Host-Microbial Interactions
DUKE UNIVERSITY MEDICAL CENTER
Center for

TRAINING PROGRAMS

Mitchell Meritorious Research Travel Awards

Current Awardees
2016 Mitchell Meritorious Research Travel Awards

Name:

Dia Beachboard

Lab:

Horner

Conference:

American Society for Virology Conference   

Date:

June 24-28, 2017

Location:

Madison, WI

Purpose:

Talk

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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.

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Name:

Xiou Cao

Lab:

Aballay

Conference:

47th Annual Meeting, Neuroscience 2017

Date:

November 11-15, 2017

Location:

Washington, DC

Purpose:

To present a poster

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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.

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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

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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.

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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

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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.

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Name:

Yi-Hong Ke

Lab:

Vilgalys

Conference:

Workshop on Molecular Evolution

Date:

July 20-30, 2017

Location:

Woods Hold, MA

Purpose:

Training and Networking

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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.

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Name:

Charmaine Mutucumarana

Lab:

Permar

Conference:

IAS 2017

Date:

July 23-26, 2017

Location:

Paris, France

Purpose:

To present a poster

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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

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 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

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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.

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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

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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.

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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

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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.

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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

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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 pH. Interestingly, 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.

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Name:

Caitlin Esoda

Lab:

Kuehn

Conference:

ASM Microbe 2017

Date:

June 1-5, 2017

Location:

New Orleans, LA

Purpose:

To present a poster

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Title: A 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.

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Name:

Ci Fu  

Lab:

Heitman

Conference:

29th Fungal Genetics Conference

Date:

Mar14-19, 2017

Location:

Pacific Grove, CA

Purpose:

To present a poster

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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.

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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

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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
secretion.
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.

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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

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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.

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Name:

Jorge Alejandro Rojas

Lab:

Vilgalys

Conference:

The 29th Fungal Genetics Conference

Date:

March 14-19, 2017

Location:

Pacific Grove, CA

Purpose:

Poster presentation

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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.

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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

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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 typhimurium, Listeria 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.

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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

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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

VazquezChristine_CHoMI.jpg

 

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

RZ_portrait.jpg

 

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.
Methods:
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.
Results:
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.
Conclusions:
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: 2.09x106 vs. 2.29x105
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

(N/A)

___________________________________________

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

___________________________________________

2015 Mitchell Meritorious Research Travel Awards

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.

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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.

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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.

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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.

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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 .

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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 4 th 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 .


Past Mitchel Travel Awardees

Past Travel Awardees