parallels to other immune-related programmed cell deaths, including mammalian pyroptosis, as these same regulators have a role in apoptotic regulation in mammals.


Name: Hannah Brown
Lab: Alspaugh
Conference: 10th International conference on Cryptococcus and Cryptococcosis
Date: March 26-30, 2017
Location: Foz do Iguacu, Brazil
Purpose: To present a poster

Identifying Upstream Components of a Fungal Alkaline Response Pathway
C. neoformans grows best in the acidic conditions found in its natural environment, but must adapt to
relatively alkaline conditions in the human host. This pathogen utilizes components of the fungal-specific
Rim pathway to regulate adaptation to alkaline pHInterestingly, the upstream components of the C.
neoformans Rim pathway, including much of the membrane pH-sensing complex, remain undefined. Our
lab has recently identified a novel upstream component of the C. neoformans Rim pathway, Rra1. Rra1
shares structural similarity to known pH sensing membrane receptors, suggesting that this protein may
function as a component of the Rim pathway pH sensing complex. This also suggests that there are
additional basidiomycete-specific upstream components of the C. neoformans Rim pathway that sense and
respond to changes in pH. Using a random insertional mutagenesis approach, I will identify novel proteins in
the C. neoformans Rim pathway that are responsible for sensing changes in pH. In preliminary studies, I
have screened through a newly created library of 10,000 insertional mutants, identifying strains that are
hyper-sensitive to alkaline pH. I have also defined which of these pH-sensitive strains have a growth-related
phenotype that is rescued by the expression of a constitutively active Rim101 protein. In this way, I have
enriched my mutant pool for strains containing mutations specific for Rim signaling defects, rather than nonspecific
pH sensitivity. I have also completed a similar screen of a library of 2100 known deletion mutants. I
will further prioritize mutants from both screens through phenotypic analysis of altered growth and virulence
similar to a Rim pathway mutant. Simultaneously, I will identify proteins interacting with known components
of the C. neoformans Rim pathway through protein immunoprecipitation of epitope tagged strains. My
preliminary studies suggest a previously undefined association between membrane lipid composition and
pH sensing. These results will elucidate the molecular interactions that drive environment-sensing in
basidiomycetes, which will provide insight into clinically relevant drug targets that can weaken C.
neoformans virulence in the human host.


Name: Caitlin Esoda
Lab: Kuehn
Conference: ASM Microbe 2017
Date: June 1-5, 2017
Location: New Orleans, LA
Purpose: To present a poster

Title: Pseudomonas aeruginosa leucine aminopeptidase modulates bacterial biofilm formation on a host cellular substrate
Abstract: Pseudomonas aeruginosa is a Gram negative bacterium known for its ability to cause infection in variety of immunocompromised patients, most notably those suffering from cystic fibrosis (CF). While numerous P. aeruginosa virulence factors have been identified, the role that many secreted bacterial proteins play in pathogenesis remains unknown. Through the study of bacterial outer membrane vesicles, our lab has identified a secreted leucine aminopeptidase, PaAP, that is highly expressed in clinical isolates form CF patients. This expression profile indicates that the aminopeptidase may play a role in P. aeruginosa virulence during chronic CF-associated infections. Central to P. aeruginosa pathogenesis is the bacterium’s ability to form robust antibiotic-resistant biofilm colonies on the surface of lung epithelial cells. We have found that deletion of this aminopeptidase leads to the formation of more robust early biofilm structures on human epithelial cell surfaces in a co-culture infection model. These data indicate that PaAP may serve as a type of “anti-virulence” factor, modulating bacterial pathogenesis mechanisms to aid in the establishment of long term host-bacterial interactions. Additionally, we have demonstrated that PaAP is able to interact directly with human lung epithelial cell components, providing a potential mechanism of action for this effect. Current work for this study is focused on defining PaAP’s host interaction partners, as well as the mechanism behind it’s modulation of bacterial biofilm formation.


Name: Ci Fu
Lab: Heitman
Conference: 29th Fungal Genetics Conference
Date: Mar14-19, 2017
Location: Pacific Grove, CA
Purpose: To present a poster

Cryptococcus neoformans and Cryptococcus deneoformans are two sibling species belong to the opportunistic human fungal pathogen Cryptococcus species complex. C. neoformans mainly undergoes bisexual reproduction, whereas C. deneoformans undergoes both unisexual and bisexual reproduction. During both sexual cycles, a dramatic yeast-to-hyphal morphological transition takes place and generates either monokaryotic or dikaryotic hyphae, and this process is regulated by a common set of genetic circuits. The unisexual cycle can also generate genotypic and phenotypic diversity de novo. Despite the similarities between unisexual and bisexual cycles, there are still genetic and morphological differences, such as an absence of an opposite mating partner and monokaryotic instead of dikaryotic hyphae. The natural populations for both species are mainly α mating type; however, the unisexual cycle is well established in C. deneoformans but not in C. neoformans, suggesting differences in mating mechanisms may contribute to this. To understand what distinguishes the different modes of sexual cycles, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and nuclear fusion. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both Cryptococcus species, and showed that they play conserved roles in cell fusion and karyogamy during sexual reproduction. The involvement of Prm1 and Kar5 indicates that there are fundamental differences between unisexual and bisexual reproduction, and between bisexual reproduction of the two sibling species. Cell fusion and karyogamy are largely dispensable for unisexual reproduction, and the unisexual cycle achieves diploidization early during hyphal development, likely through endoreplication. During the bisexual cycles, in C. deneoformans, karyogamy occurs early during the hyphal development whereas it occurred inside the basidium in C. neoformans.


Name: Masashi Kanayama
Lab: Shinohara
Conference: the 39th Annual Meeting of the Molecular Biology Society of Japan (MBSJ)and the 45th Annual Meeting of the Japanese Society for Immunology (JSI)
Date: Nov 30th, 2016- Dec 2nd, 2016 (MBSJ) and Dec 5th-7th, 2016 (JSI)
Location: Yokohama, Japan (MBSJ) and Okinawa, Japan (JSI)
Purpose: To present a poster

Strategy of Zygomycete fungi to evade anti-fungal immune responses

Zygomycosis (also called as mucormycosis) is a severe fungal infection caused by Zygomycetes
such as Mucor circinelloides and Rhizopus oryzae. Despite the rapid progression, dismal prognosis, and
high mortality of zygomycosis, the pathogenesis is poorly understood. Therefore, diagnosis and
treatments for the infection are extremely limited. Here, we demonstrate that infections with
Zygomycetes do not induce strong innate immune responses both in vivo and ex vivo. This is may be a
reason mice infected with Zygomycetes die significantly quicker than mice infected with other fungal
pathogens such as Candida and Cryptococcus. As an immune evasion mechanism of Zygomycetes, we
found that the fungi secrete a protein (termed ZIRF, Zygomycetes Immune Regulatory Factor), which
strongly inhibits TLR2-mediated signaling in mouse and human macrophages. Zygomycetes culture
supernatants, which include ZIRF, inhibit host innate immune responses in vivo and ex vivo. ZIRF
secretion by Zygomycetes is inducible by glucose. This may attribute to the high risk of Zygomycosis in
diabetic patients. We also found that the virulence of Zygomycetes correlates with amounts of ZIRF
In sum, ZIRF appears to be a pathogenic factor to evade host anti-fungal immunity.
Understanding the impact and mechanism of ZIRF-mediated host immune regulation may serve to
develop novel diagnostic and therapeutic approaches to treat Zygomycosis. In addition, ZIRF may be
able to treat autoinflammatory and autoimmune diseases.


Name: Dora Posfai
Lab: Derbyshire
Conference: Malaria Gordon Research Conference
Date: July 2-7, 2017
Location: Les Diablerets, Switzerland
Purpose: Poster presentation and maybe a talk

Manipulation of host aquaporin-3 during liver stage Plasmodium infection
The liver stage of the Plasmodium life cycle remains an elusive part of the parasite’s life cycle, and a stage that is
ideal for the development of prophylactic treatments for malaria – a disease that kills over 450,000 people every
year. When Plasmodium parasites are first inoculated into a human host by an infected mosquito, they must
travel to the liver where they undergo morphological changes and rapid asexual replication. The parasites are
then released from the liver and only after this transformation are they able to infect red blood cells and cause the
symptoms of malaria. While the liver stage is obligatory in the Plasmodium life cycle, our understanding of hostparasite
interactions are limited. Through global transcriptional analysis I have identified host aquaporin-3
(AQP3) as a gene that is highly over-expressed upon P. berghei infection and localizes to the parasitophorous
vacuole, the interface between the host and pathogen. I have shown that depletion of AQP3 significantly reduces
parasite load and that treatment of hepatocytes with auphen, a known AQP3 inhibitor of glycerol permeability
halted parasite development. This data suggests glycerol is an important nutrient for the development of
Plasmodium parasites. Because of the high expression in infected cells, this protein is a promising target for the
development of a malaria treatment and mouse studies are in progress to determine if auphen is a effective in
clearing Plasmodium infections in vivo.


Name: Jorge Alejandro Rojas
Lab: Vilgalys
Conference: The 29th Fungal Genetics Conference
Date: March 14-19, 2017
Location: Pacific Grove, CA
Purpose: Poster presentation

A phylogenetic approach based on PCR target enrichment and high throughput
sequencing for fungi: The Ilyonectria complex
Alejandro Rojas, Khalid Hameed and Rytas Vilgalys
Biology Department, Duke University, Durham, NC
The use of high throughput sequencing (HTS) has facilitated the study of
the systematics and phylogenetics of organisms. Nonetheless, non-model
organisms and complex systems in fungi require large datasets to study the
phylogenetic relationships among their members, which has been traditionally
approach by sanger sequencing multiple genes to resolve their taxonomy. The
use of HTS could provide large amounts data for multiple genes suitable for
phylogenetics analyses. The present study used ten unlinked genes, eight nuclear
genes (LSU, SSU, ITS, RPB1, RPB2, TUB, ACT and EF1-a) and two
mitochondrial genes (ATP6 and mitSSU) that can be amplified from a single
individual, using a barcoding approach per individual. We describe a novel
approach using PacBio sequencing to obtain ~1kb sequences, using a multilocus
sequence typing (MLST) approach and the downstream analysis to genotype 100
individuals in single sequencing run, providing enough loci to characterize a
species complex. This approach was applied to Ilyonectria complex, an endophyte
present in cottonwood (Populus trichocarpa and Populus deltoides), but also
associated with black-foot disease on grapevines. The genus Ilyonectria was
recently separated from other Cylindrocarpon-like anamorphs, and 17 species
have been described up to now, most of them associated with disease. The HTSMLST
approach was used on endophytic and pathogenic isolates of Ilyonectria to
resolve the species complex associated with Populus. The methodology could be
applied to study the phylogenetics and systematics of other fungal groups.


Name: Elyse Schmidt
Lab: Taylor
Conference: Keystone Symposia: Mitochondrial Communication (A4)
Date: January 14-18, 2017
Location: Taos, New Mexico, USA
Purpose: To present poster and possible talk

The Role of Irgm1 in Macrophage Metabolism and Mitochondrial Function 
The Immunity Related GTPases (IRGs) are a family of IFN-γ induced proteins that mediate diverse immune responses. In mice, the absence of one particular IRG, Irgm1, has a profound impact on susceptibility to bacterial pathogens, such as Salmonella typhimuriumListeria monocytogenes, and Mycobacteria sp. Previous work has attributed the bacterial susceptibility in Irgm1-deficient mice to reduced bactericidal activity of immune cells, leading to unrestricted bacterial growth, increased inflammation, and ultimately death of the host. In this study, we address an alternative hypothesis – that Irgm1-deficiency drives a set of metabolic and mitochondrial alternations that then impact inflammatory cytokine production in our macrophage model.
The initial discovery that our IFN-γ stimulated Irgm1-deficient macrophages had increased mitochondrial fragmentation compared to WT suggested these cells had an altered mitochondrial metabolism. Metabolic studies demonstrated that these cells had an increased glycolytic rate, reduced oxidative phosphorylation, and an accumulation of long-chain acylcarnitines – all hallmarks of classical inflammatory macrophage activation. We hypothesized that these cells may exhibit other hallmarks of classical activation, such as the production of inflammatory cytokines. We found increased secretion of the cytokines RANTES and MCP-1 in Irgm1-deficient macrophages, and that this increased secretion was muted by inhibiting glycolysis, ROS production, or fatty acid synthesis. Taken together, these findings suggest that Irgm1 plays a key role in modulating the metabolism and signaling of immune cells in the murine innate immune system.


Name: Justin Silverman
Lab: David
Conference: CoDA Work 2017: The 7th International Workshop on Compositional Data Analysis
Date: June 5-9, 2017
Location: Abbadia San Salvatore, Italy
Purpose: To present poster and talk

A phylogenetic transform enhances analysis of compositional microbiota data
High-throughput DNA sequencing technologies have revolutionized the study of
microbial communities (microbiota) and have revealed their importance in both human
health and disease. However, due to technical limitations, data from microbiota surveys
reflect the relative abundance of bacterial taxa and not their absolute levels. It is well
known that applying common statistical methods, such as correlation or hypothesis
testing, to relative abundance data can lead to spurious results. Here, we introduce the
PhILR transform, a data transform that utilizes microbial phylogenetic information. This
transform enables off-the-shelf statistical tools to be applied to microbiota surveys free
from artifacts usually associated with analysis of relative abundance data. Using
environmental and human-associated microbial community datasets as benchmarks,
we find that the PhILR transform significantly improves the performance of distancebased
and machine learning-based statistics, boosting the accuracy of widely used
algorithms on reference benchmarks by 90%. Because the PhILR transform relies on
bacterial phylogenies, statistics applied in the PhILR coordinate system are also framed
within an evolutionary perspective. Regression on PhILR transformed human microbiota
data identified evolutionarily neighboring bacterial clades that may have differentiated to
adapt to distinct body sites. Variance statistics showed that the degree of covariation of
bacterial clades across human body sites tended to increase with phylogenetic
relatedness between clades. These findings support the hypothesis that environmental
selection, not competition between bacteria, plays a dominant role in structuring humanassociated
microbial communities.


Name: Jogender Singh
Lab: Aballay
Conference: 21st International C.elegans Conference
Date: June 21-25, 2017
Location: UCLA
Purpose: To present poster

Protein aggregation is a major contributor to aging and age-related diseases. The unfolded protein response (UPR) is a stress response pathway that is activated upon increased unfolded and/or misfolded proteins in the endoplasmic reticulum (ER), and enhanced ER stress has beneficial effects on health and lifespan. To understand the effect of mutations on the upregulation of the UPR and subsequent effects on immunity and lifespan in animals, we conducted a forward genetic screen in Caenorhabditis elegans for mutants exhibiting high levels of UPR. We found that UPR upregulation resulted from mutations in the lipoproteins, vitellogenins, which are homologs of human apolipoprotein B-100. Lipoprotein aggregation adversely affected the lifespan of organisms and their response to pathogen infection, effects that are mitigated by the resultant upregulation of the UPR. Our studies suggest that lipoprotein aggregation plays a major role in health and lifespan and that lipoprotein accumulation may contribute to immunosenescence.


Name: Christine Vazquez
Lab: Horner
Conference: Viruses and Cells Gordon Research Conference
Date: May 14-19, 2017
Location: Lucca, Italy
Purpose: To present a poster and talk

Identification of a Riplet-dependent, RIG-I-independent antiviral response regulated by hepatitis C virus NS3-NS4A during infection 
Hepatitis C virus (HCV), a positive-strand RNA virus, has multiple ways to evade the host innate immune system. This evasion is largely mediated by the actions of the HCV NS3-NS4A complex, consisting of a serine protease (NS3) and its membrane targeting subunit (NS4A). NS3-NS4A cleaves multiple host proteins, including MAVS, an adaptor in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I, to block innate immunity. To identify how NS3-NS4A targets Riplet and MAVS for immune control, we performed directed mutagenesis of NS4A. We identified an amino acid (Tyrosine 16, Y16) in the NS4A transmembrane domain that is required for NS3-NS4A cleavage of Riplet, but is not required for cleavage of MAVS. A virus containing the mutation Y16F has decreased replication in the human hepatoma Huh7 cells, which have functional RIG-I signaling. However, this mutation does not impact HCV replication in the related Huh-7.5 cells, which lack functional RIG-I signaling. To determine if RIG-I was the factor mediating this differential replication, we generated Huh7 RIG-I-knockout (KO) cells. Surprisingly, replication of HCV NS3-NS4A Y16F was not restored in the RIG-I KO cells. This suggests that HCV NS3-NS4A Y16F is unable to regulate a RIG-I-independent pathway that limits replication. Additionally, HCV NS3-NS4A Y16F was unable to replicate to the levels of WT in MAVS KO cells, suggesting that the NS4A mutant virus is unable to regulate a MAVS-independent signaling pathway. We found that Huh-7.5 cells have decreased Riplet expression compared to Huh7 cells, suggesting that Riplet restricts HCV NS3-NS4A Y16F replication in Huh7, but not Huh-7.5 cells. To test this, we over-expressed Riplet in Huh-7.5 cells and found that this resulted in differential replication between the wild-type and mutant viruses. Taken together, these data suggest that targeting of Riplet by NS3-NS4A is important for HCV immune evasion and that NS3-NS4A cleaves Riplet to evade a Riplet-dependent, RIG-I-independent antiviral response.


Name: Raul Zavaliev
Lab: Dong
Conference: Intercellular communication in development and disease
Date: July 10-15, 2017
Location: Berlin, Germany
Purpose: Talk and poster presentations

The main route of cell-to-cell communication in plants is through nanoscale channels termed plasmodesmata (Pd). However, the precise role of Pd and cell-cell communication during response to non-viral pathogens is not known. In the present study we employed a previously developed system of controlled Pd manipulation for determining the role of intercellular transport during response to virulent bacterial pathogen Pseudomonas syringae in a model plant Arabidopsis. Our findings show that transport through Pd is critical for the establishment of the systemic acquired resistance (SAR), which is associated with long-distance immune signal translocation from infected to non-infected leaves. In addition, we found that Pd transport is important for short-distance immune signal translocation, as Pd closure in infected leaves leads to increased susceptibility. Such response could be suppressed by application of immune hormone salicylic acid (SA) which restored the resistance to pathogen. By performing tissue specific Pd closure, we found that Pd in mesophyll and vasculature tissues, but not in epidermal tissue, are important for immune response to bacterial pathogen. Further dissection of the dynamics of cell-to-cell communication during infection indicated that the immune signal translocation follows a circadian rhythm, having a 24h period. Gene expression analysis revealed that Pd closure during infection alters the temporal expression pattern of defense genes responsible for accumulation of SA, as well as the circadian regulator of those genes. In conclusion, our data reveal that the immune signaling is differentially regulated both at the temporal and cell type levels. Therefore, we will further focus on determining how the function of the master immune regulator NPR1 is controlled temporally as well as in different cell types.



Name: Katherine Bonnington
Lab: Kuehn
Conference: Bacterial Cell Surfaces Gordon Research Conference
Date: June 26-July 1, 2016
Location: West Dover, VT
Purpose: To present poster

Outer membrane vesicle production potentiates outer membrane remodeling and maintenance in Gram-negative bacteria 
The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell’s successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism to remove unfavorable lipopolysaccharide (LPS) glycoforms except by slow dilution through cell growth. As all Gram-negative bacteria constitutively shed outer membrane vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally-disadvantageous LPS species. As a well-characterized model system, we examined the activation of PhoP/Q and PmrA/B two component systems (TCS) in Salmonella enterica. In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCS modify the LPS lipid A and core, lengthen the O-antigen, and up-regulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induces the addition of modified species of LPS to the OM, down-regulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly-budded OMVs following two sets of rapid environmental shifts or a mock-shifts over time, revealed retention of lipid A species masking the negatively-charged phosphate moieties and the selective loss of palmitoylated species after exposure to moderately acidic environmental conditions.


Name: Joanne Dai
Lab: Luftig
Conference: 17th International Symposium on EBV and Associated Diseases
Date: August 8-12, 2016
Location: Zurich, Switzerland
Purpose: To present poster and talk

EBV persists in resting memory B cells that circulate in the infected host. The germinal center (GC) model of persistent infection posits that, in vivo, the infected cell transits through the germinal center, avoids elimination by apoptosis, and emerges as a latently-infected, quiescent memory B cell. However, some findings suggest that EBV can bypass the GC by directly infecting memory B cells. Nonetheless, we have observed that in vitro infection of naïve and memory B cells mimics several aspects of GC B cells, indicating that a GC-like reaction is inherent to the viral life cycle.
BH3 profiling is used to query apoptotic sensitivity and regulation. This involves exposing intracellular mitochondria and inducing cytochrome C release by adding proapoptotic peptides. To confirm BH3 profiling results, we treated EBV-infected B cells and uninfected GC B cells with ABT-737, a BH3 mimetic which activates intrinsic apoptosis by inhibiting BCL-2, BCL-xL, and BCL-W, but fails to inhibit MCL-1 and BFL-1.
Both human GC B cells and EBV-infected B cells depend upon MCL-1 for survival and, consequently, are resistant to ABT-737. This apoptosis resistance is absent in uninfected B cells that have been stimulated by mitogens to proliferate, suggesting that MCL-1 dependence is virus-specific. We also observed GC-levels of CD38 expression in infected cells and upregulation of several markers that are associated with B cell maturation, such as PAX5, IRF4, BLIMP1, and XBP1. However, there is a significant downregulation of BCL-6, which is necessary for the GC reaction, and CXCR4, indicating that some aspects of the GC are adverse for EBV outgrowth.
These findings show that, to an extent, GC mimicry is important in EBV outgrowth in vitro, with the notable exception of BCL-6. The role of MCL-1 in mediating survival in not just EBV-infected B cells but also in normal GC B cells make it an especially interesting candidate to target in EBV-associated malignancies and GC-derived lymphomas.


Name: Eric Feeley
Lab: Coers
Conference: International Cytokine and Interferon Society
Date: October 16-19, 2016
Location: San Francisco, CA
Purpose: To present poster

The Guanylate binding proteins (Gbps) are a family of GTPases that are strongly upregulated upon interferon-γ stimulation. The Gbp proteins are important for restriction of several pathogens as well as playing a critical role in inflammasome activation. Gbps have been shown to localize to several intracellular pathogens, such as Legionella pneumophilla and Toxoplasma gondii. However the mechanism by which the Gbps are recruited to the pathogen are not well understood. My work has focused on identifying how the Gbp proteins are selectively recruited to pathogens.
I’ve identified the galectin family of proteins as important players in the recruitment of Gbps to the pathogen. Galectins are glycan binding proteins that are primarily expressed in the cytoplasm and on the cell surface of cells. The galectin proteins have a high binding affinity for beta-galctosides. I’ve shown that Galectin 3, in particular, is critical for the proper recruitment of Gbps to pathogens. Galectin 3 has also been shown to be important for numerous other cellular functions, including cell adhesion, autophagy, apoptosis, inflammation, and innate immune function. Intracellular galectin has been shown interact with damaged vacuoles. Because of galectins role in sensing disrupted vacuoles, we proposed that recognition of the disrupted pathogen vacuole may be a key component in the recruitment of Gbps to the pathogen. I’ve shown that macrophages deficient in galectin 3, either by shRNA knockdown or cells derived from galectin 3 knockout mice, have a reduced ability to target Gbps to pathogens. We propose that this damage sensing ability is broadly applicable for the recruitment of host defense proteins.


Name: Nandan Gokhale
Lab: Horner
Conference: 23rd International Symposium on Hepatitis C Virus and Related Viruses
Date: October 11-15, 2016
Location: Kyoto, Japan
Purpose: To present poster and talk

The role of N6-methyladenosine in hepatitis C virus infection 
RNA regulatory controls play a critical role in the life cycle of RNA viruses such as hepatitis C virus (HCV). The RNA modification N6-methyladenosine (m6A) is the most abundant modification of cellular RNA, and regulates RNA structure, stability, translation, and localization, likely through interaction with specific m6A-binding proteins. However, the role of m6A in the life cycle of cytoplasmic, positive-sense RNA viruses such as HCV is completely unknown. We have demonstrated that the HCV RNA genome is modified by m6A in infected hepatocytes, and have identified sites of m6A-modification on the viral genome. To test the effect of m6A on HCV RNA, we mutated a subset of these candidate sites to abrogate methylation, but maintain coding potential. We found that these mutations specifically increase HCV virion production without affecting viral RNA replication. Additionally, siRNA depletion of the cellular m6A-methyltransferases increases viral titers, while depletion of the m6A-demethylase decreases viral titers, suggesting that m6A negatively regulates HCV particle production. Interestingly, during HCV infection, the m6A-binding YTH-domain family proteins YTHDF1, YTHDF2 and YTHDF3 relocalize to cytoplasmic lipid droplets, which are known sites of HCV virion assembly. This change in localization of YTHDF proteins requires productive HCV infection and is not observed in cells replicating subgenomic viral RNAs. Critically, siRNA depletion of each YTHDF protein increases viral titers, without affecting HCV RNA replication, demonstrating that these proteins specifically impact HCV particle production. We found that all three YTHDF proteins bind to HCV RNA, and by using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we have identified candidate binding sites of these proteins on HCV RNA, some of which overlap with sites of m6A modification. Taken together, our data suggest that m6A and the YTHDF proteins negatively regulate HCV assembly through protein-viral RNA interactions.


Name: Cody Nelson
Lab: Permar
Conference: International Herpesvirus Workshop (IHW)
Date: July 23-27, 2016
Location: Madison, WI
Purpose: To present poster and talk

Neutralizing antibodies induce cytomegalovirus genetic bottleneck and protect
against severe congenital disease in a novel rhesus monkey model
Cytomegalovirus (CMV) is the most common cause of congenital infection worldwide,
frequently resulting in infant hearing loss, brain damage, or neurodevelopmental delay.
One potential therapeutic intervention for the elimination of congenital CMV is a
protective maternal vaccine that prevents pregnant women from acquiring CMV and
subsequently transmitting the virus to their child in utero. Previously we demonstrated
that rhesus CMV (rhCMV) can cross the placenta in seronegative pregnant macaques
following I.V. inoculation with a fibroblast-tropic rhCMV strain (180.92) and two
epithelial-tropic strains (UCD52/UCD59), thereby providing a valuable animal model for
investigation of congenital CMV vaccine efficacy. In our initial study, CD4+ T-cell
depletion prior to viral inoculation resulted in a high rate of congenital rhCMV
transmission (4/4 dams) and fetal abortion (3/4 dams). We now report that passive
infusion of highly-neutralizing rhesus IgG (100mg/kg; IC50=24.9ng/mL) prior to I.V.
rhCMV challenge of CD4+ depleted dams reduced peak viral load in maternal plasma
by an order of magnitude (mean 14-days post-infection: 2.09×106 vs. 2.29×105
copies/mL) and provided complete protection against fetal loss (0/3 dams aborted;
p=0.04), yet did not prevent placental rhCMV transmission (2/3 dams transmitted).
Pharmacokinetic analysis demonstrated that neutralization titers of maternal plasma
were highest immediately following IgG infusion, but decreased rapidly to baseline over
7-10 days. Interestingly, amplicon deep sequencing of rhCMV glycoprotein L (gL) and
glycoprotein B (gB) from maternal plasma revealed that infused IgG created a genetic
bottleneck by 2 weeks post-infusion/inoculation, limiting viral replication to a single viral
strain (UCD52) and decreasing the diversity of haplotypes at both loci. Our data suggest
that the presence of highly-neutralizing rhCMV antibodies at the time of inoculation may
influence early viral evolution and limit variability of the virus population. A better
understanding of how antibody-mediated selection impacts placental rhCMV
transmission and fetal outcome will inform future vaccine development efforts.


Name: Hilary Renshaw
Lab: Steinbach
Conference: Molecular Mycology Course at Marine Biological Laboratory
Date: July 12-28, 2016
Location: Woods Hole, MA
Purpose: To present poster and talk

Distinct Roles of Myosins in Hyphal Morphology and Virulence in Aspergillus fumigatus 
Background/Objective: Polarized hyphal growth and septation facilitate invasion of host tissue by A. fumigatus. To better understand these fundamental cellular processes, we have chosen to study myosins, a group of motor proteins categorized into classes based on their structure. A. fumigatus contains one class II (myoB) and one class V (myoE) myosin. The role of myosins in the growth and virulence of a human pathogen has never been explored. Because of myosins’ involvement in critical cellular processes, an understanding of their precise functions and regulation in A. fumigatus will lead to fundamental knowledge of pathogenesis which may help identify novel drug targets.
Methods: We generated two myosin single deletion strains (ΔmyoB and ΔmyoE) and a double deletion strain (ΔmyoB ΔmyoE). Radial growth was assessed on solid media daily for five days and conidiation quantified. Transmission electron microscopy was used to visualize septa and hyphal morphology. Conidia viability was assessed using bis-(1,3-Dibarbituric acid)-trimethine oxanol (DiBAC). Virulence was determined using a persistently immunosuppressed murine model of invasive aspergillosis. Phosphorylated resides were determined by GFP-Trap protein purification of MyoE-GFP, phosphopeptide enrichment, and LC/MS-MS.
Results: While the ΔmyoB showed no significant difference in radial extension, the ΔmyoE and ΔmyoB ΔmyoE strains resulted in a significant defect (p<0.001 at day 5). Both MyoB and MyoE are required for full conidiation (p<0.0001). Deletion of myoE resulted in hyperbranching and loss of polarity. TEM revealed that the cell walls of both the ΔmyoB and the ΔmyoE strains appear normal but the ΔmyoB strain contains incomplete, thicker septa. Septa in the ΔmyoE strain appear wild-type; however staining revealed that deletion of myoE resulted in hyperseptation (p<0.001). In our murine model, both the ΔmyoE strain was hypovirulent (p<0.01). Deletion of myoB resulted in a 2-fold increase in inviable conidia (p<0.05), and deletion of myoB or myoE resulted in significantly delayed germination. Because of the significant radial extension and hypovirulence in the ΔmyoE strain, we became interested in MyoE as a drug target and wanted to understand its regulation. We determined that MyoE is phosphorylated at eight residues encompassing each of its four domains.
Conclusion: We demonstrated that myosins have distinct roles in hyphal morphology and virulence. MyoB and MyoE are required for conidiation. MyoE has a role in preserving hyphal polarity and/or suppressing new growth foci. MyoB is important for proper septa formation, while MyoE may have a role in septa frequency. MyoE is required for virulence in a murine model. We are currently exploring the importance of posttranslational modification (focusing on phosphorylation and acetylation) as a regulation mechanism of MyoE to better understand its role in virulence.


Name: Aaron Smith
Lab: Thiele
Conference: Molecular Mycology Course at Marine Biological Laboratory
Date: July 12-28, 2016
Location: Woods Hole, MA
Purpose: To present poster

Copper is an essential trace element in virtually all forms of life, yet is toxic if left unregulated due to the generation of reactive oxygen species (ROS) via Fenton chemistry or displacement of iron from native solvent accessible Fe-S clusters in proteins. Copper plays a vital role in the pathogenicity and virulence of the human fungal pathogen Cryptococcus neoformans, as it is a cofactor for many virulence determinants such as Cu,Zn-SOD and laccase. However, during the onset of infection within the lung, macrophages exploit the toxic properties of copper by bombarding the invaders with excessive levels of copper within the phagosome upon phagocytosis. C. neoformans responds by upregulating the expression of copper detoxifying metallothioneins which buffer the cytosolic concentrations of excess copper. This balancing act between copper acquisition and detoxification of C. neoformans plays an essential role on the successful establishment of infection within the lung and eventual dissemination to the brain, resulting in lethal meningoencephalitis. Once disseminated to the brain, the pathogen must rely on high affinity copper transporters, Ctr1 and Ctr4, to acquire the scarce micronutrient from this environment.
My current research is focused on understanding the molecular mechanisms involved in competition for copper at the host-pathogen axis. Specifically, I am interested in the acquisition and mobilization of copper within host macrophages upon phagocytosis of C. neoformans. Alternatively, the toxic levels of copper shunted to the phagosome present an acute environmental stimulus that may trigger a copper dependent response in C. neoformans that ultimately results in an outcome of successful establishment of infection. Additionally, recent reports suggest C. neoformans alters many aspects of phagolysosomal maturation, enhancing pathogen survival. I am currently investigating the copper dependent secretome of C. neoformans to identify secreted proteins involving the manipulation and modulation host cellular processes. In a broad sense, my current research project will broaden our current understanding on copper homeostasis between the host and pathogen as well as identify new determinants in C. neoformans virulence.


Name: Ashley Sobel Leonard
Lab: Koelle
Conference: Viral Genomics and Evolution
Date: June 8-10, 2016
Location: Cambridge, UK
Purpose: To give talk

Knowledge of intra-host viral evolution in humans remains limited, although nextgeneration
sequencing (NGS) now offers the unprecedented ability to characterize viral
evolution at this scale. Here, we use serial NGS samples of influenza virus collected from
two human challenge studies to characterize viral evolution during a single infection. In
these studies, 38 healthy volunteers were challenged with egg-passaged influenza A
subtype H3N2 via direct intranasal inoculation. Nasal wash samples were collected over
the following 7 days, and deep sequencing of isolates from these samples and the viral
inoculum was conducted. Analysis of this inoculum indicated that it was genetically
diverse, and enriched with variants suggestive of egg-adaptation. Using measurements of
genetic distance, we show that the viral populations in the challenged subjects underwent
considerable directional evolution towards the reference strain, with egg-adapted viral
variants rapidly declining in frequency in all but one subject. We further infer an in vivo
viral fitness landscape from these serially-sampled NGS data. We inferred these fitness
landscapes by reconstructing viral haplotypes and using haplotype frequency changes
between sampling time points to estimate haplotype-specific fitness values. Analysis of
the resulting fitness landscape suggested the presence of an epistatic interaction between
two variants on the HA gene segment. One of these variants, H172Q on the HA1 peptide,
alters antigenicity and occurs at a key epitope site. The second variant, G75R on the HA2
peptide, affects HA protein stability. The reconstructed fitness landscape indicates that
haplotypes containing the reference amino acids at these sites had higher fitness than
haplotypes containing either variant amino acid. The haplotype containing the both
variant amino acids was significantly less fit than those containing only one variant
amino acid, suggesting epistasis. Moreover, the observation that these variants were
enriched during egg-passage suggests that the fitness landscape for the virus differs
substantially between the in ovo and in vivo environments. Our finding of a putative
epistatic interaction provides further support for epistasis in facilitating influenza’s
adaptive evolution. Together, our results indicate that, given sufficient viral genetic
diversity at the onset of infection, influenza virus can rapidly adapt to a novel host
environment, even during the limited duration of an acute infection.


Name: Max Villa
Lab: David
Conference: Microbial Diversity Course
Date: July 1 – August 17, 2016
Location: Woods Hole, MA
Purpose: To give train and network



Name: Liuyang Wang
Lab: Ko
Conference: American Society of Microbiology Microbe Meeting
Date: June 16-20, 2016
Location: Boston, MA
Purpose: To present a poster and talk

Background: Sepsis is a systemic, deleterious inflammatory response to infection. Sepsis is a
leading cause of death, and identifying sensitive and specific biomarkers could improve diagnosis,prognosis, and treatment. This study reports a novel and robust biomarker for sepsis death based onanalysis combining multiple “omics” datasets.
Methods: Three patient datasets were used to examine the association between genetic,
transcriptional, metabolite, and cytokine markers and sepsis: A GWAS analysis was performed from218 cases of non-typhoidal Salmonellae (NTS) bacteremia patients and 3000 controls. Metabolites,RNA-seq, and cytokine levels were examined in SIRS patients from the previously publishedCAPSOD study and an independent cohort with temporal data, the VAP study (157 samples).
Results: A pathway-based GWAS of NTS bacteremia showed a strong statistical enrichment forSNPs near genes of the methionine salvage pathway. Measurement of the pathway’s substrate, 5′-methylthioadenosine (MTA), in two independent cohorts of sepsis patients demonstrated that plasmalevels are increased in nonsurvivors compared to survivors and controls. High plasma MTA wascorrelated with high levels of inflammatory cytokines (IL-6 and IL-8), suggesting elevated MTA couldmark a subset of patients with excessive inflammation. Finally, we evaluated a machine-learningmodel by combining MTA and other clinical variables and measured approximately 80% accuracy(based on AUC) in cross-validation and testing of independent samples. Notably, MTA alone wasnearly as successful in predicting sepsis outcome.
Conclusions: Based on integrative analysis of panomics approaches, we identified and validated thatMTA is a robust prognostic biomarker of sepsis death. Our approach combining genetic associationdata with biomolecule measurements can shape our understanding of sepsis and lead to the development of accurate biomarkers.


Name: John Withers
Lab: Dong
Conference: International Society for Molecular Plant-Microbe Interactions
Date: July 17-21, 2016
Location: Portland, OR
Purpose: To present a poster and talk

Posttranslational Modifications of NPR1: Dynamic Regulation of Immune Responses through the Interplay of Sumoylation and Phosphorylation
NPR1 is a master regulator of salicylic acid (SA)-mediated basal and systemic acquired resistance in plants. NPR1 translocation into the nucleus is required for 99% of the transcriptomic changes triggered by SA, but how this single protein orchestrates genome-wide transcriptional reprogramming remains a major question. Since NPR1 transcription is not dramatically affected by pathogen challenge, studies of NPR1 have been focused on protein interactors and post translational modifications (PTMs). In response to increases in cellular SA levels, NPR1 activity and degradation are dynamically regulated by the interplay between sumoylation and phosphorylation. Sumoylation of NPR1 is required for phosphorylation at Ser11/Ser15 and switches its association from WRKY to TGA transcription factors, which repress and activate defense genes, respectively. In the absence of SA accumulation, sumoylation of NPR1 is inhibited through phosphorylation at Ser55/Ser59, keeping NPR1 stable and quiescent. Through phosphoproteomic analyses, we have identified a novel phosphorylation site in NPR1. While this phosphorylation event does not affect interactions with previously characterized NPR1 protein partners, it dramatically affects NPR1-dependent defense gene expression. Our results indicate that this PTM may be functioning to regulate the transcriptional activity of NPR1 at defense gene promoters



Name: Argenia Doss
Lab: Aballay
Conference: 20th International C. elegans Meeting
Date: June 24-28, 2015
Location: University of California, Los Angeles Los Angeles, California
Purpose: To present poster and talk

Neural regulation of innate immune response using optogenetics
Previously, our laboratory showed that OCTR-1, a G protein-coupled catecholamine receptor, suppresses the innate immune response in Caenorhabditis elegans . As a result, animals expressing the loss-of-function oct r-1 allele, ok371, exhibited an enhanced resistance to killing by the human opportu nistic pathogen Pseudomonas aeruginosa . Despite OCTR-1 being expressed in five neurons, d ata from our laboratory suggests that OCTR-1 functions in chemosensory neur ons ASI and/or ASH to suppress innate immunity. However, the specific role of ASI and ASH neurons is unclear. Unpublished data from our laboratory showed that AS I neurons do not affect the susceptibility of C. elegans to P. aeruginosa . Therefore, we examined the role of ASH neurons during infection. With the use of optogenet ics, we were able to assess whether activation of ASH neurons via Channelrhodopsin-2 al ters the susceptibility of C. elegans to P. aeruginosa -mediated killing. Preliminary data from our experi ments revealed that activation of ASH neurons during exposure to P. aeruginosa alters the susceptibility of C. elegans to P. aeruginosa -mediated killing. This suggests that ASH neurons c an regulate the innate immune response in C. elegans and OCTR-1 may suppress innate immunity by acting through ASH neurons. Ongoing experiments are directed towards determining how ablation of ASH neurons and OCTR-1 expression i n ASH neurons alters immune response. Currently, our data suggests that ASH neu rons, and less likely ASI neurons, possess an immunoregulatory function. Data from these studies will help clarify the cellular process(es) involved in OCTR-1 ’s regulation of innate immune response in C. elegans.


Name: Barbara S. Sixt
Lab: Valdivia
Conference: 10th Cold Spring Harbor meeting on Microbial Pathogenesis and Host Response
Date: Sept 8 – Sept 12, 2015
Location: Cold Spring Harbor, NY
Purpose: To present poster and talk

A genetic screen identifies the inclusion membrane protein CpoS as a suppressor of host cell death during Chlamydia trachomatis infection 
Chlamydia trachomatis is the etiologic agent of endemic blinding trachom a and a leading cause of bacterial sexually transmitted disease worldwide. I ts obligate intracellular lifestyle and its complex developmental cycle have significantly hampered the development and application of molecular genetic approaches to study its virulence traits and pathog enesis. Hence, while C. trachomatis is well known to modulate the programmed cell death machinery of its host cell, the identity and function(s) of bacteri al anti-death factors are unknown. Our study aimed to exploit recently developed tools for genetic manipulation of chlamydiae to obtain and characteri ze strains with defects in the control of host cell death. In a screen of a library of chemically mutag enized C. trachomatis strain (serovar L2) we identified a strain that induces strongly elevated levels of c ytotoxicity. Our investigations revealed that cell death induction by this mutant: (1) is dependent on live replicating bacteria, (2) is initiated during mid-s tage of infection, (3) can be observed in various cell line s, (4) is of mixed (apoptotic/necrotic) nature, and (5) adversely affects establishment of intracellular Chlamydia -containing vacuoles (“inclusions”) and hence formation of infectious progeny. Through the use of recombinant analysis, plasmid-based complementation, and directed gene disruption the p henotype could be linked to the loss of CpoS ( C hlamydial P romoter o f Cell S urvival), a bacterial secreted effector protein tha t is inserted into the membrane of the Chlamydia -containing vacuole. In conclusion, our study ident ified CpoS as a novel anti- death virulence factor. The discovery of mutants wi th defects in host cell death control will help to clarify the role of this virulence trait in Chlamydia pathogenesis.


Name: Jessica R. McCann
Lab: Seed
Conference: Cold Spring Harbor Microbial Pathogenesis Meeting
Date: Sept 8-12, 2015
Location: Cold Spring Harbor, NY
Purpose: To present poster and talk

Early life colonization with nontypeable Haemophilus influenzae exacerbates juvenile airways disease in mice. 
There is mounting evidence of a connection between asth ma development and colonization with nontypeable Haemophilus influenzae bacteria (NTHi). Specifically, NTHi nasal colonization of infants within 4 weeks of bir th increases the risk of development of asthma in later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respirat ory bacterial antigens when compared to cells derived from infants who do not go on to develop asthma symptoms in childhood. We hypothesized that early life colonization with NTHi promotes immune system reprogramming and development of atypical inflam matory responses. We tested whether NTHi colonization of mice on day of life 3 i nduced or exacerbated juvenile airways disease induced using an ovalbumin (OVA) allergi c model of asthma. We found that while early life NTHi colonization did not induc e allergic airways disease using subimmunizing doses of OVA, animals that were colonized on day of life 3 and subjected to allergy induction had exacerbated airways dis ease as juveniles. Exacerbation was defined by increased cellular infiltra tion into the lung, increased inflammatory cytokines IL-5 and IL-13 in the lung lavage fluid, and increased expression of inflammatory genes in lung tissue in early life colon ized mice compared to mice that had not been colonized as neonates. We found that NTHi -colonization increased respiratory system resistance in response to increasing doses o f bronchoconstrictor medication following OVA immunization and challenge. We plan to characterize the bacterial components and immune effectors that influe nce the increased allergic airways disease phenotypes following early life colonization. O ur ultimate goal is to specifically block early life colonization with NTHi while leaving b eneficial microbiota intact, potentially protecting a subset of the population from developing asthma symptoms during childhood.


Name: Qin Yan
Lab: Fowler
Conference: 2IDWeek 2015
Date: October 7 – 11, 2015
Location: San Diego, CA
Purpose: To present poster and talk

Crif1 Is Associated with Susceptibility to Staphylococcus aureus Infection through Regulating Host Cell Apoptosis
Background: We previously showed that chromosome 8 of A/J mice was responsible for susceptibility to S. aureus infection (Ahn PLOS Pathogen s , 2010). However, the specific genes responsible for this susceptibility are unknown. Methods: Chromosome substitution strain (CSS8, chr. 8 from A/J but otherwise C57BL/6J) and N2 backcross mice (F1 [C8A] × C57BL/6J) were applied. Quantitative trait loc i (QTL) analysis of S. aureus – infected N2 backcross mice was used to identify gene regions on Chr. 8 associated with susceptibility. Genes in the significant QTL region were evaluated using whole genome expression data from 1) S. aureus – infected mice and 2) humans with S. aureus bloodstream infection (BSI). Genes identified by all 3 strategies were further analyzed for their apoptotic function with siRNA knockdown. Results: One QTL region on chr. 8 containing 161 genes was significantly associated with s usceptibility to S. aureus infection (83180780 – 88103009). Of these 161 genes, 7 were differentially expressed in both: a) S. aureus – infected A/J (susceptible) and C57BL/6J (resistant) mice; and b) patients with S. aureus BSI (n=32) and healthy subjects (n= 44) ( Asf1b, Crif1, Dnaja2, Farsa, Inpp4b, Prdx2 and Tnpo2 ). Flow cytometry analysis found increased late apoptosis in bone marrow derived macrophages (BMDMs) from A/J, CSS8 vs C57BL/6J at both naïve (25.8% and 18.7% vs. 12.6%; p<0.05) and S. aureus – infecte d status (23.2% and 16.7% vs 10.2%; p<0.05). Down – regulation of Crif1 by siRNA in BMDMs increased apoptosis (38.3%) as compared with scramble siRNA (17.3%) at the naïve status. Stimulation by S. aureus induced even higher apoptosis, with 40.6% for Crif1 siRN A and 32.3% for scramble siRNA. Finally, real – time PCR confirmed that Crif1 was down – regulated in A/J mice before and post S. aureus – infection. Conclusions: These findings suggest that Crif1 contributes to susceptibility to S. aureus infection in mice by a ffecting host cell apoptosis and indicate its involvement in human response to S. aureus infection.


Name: Rene Raphemot
Lab: Derbyshire
Conference: FASEB Molecular Pathogenesis: Mechanisms of Infectious Disease
Date: July 12-17, 2015
Location: Keystone, CO
Purpose: To present poster and talk

Discovery of novel drug targets in human liver cell s for malaria prevention
About half of the world’s population remains at risk of contracting malaria, which is responsible for approximately one million deaths and socio -economic disparities worldwide. The emergence of Plasmodium drug resistance to current artemisinin- combination therapies is raising major concerns for the tr eatment of malaria and creating a critical need to develop new antimalarial drugs. Most antimalarial drugs target the blood stage of malaria for treatment and the develo pment of drugs targeting malaria’s elusive liver stage for a prophylactic strategy is still limi ted. Indeed, drugs targeting malaria’s liver stage represent effective and potentiall y curative therapies, as they would prevent the progression to the cyclical blood stage and th e clinical manifestation of the disease. Here, we aim to identify unique human targets to prevent malaria infection of liver cells to advance the current biological understandi ng of host-parasite interactions and facilitate drug development for malaria preventio n. To identify novel host genes important for Plasmodium parasite infection of liver cells, we have developed a high- throughput small interfering RNA (siRNA) screening stra tegy for genome-wide interrogation of human liver cells. Initial siRNA screens of the druggable genome performed in HepG2 cells in 384-well plate format hav e revealed several genes that significantly decrease Plasmodium berghei infection (Z-score < −3) when knocked down. We anticipate that this siRNA screening strategy will lea d to the identification of novel host factors that are essential during malaria liver stage infection. Future plans include investigating the function of these host genes to advan ce our understanding of disease pathogenesis.


Name: Sarela Garcia
Lab: Thiele
Conference: 2015
Date: 07/26/2015 to 07/31/2015
Location: Mount Snow in West Dover, VT, USA
Purpose: To present talk and poster

Potential role of a putative lytic polysaccharide m ono-oxygenase (LPMO) in the virulence of the human fungal pathogen Cryptococcus neoformans
Copper (Cu) is an essential trace element in both h uman cells and in the human fungal pathogen Cryptococcus neoformans . C. neoformans is acquired through the lungs and crosses the blood-brain barrier where it causes let hal meningitis. We demonstrated that the C. neoformans Cu detoxification machinery is important for C. neoformans survival to host Cu in lung, yet C. neoformans uses laccase, a Cu-dependent enzyme, to make melanin in the brain as an important virulence fact or. Under Cu limiting conditions C. neoformans induces transcription of Cu importer genes, as well as BIM1. BIM1 deletion results in a growth defect under Cu deficiency cond itions, suggesting a role in either Cu acquisition or in adaptation to Cu deficiency. BIM1 is predicted to be a lytic polysaccharide mono-oxygenase (LPMO) enzyme that fu nctions in chitin degradation and that requires Cu for activity. Chitin is an es sential cell wall homopolysaccharide that is absent in mammals. Despite the apparent lack of substrate in mammalian hosts, LPMOs from other organisms are virulence factors du ring infection. Preliminary studies in our laboratory suggest that the Cu-deficiency re sponse in C. neoformans is important for brain colonization. We describe initial studies to ascertain if Bim1 function is critical for virulence.


Name: Shannon Esher
Lab: Alspaugh
Conference: Molecular Mycology: Current Approaches to Fung al Pathogenesis
Date: June 14-30, 2015
Location: Marine Biological Laboratory, Woods Hole, MA
Purpose: To present talk and poster

Identifying the role of an uncharacterized, Cryptococcus species-specific gene, in regulating the cell wall in response to the host
Cryptococcus neoformans is an opportunistic fungal pathogen that causes life- threatening disease in immunocompromised hosts. The C. neoformans cell wall is a dynamic structure that this fungus carefully controls in response t o its environment. Upon entering the host, C. neoformans dramatically alters its cell wall to facilitate immune avoidance and regulate the host-pathogen interface. We have identified MAR1 , a novel Cryptococcus species-specific gene that is an important regulator of these host-indu ced cell wall changes. We have generated a mar1∆ mutant strain and shown that it has sensitivities to hig h temperature and alkaline pH, in addition to a capsule defect. Interestingly, this capsule defect is due to a defect in polysaccharide attachment to the cell wall, rather than polysaccharide biosynthesis. Using cell wall staining and biochemical assays, we have observed an increase in the immunogenic cell wall components, chitin and chitooligomers, specifically in host-mimicking conditions. By co- culturing C. neoformans with macrophages and assaying the amount of the inflam matory cytokine TNF  secreted, we have shown that the mar1∆ mutant induces 4-5 times more TNF  production than wild type cells. These results suggest that Mar1 is regulating important cell wall changes in response to the host. In its absence, the aberr ant cell wall contains, and likely exposes, more chitin and chitooligomers, resulting in in creased macrophage activation. Future studies will elucidate how Mar1 is regulating these cell wall changes, and what implications they have on the host immune response in vivo .


Name: Yangnan Gu
Lab: Dong
Conference: The 4th international conference on


biotic plant interactions

Date: Aug1-3, 2015
Location: Nanjing, China
Purpose: To present a poster

The 4th International Conference on Biotic Plant Interactions 
In plants, effector-triggered immunity (ETI) is a major defense mechanism and often associated with programmed cell death (PCD). Although NB-LRR recepto rs involved in ETI have been extensively studied, how they activate PCD and disease resi stance remains elusive as plants lack close homologs of caspases involved in PCD in animals. Throug h a genetic screen, the Arabidopsis membrane protein CPR5 was identified as a negative regul ator of ETI and its associated PCD. We subsequently demonstrated that CPR5 i s a novel membrane-bound nuclear pore component and physically associates with core cell cy cle components CYCLIN- DEPENDENT KINASE INHIBITORs (CKIs). Significantly, m utations in CKIs completely suppress autoimmune and spontaneous cell death phenotype of cpr5 mutant. At the molecular level, interaction of CKIs with CPR5 was regulated by ET I signaling. CKIs were released by CPR5 upon ETI induction to activate another core cell cycle regulator, E2F. Indeed, we found that ETI and PCD responses induced by both TIR-NB-LRR and CC-N B-LRR classes of immune receptors are compromised in cki and e2f mutants. We further show that E2F is deregulated during ETI probably through CKI-mediated hyperphosphorylation o f RETINOBLASTOMA- RELATED 1 (RBR1). Our study demonstrates a nuclear pore a ssociated cell cycle regulation event responses to pathogen effector and contributes to im munity and host PCD, illustrating an interesting noncanonical role of canonical cell cycle regula tors in plant immunity.