Current Awardees

Current Awardees

2015 Mitchell Meritorious Research Travel Awards

Name: Katherine Bonnington
Lab: Kuehn
Conference: Gordon Research Conference in Glycobiology, 2015
Date: March 1 – 6, 2015
Location: Lucca (Barga), Italy
Purpose: To present poster and talk

Abstract:

Lipopolysaccharide binding by heat-labile enterotoxin influences outer membrane dynamics in enterotoxigenic E. coli
SNumerous bacterial toxins are lectins, most notably due to their ability to bind sugars on eukaryotic surfaces before being taken up by a host cell. The heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) binds not only to ganglioside GM1, but also to Gram-negative lipopolysaccharide (LPS) via distinct sites on its donut-shaped pentamer of B subunits. Though the binding of LTB to host GM1 is well-characterized, the kinetics, specificity, and physiological effects of LTB binding to LPS on a bacterial membrane were heretofore unknown. We have found that LTBbinds the inner residues of an R1 type LPS core and are currently elucidating the importance of each residue to binding using STD-NMR. Although the strength of LTB-LPS binding, determined via ITC and SPR, is less than LTB-GM1, the downstream effects of this multivalent binding on bacterial model membranes are significant. For instance, increasing ratios of LTB:LPS reveals a trend of decreasing melting temperatures of the LPS acyl chains. Moreover, addition of LTB to giant unilamellar vesicles (GUVs) containing fluorescently-labeled LPS causes LPS clustering and shape deformations visible by confocal microscopy. Corroborating these in vitro findings with an in vivo model, outer membrane vesicle (OMV) production of LT+ ETEC (WT) is significantly higher than LT- ETEC (ΔeltA) and ETEC (ΔgspD) with LT secretion halted in the periplasm. Greater levels of expression of WT LTB, but not of LTB W88F (a point mutant deficient in LPS binding), increase OMV production in LT- ETEC. As such, cell surface-binding multivalent lectins such as LT may play greater roles in modulating membrane dynamics, even for the rigid Gram-negative outer membrane, than have been previously appreciated.

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Name: Yuxuan Miao
Lab: Abraham
Conference: Innate Immunity and Determinants of Microbial Pathogenesis (Z3)
Date: April 9-24, 2015
Location: Olympic, CA
Purpose: To present poster and talk

Abstract:

An intracellular TRP channel senses pathogen-mediated lysosome neutralization triggering expulsion of bacteria within exosomesYuxuan Miao
Vertebrate cells have evolved an elaborate cell-autonomous defense program to monitor various subcellular compartments for infection and to evoke a counter response. This program is activated by pathogen associated pattern molecules and also by specific actions employed by intracellular pathogens to alter cellular microenvironments. Here, we describe extracellular expulsion of uropathogenic E.coli from infected bladder epithelium cells (BECs) as a distinct cellular defense response which is triggered when intracellular UPEC encapsulated in autophagosomes reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of bacteria. Interestingly, upon extracellular discharge, the expelled bacteria are still encased in membranous vesicles which resemble exosomes, the key export machinery for the cell. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.

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Name: Blake Billmyre
Lab: Heitman
Conference: 28th GSA Fungal Genetics Meeting
Date: March 17 – 22, 2015
Location: Asilomar, CA
Purpose: To present poster and talk

Abstract:

Evolution of an outbreak: Hypermutators and theCryptococcus gattii outbreak
Blake BillmyreOver the past fifteen years, an ongoing outbreak of the human fungal pathogenCryptococcus gattii has occurred in the Pacific Northwest of the United States and Canada. This outbreak is comprised of three subtypes of the VGII molecular type of C. gattii, based on previous work using multilocus sequence typing (MLST), including the VGIIa/major, the VGIIb/minor, and the VGIIc/novel lineages. In this study, we performed whole genome sequencing and analysis of previously published genomes to analyze a total of 53 VGII isolates. We were able to establish that each of these clonal lineages was related through sexual recombination in the past, but that recent sexual crosses did not appear to contribute to the establishment of the outbreak lineages. Instead we found that VGIIa and VGIIb were likely introduced independently from South America and Australia respectively, while VGIIc may have arisen in the United States. All three of these groups are highly clonal. Interestingly, we found that the major component of the outbreak, VGIIa, has a clonal sublineage with diminished virulence that harbors a single base pair deletion in the coding region of the gene encoding the DNA mismatch repair component Msh2. Strains with this nonsense mutation in MSH2 have an increased mutation rate, ~5-fold in typical genes, but an even more dramatic hypermutator phenotype (~100-fold elevation) in genes containing a homopolymer run within their coding regions. These genes occur frequently throughout the C. gattii genome, with 4% of the gene set containing a run of 8 identical bases or longer within the coding region. One of these mutations in a homopolymer run resulted in an unselected drug resistance phenotype to both FK506 and rapamycin via inactivation of the gene encoding the FKBP12 homolog. A de novo deletion of MSH2and crosses with a congenic strain both demonstrated that the hypermutation trait segregates with the msh2 mutation. We hypothesize that the VGIIa/major strains responsible for the majority of the Pacific Northwest outbreak may have undergone microevolution mediated by a transient hypermutator state in adapting to a new environment to cause disease. This model has been previously demonstrated in virulences trajectories of bacterial pathogens and also operates in human colon cancer tumors but has not been previously observed in a fungal pathogen. Studies in progress seek to address whether the mutator state is an ancestral or derived lineage.

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

Abstract:

Role of natural genetic variation in the control of susceptibility to bacterial infections in Caenorhabditis elegans
Natalia MartinSeveral studies have shown that specific neuronally expressed G-protein couple
receptors (GPCR) and neurons modulate the immune response against pathogen infection 1-3. For instance, different mutants in the gene npr-1, which encodes a GPCR of the neuropeptide Y receptor family, were shown to be more susceptible to the human opportunistic pathogen Pseudomonas aeruginosa than the wild-type laboratory strain (N2) 1. Remarkably, most of C.
elegans wild isolates present a partial loss-of-function isoform of NPR-1. However, they suppress the enhanced susceptibility to pathogen phenotype of npr-1 mutants. We are taking advantage of this natural genetic variability among C. elegans wild
isolates to further identify key components of the NPR-1 pathway.
In order to characterize the mechanism that compensates the effect of the npr-1 mutation in an isolate from Germany, we used interference RNA and identified the requirement of C. elegansimmune pathways for this suppression mechanism. Furthermore, we performed full-genome microarrays to find cluster of genes up-regulated in the German isolate versus an npr-1 deficient strain in a wild-type background. This allowed us to determine the involvement of core cellular activities on the German isolate resistance to P. aeruginosa infection. To identify the suppressor mutation, we used a combination of whole genome sequencing with a very finegrained single nucleotide polymorphism mapping strategy 4. The identification of the responsible gene using this approach is underway.

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Name: Shannon Esher
Lab: Alspaugh
Conference: 28th Fungal Genetics Conference
Date: March 17 – 22, 2015
Location: Pacific Grove, CA
Purpose: To present poster and talk

Abstract:

Identifying the role of a Cryptococcus species-specific cell wall gene in response to the host
Shannon EsherCryptococcus 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 to 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 identifiedMAR1, a novel Cryptococcus species-specific gene that is an important regulator of these host-induced cell wall changes. We have generated a mar1∆ mutant strain and shown that it has sensitivities to elevated 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 induction of the inflammatory cytokine TNFa, we have shown that the mar1∆mutant induces 4-5 times more TNFa production than wild type cells. These results suggest that Mar1 is regulating important cell wall changes in response to the host. In the absence of the Mar1 protein, the aberrant cell wall contains, and likely exposes, more chitin and chitooligomers, resulting in increased 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: Ryan Finethy
Lab: Coers
Conference: Chlamydia Basic Research Society 2015
Date: March 29 – April 1, 2015
Location: New Orleans, LA
Purpose: To present talk and poster

Abstract:

TBA
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Name: Masashi Kanayama
Lab: Shinohara
Conference: 2015 Gordon Research Seminar on Immunology of Fungal Infections
2015 Gordon Research Conference on Immunology of Fungal Infections
Date: January 17 – 23, 2015
Location: Galveston, TX
Purpose: To present talk and poster

Abstract:

Autophagy enhances NFκB activity in specific tissue resident macrophages by sequestering A20 to boost early anti-fungal immunity
Kristen SmithTo avoid developing spontaneous inflammation, molecular signaling that triggers immune responses must be well restrained in a steady state. However, in acute infections, such restraints may prevent quick and efficient anti-microbial responses. Autophagy is a common cellular process in eukaryotes to degrade cytoplasmic contents and has been known to regulate several immune responses. Here, we report a novel role of autophagy in host fungal clearance by enhancing early immune responses through increased nuclear factor kappa B (NFκB) activity. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80hi tissue resident macrophages in the spleen, kidney and peritoneal cavity. We identified that p62, an autophagic adopter protein, mediates A20 sequestration by autophagosome. This allows the macrophages to release high levels of chemokines to effectively attract neutrophils to infected sites. Indeed, mice lacking autophagy in myeloid cells showed higher susceptibility to Candida albicans infection with impaired neutrophil recruitment. Thus, autophagy acts to break A20-dependent NFκB suppression in F4/80hi tissue resident macrophages and contributes to the initiation of efficient innate immune responses.

Autophagy in myeloid cells inhibits development of spontaneous pulmonary inflammation
Autophagy is a cellular process to remove intracellular components such as pathogenic microorganisms and damaged mitochondria.   Autophagy is also known to regulate several immune responses to control inflammation and to protect hosts from pathogens. However, it is still unclear whether and how autophagy plays a role to maintain immune homeostasis. In this study, we found that deficiency of ATG7, which is an essential protein for autophagy, in myeloid cells induced spontaneous inflammation in the lungs but not in other organs in 3-week oldAtg7fl/flLysM-Cre (Atg7 CKO) mice. Pulmonary inflammation in the Atg7 CKO mice was mediated by innate immune cell infiltration in the lung, initiated 2-3 weeks after birth. The lung is constantly exposed by environmental pathogens such as bacteria, viruses, and fungi. Indeed, in the lung of 3 weeks old Atg7 CKO mice, bacterial and fungal burdens were significantly increased compared to those in WT mice, suggesting bacteria and fungi as environmental triggers of inflammation.  Indeed, intranasal instillation of antibiotics reduced the bacterial burden in the lung, and prevented spontaneous lung inflammation in Atg7 CKO mice. In addition, we found that alveolar macrophages (AMs) from Atg7CKO mice showed higher reactive oxygen species (ROS) production and enhanced sensitivity in detecting low concentrations of pattern recognition receptor ligands. Blockade of ROS inhibited the production of pro-inflammatory cytokines and chemokines in AMs; then protected Atg7 CKO mice from developing spontaneous pulmonary inflammation. Taken together, autophagy in myeloid cells plays a critical role in keeping pulmonary immune homeostasis by controlling loads of microbes in the lung.