Past Awardees
| Name: | Anthony Piro |
| Lab: | Coers |
| Conference: | Chlamydia Basic Research Society, 2013 |
| Date: | March 19 – 22, 2013 |
| Location: | San Antonio, TX |
| Purpose: | To present poster and talk |
Abstract:
Developing mouse models of chronic Chlamydia trachomatis infections
Background and Significance: Infection with Chlamydia trachomatis results in modulation of
intracellular tryptophan stores as part of a protective immune response in humans.
C. trachomatis is a natural tryptophan auxotroph and an obligate intracellular pathogen, and thus, the degradation of intracellular tryptophan stores through the Interferon-inducible enzyme indole-2,3-dioxygenase (IDO) deprives the microbe of an essential nutrient. In response to IDOmediated tryptophan starvation C. trachomatis enters a persistent state of diminished growth. We and others have previously hypothesized that IDO-induced C. trachomatis “persister” cells are refractory to adaptive immune clearance and thereby play a critical role in the establishment of chronic C. trachomatis infections.
Objectives: The existence of a persistent form of C. trachomatis has been extensively demonstrated in tissue culture models. Because mouse (unlike human) urogenital epithelial cells
fail to induce robust IDO expression upon stimulation with Interferons, there is as yet no in vivo model that recapitulates the interactions of C. trachomatis persister cells with the immune
system. To overcome these experimental limitations, we aimed to generate mouse models that recapitulate the IDO-dependent induction of C. trachomatis persister cells as observed in humans.
Methods: We used standard gene targeting technology to generate two transgenic mouse lines expressing human IDO under control of 1) a Cre-inducible and 2) an Interferon-inducible
promoter.
Results: We successfully engineered mice that feature strong expression of hIDO upon expression of Cre recombinase or stimulation with Interferon, respectively.
Conclusions: We generated mouse models engineered with key features of the human immune response. We will employ these novel animal models to test the hypothesis that C. trachomatis
co-opts IDO-driven immune responses to coordinate its escape into persistence.
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| Name: | Brian Head |
| Lab: | Aballay |
| Conference: | 2013 FASEB Meeting on Molecular Pathogenesis: Mechanisms of Infectious Disease |
| Date: | July 21 – 26, 2013 |
| Location: | Snowmass, CO |
| Purpose: | To present poster and talk |
Abstract:
Whole genome expression analysis of C. elegans upon recovery from Salmonella enterica infection
C. elegans are capable of mounting an immune response to a variety of pathogens. In the last decade, several major steps in the C. elegans innate immune response have been dissected including: 1) host surveillance pathways detect a pathogen and/or loss of homeostasis, 2) various intracellular signaling modules are activated in distinct cell types, and 3) immune effector molecules are produced at the site of infection (primarily the intestinal and hypodermal epithelia). Moreover, stress response pathways are activated to cope with cellular damage from both host immune effectors and pathogen-derived molecules.
Much less is known about the physiology of the animal during recovery from an infection. Thus, we are utilizing the C. elegans-pathogen model to study recovery from infection by Salmonella enterica. After generating a “mild” Salmonella infection in the worm, we alleviate this infection by a combination of antibiotic treatment plus a change in food source. We demonstrate that “recovered” worms have a reduced bacterial burden and enhanced survival. To better understand the transcriptional response during recovery, we have utilized whole genome microarray technology. A variety of expected and novel gene clusters are altered during recovery. Representative genes from several clusters and other interesting candidates were confirmed by qRT-PCR. We are in the process of testing these genes for effects on recovery using standard reverse genetic techniques.
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| Name: | Jeffrey Barker |
| Lab: | Valdivia |
| Conference: | 6th Biennial Chlamydia Basic Research Society Meeting |
| Date: | March 19 – 22, 2013 |
| Location: | San Antonio, TX |
| Purpose: | To present poster and talk |
Abstract:
A STING-mediated type I interferon response is induced by cytosolic sensing of the Chlamydia second messenger cyclic di-AMP
A major mechanism for the initiation of Type I IFN responses in mammalian cells is through the detection of microbial nucleic acids by the cytosolic adaptor protein STING (Stimulator of IFN Genes). Chlamydia trachomatis induces Type I IFN in a process that is dependent on STING yet the IFN inducing ligands remains unknown. We found that Chlamydia synthesizes the newly identified bacterial metabolite cyclic di-adenosine monophosphate (c-di-AMP) and that this nucleic acid is the most prominent ligand for STING-mediated activation of IFN responses. We used primary mouse lung fibroblasts and HEK293t cells to monitor IFNb expression in response to Chlamydia infection, c-di-AMP and other Type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment lead to the induction of Type I IFN responses in cells expressing STING, but not in cells expressing a STING variant that cannot sense cyclic di-nucleotides, even though these variants were capable of responding to cytoplasmic DNA and dsRNA. The failure to induce Type I IFN responses to Chlamydia and c-di-AMP correlated with the lack of transport of STING from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing Type I IFN responses in Chlamydia infected cells.
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| Name: | Yun Cai |
| Lab: | Aballay |
| Conference: | 19th International C. elegans Meeting |
| Date: | June 26 – 30, 2013 |
| Location: | University of California, Los Angeles |
| Purpose: | To present poster and talk |
Abstract:
Antibiotics can modulate the immune system to enhance survival of Caenorhabditis elegans during Yersinia pestis infection
Caenorhabditis elegans has been intensely used as a desirable and efficient model for different genetic and chemical screens. To determine drugs that have a beneficial effect on the immune system, we studied the effect of diverse chemical compounds on the expression of gene F35E12.5,which is a reporter of the activation of the p38/PMK-1 signaling pathway. Using strain AY101, in which a green fluorescence protein reporter gene was fused to the promoter of gene F35E12.5, we identified 45 compounds which can strongly up-regulate F35E12.5. Sixteen of them are antimicrobial agents, including Quinolones, Azole antifungal agents, Tetracyclines, and Polymyxins. We found that animals treated with antibiotics were more resistant to Y. pestiscompared to the untreated ones. In addition, treated animals were also more resistant to control animals when they were infected with antibiotic resistant Y. pestis, indicating that the resistance to infection was not only due to the bactericidal effect of the drug. Our studies identified a new mechanism by which certain antibiotics confer protection to bacterial infections.
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| Name: | Danielle Pilla |
| Lab: | Coers |
| Conference: | 2013 FASEB Meeting on Microbial Pathogenesis: Mechanisms of Infectious Disease |
| Date: | July 21 – 26, 2013 |
| Location: | Snowmass, CO |
| Purpose: | To present poster and talk |
Abstract:
Guanylate binding proteins promote pyroptosis in Legionella pneumophila infected macrophages
The cytokine, Interferon-gamma (IFNγ), is a critical factor in the regulation of innate and adaptive immune responses. IFNγ induces the expression of about 2,000 genes including those involved in the production of nitric oxide (Nos2) and superoxide radicals (Nox2). In mice, IFNγ also controls the expression of Immunity Related GTPases (IRGs) which localize to pathogen-containing vacuoles and promote the degradation of these vacuoles. We found that bone marrow-derived macrophages with a quadruple deficiency in Nos2, Nox2 as well as two regulatory IRG genes named Irgm1 and Irgm3 (QKO BMM) had a partial defect in IFNγ-mediated resistance to infections with the intracellular pathogen Legionella pneumophila. These observations suggested to us that additional IFNγ-induced host defense pathways were active in these cells. To identify these pathways, we first aimed to identify additional host proteins that target Legionella-containing vacuoles (LCVs). We found that at least 3 members of the guanylate binding protein (GBP) family target LCVs in in IFNγ-activated QKO BMM. GBPs constitute a second family of IFNγ-inducible GTPases that are important for blocking infections with various pathogens. Previous research has described a role for one member of this protein family in activating the NRLP3 inflammasome and pyroptosis, a pro-inflammatory cell death pathway involved in host immunity. By measuring LDH release in macrophages infected with L. pneumophila, we found that IFNγ induces pyroptosis in wildtype cells but not in BMMs deficient in a cluster of 5 GBP genes on chromosome 3. Pyroptosis induced by L. pneumophila in IFNγ-activated BMM was independent of the canonical inflammasome components NLRP3, NLRC4, and Asc suggesting that GBPs provide host resistance to L. pneumophila infections through a novel cell death pathway.
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2012 Bacteriology Meritorious Research Travel Award Recipients
| Name: | Adam Kulp |
| Lab: | Kuehn |
| Conference: | The 2011 Madison Molecular Genetics of Bacteria and Phages Meeting |
| Date: | August 2 – 7, 2011 |
| Location: | Madison, WI |
| Purpose: | To present talk and poster |
Abstract:
The recognition of β-strand motifs by RseB is required for σE activity in Escherichia coli
Background: The envelope of Gram negative bacteria is a complex and essential barrier. Bacteria have evolved several stress response pathways to protect and maintain the envelope. One of the best studied envelope stress response pathways is the SigmaE response, which recognizes misfolded outer membrane proteins (OMPs). The SigmaE pathway is activated by the recognition of the C-termini of OMPs in the periplasm. The inner membrane protein DegS binds the C-terminus of a misfolded OMP, while the periplasmic protein RseB reportedly recognizes other features of the OMP. Although the interaction between DegS and the OMP C-terminus is well studied, little is known about how RseB recognizes OMPs.
Specific Aims: We sought to determine how RseB recognizes misfolded OMPs and how recognition of OMP motifs provides specificity for the SigmaE envelope stress response.
Methods: C-terminal segments of various OMPs were expressed in the periplasm of Escherichia coli wildtype and rseB-deficient reporter strains. By mutating the OMP segments and comparing the different stress response levels, we could precisely explore RseB recognition motifs in OMPs.
Results: We discovered that RseB recognizes beta-strand motifs of misfolded OMPs and that these motifs help determine the strength of the SigmaE response in an RseB-dependent manner. If a beta-strand motif is not recognized by RseB, then the SigmaE response is not induced. Instead, another envelope stress response pathway, the CPX pathway, is induced.
Conclusions: In addition to the well-studied recognition of a C-terminal phenylalanine motif by DegS, we have identified and characterized the recognition of beta-strand motifs by the periplasmic protein RseB. Both of these OMP motifs are essential for SigmaE activation in wild-type strains, and both motifs determine the strength of the ensuing stress response. The dual requirements make the SigmaE stress response pathway very specific to misfolded OMPs, likely to prevent inappropriate activation.
| Name: | Kristen Smith |
| Lab: | Lee |
| Conference: | Keystone Symposia on Molecular and Cellular Biology |
| Date: | March 13 – 18, 2013 |
| Location: | Whistler, British Columbia |
| Purpose: | To present talk and poster |
Abstract:
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen with several known survival mechanisms, one of which is its ability to inhibit apoptosis in host alveolar macrophages. It has been shown that apoptotic bodies from cells infected with Mtb are able to induce T cell-mediated immune responses against the pathogen. By screening an Mtb H37Rv transposon library containing 10,000 mutants, we have identified 22 pro-apoptotic mutants. The primary screen identified mutants associated with increased membrane damage during infection, as apoptotic bodies will lyse if there are no antigen-presenting cells to remove them. This was done by measuring the release of lactate dehydrogenase, an enzyme that is present in the cytoplasm of healthy cells but is also found in the supernatant after a loss of membrane integrity. Candidates from the primary screen were further tested to verify a pro-apoptotic phenotype. We are currently studying the immunogenicity of these mutants in vivo. We hypothesize that these mutants will be more immunogenic than parental strain H37Rv, since it has been shown that apoptotic bodies from Mtb infected cells are engulfed by dendritic cells, which in turn present mycobacterial antigens to CD8+ T cells. The inability of BCG to activate CD8+ T cells has been postulated as one of the reasons BCG has limited effectiveness as a vaccine. Accordingly, the mutants identified in this work have potential as novel vaccine candidates.
| Name: | Samantha Bowen |
| Lab: | Abraham |
| Conference: | Keystone Symposia on Molecular and Cellular Biology |
| Date: | March 30 – April 4, 2012 |
| Location: | Big Sky, MT |
| Purpose: | To present talk and poster |
Abstract:
Development of reflux nephropathy is dependent upon sustained pyelonephritis
Reflux nephropathy (RN), or renal scarring, is a serious complication associated with vesicoureteral reflux (VUR), a pediatric congenital disorder in which malformation of the urinary tract allows urine to flow retrogradely from the bladder to the kidneys. RN is characterized by the formation of fibrotic scars, rearrangement of kidney architecture, and loss of filtration function, but its development is poorly understood. Some studies have suggested that the reflux of sterile urine alone can induce RN pathology. However, most patients are diagnosed with VUR following presentation with febrile urinary tract infection (UTI), suggesting a potentially significant role for infection, particularly pyelonephritis (kidney infection), in RN pathogenesis. Objective: (1) To determine whether the presence of VUR induces pyelonephritis following infection of the bladder, and (2) to determine if pyelonephritis in conjunction with VUR is necessary to induce RN pathology in a naturally refluxing mouse model, the C3H/HeN mouse. Methods: UTIs were induced in refluxing C3H/HeN mice and non-refluxing control C57BL/6 mice by instilling bladders with the clinical uropathogenic E. coli strain CI5, followed by sacrifice at 6h, 2d or 6d post-infection. Uninfected controls were sham-treated with saline. Some C3H/HeN mice were treated with 5 mg/kg trimethoprim and sulfamethoxazole 2x per day for 2d following infection. Colony-forming units were quantitated for bladders and kidneys, and kidneys were also examined and scored for histology. Results: Refluxing C3H/HeN mice developed pyelonephritis that persisted for at least 6d following bladder infection due to the presence of VUR, while control non-refluxing C57BL/6 mice did not develop pyelonephritis. Following antimicrobial treatment to abolish kidney infection, C3H/HeN mice redeveloped pyelonephritis seeded from a protected bladder bacterial population. Significant inflammation, fibrosis, and scarring was observed in infected C3H/HeN kidneys. Sham-infected refluxing C3H/HeN mice and infected non-refluxing C57BL/6 control mice did not exhibit renal scarring pathology. Additionally, acute pyelonephritis, induced by altering the infection protocol to force bacteria into the kidneys in the absence of reflux, resolved quickly and without persistence in non-refluxing C57BL/6 mice, and did not provoke noticeable renal inflammation or fibrosis in contrast to refluxing C3H/HeN mice. Conclusions: The presence of VUR induces persistent pyelonephritis accompanied by significant RN pathology characterized by fibrotic scarring, while sterile reflux and acute pyelonephritis are insufficient to provoke RN pathogenesis. Thus, the co-occurrence of UTI and VUR represents both a detrimental clinical scenario and an important therapeutic target.
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2010 Bacteriology Meritorious Research Travel Award Recipients
| Name: | Adam Kulp |
| Lab: | Kuehn |
| Conference: | American Society for Microbiology 110th General Meeting |
| Date: | May 23 – 27, 2010 |
| Location: | San Diego, CA |
| Purpose: | To present talk and poster |
Abstract:
Models of outer membrane vesicle biogenesis
Outer membrane vesicles (OMVs) are made by all Gram-negative bacteria. Many studies have investigated the structure and functions of OMVs from various species of bacteria, but very little known about the mechanism of OMV formation. It is known that vesiculation is increased in response to the presence of misfolded outer membrane proteins (OMPs) in Escherichia coli, indicating that the regulatory mechanism may be linked to envelope stressors. Also, studies of Pseudomonas aeruginosa OMVs indicate that certain membrane bound molecules, specifically PQS and B-band LPS, may be directly involved in vesiculation. To further elucidate the mechanism of vesiculation we examined the role of misfolded OMPs, PQS and B-band LPS on the production of OMVs in E. coli. Our findings indicate that OMVs are produced in response to misfolded OMPs in a manner that is specific to the OMP but independent of the SigmaE pathway, and that vesiculation is not directly driven by PQS and B-band LPS in E. coli.
| Name: | Devin Bolz, PhD |
| Lab: | Aballay |
| Conference: | Gordon Research Seminar and Conference: Microbial Toxins & Pathogenicity |
| Date: | July 10 – 16, 2010 |
| Location: | Waterville Valley, NH |
| Purpose: | To present talk and poster |
Abstract:
The role of PMK-1/p38 MAPK in the immune response against Yersinia pestis in natural hosts
Yersinia pestis has acquired a variety of complex strategies that enable the bacterium to overcome defenses in different hosts and ensure its survival and successful transmission. To identify host factors involved in the early response to Y. pestis infection, we utilized the genetically tractable nematode Caenorhabditis elegans. A full-genome microarray analysis of C. elegans infected with Y. pestis revealed enrichment in genes that are markers of innate immune responses and regulated by a conserved PMK-1/p38 mitogen-activated protein kinase. Consistent with a role in regulating expression of immune effectors, inhibition of PMK-1/p38 by mutation or RNAi enhances susceptibility to Y. pestis. Further studies of mosaic animals where PMK-1/p38 is exclusively inhibited or overexpressed in a tissue-specific manner indicate that PMK-1/p38 controls expression of a CUB-like family of immune genes at the cell-autonomous level. Given the conserved nature of PMK-1/p38 MAPK-mediated signaling and innate immune responses, PMK-1/p38 MAPK may play a role in the immune response against Y. pestis in natural hosts.
| Name: | Nicole Spahich |
| Lab: | St. Geme |
| Conference: | 2010 American Society of Microbiology General Meeting |
| Date: | May 23 – 27, 2010 |
| Location: | San Diego, CA |
| Purpose: | To present talk and poster |
Abstract:
Mutation of LPS synthesis genes disrupts synthesis and secretion of the Haemophilus influenzae Hap adhesin
Nontypable Haemophilus influenzae is a major cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. An important step in H. influenzae colonization is adherence to host epithelial cells, mediated by surface-exposed proteins such as the Hap adhesin. Hap is a conventional, monomeric autotransporter, consisting of a signal peptide, an adhesive passenger domain, and an outer membrane beta barrel that facilitates presentation of the passenger domain on the bacterial surface. In an effort to define the cellular machinery involved in the synthesis and secretion of Hap, we created a mariner transposon mutant library in H. influenzae strain Rd expressing a Hap mutant with enhanced adhesive capabilities (Rd/HapS243A). Several mutants with transposon insertions in LPS synthesis genes were found to be non-adherent, suggesting an effect on Hap function. To explore the relationship between LPS structure and Hap synthesis and secretion, we insertionally inactivated multiple genes involved in synthesis of the LPS oligosaccharide core in H. influenzae strain Rd/HapS243A. We found that mutation of rfaF, pgmB, lgtC, kfiC, orfE, and rfbP resulted in a loss of Hap in the bacterial outer membrane, a decrease in hap transcript, and a decrease in bacterial adherence to Chang epithelial cells. In contrast, these mutations had no effect on the quantity of the H. influenzae P5 and IgA1 protease outer membrane proteins, suggesting a Hap-specific effect. Curiously, mutation of the lic2A and rfbB LPS synthesis genes in strain Rd/HapS243A did not affect Hap expression. Mutation of rfaF, pgmB, lgtC, kfiC, orfE, or rfbP along with disruption of the periplasmic protease gene htrA resulted in accumulation of Hap in the periplasm. We hypothesize that mutation of certain LPS synthesis genes results in an inability of Hap protein to be inserted into the outer membrane, causing an accumulation of protein in the periplasm and down-regulation of hap transcription. We speculate that the interrelationship between the variability of LPS structure and the quantity of Hap in the outer membrane influences H. influenzae pathogenicity.
| Name: | Hector Alex Saka, Ph.D. |
| Lab: | Valdivia |
| Conference: | 2010 FASEB Summer Research Conference |
| Date: | July 25-30, 2010 |
| Location: | Steamboat Springs, Colorado |
| Purpose: | To present talk and poster |
Abstract:
Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids
Lipid droplets (LD) are dynamic, ER-derived organelles that constitute the main store of neutral lipids in eukaryotic cells. They are primarily composed of cholesterol esters and triacylglycerols, surrounded by a monolayer of phospholipids and LD-associated proteins. The obligate intracellular pathogen Chlamydia trachomatis (CT), the most common cause of sexually transmitted infections and the leading cause of preventable blindness worldwide, recruits and translocates LDs into the lumen of the chlamydial parasitopherous vacuole. To define the molecular basis for this manipulation of LD by Chlamydia, LDs associated proteins from infected and uninfected HeLa cells were analyzed by mass spectrometry. A state-of-the- art technique for proteomics analysis by bottom-up LC-MS/MS and LC-MSE (Waters nanoAcquity/Synapt-HDMS instrumentation) was used, allowing for the identification and absolute quantitation of 242 proteins (2 or more peptides to match, detected in at least 3 independent runs), including most of the already recognized LD-associated proteins (i.e., perilipins, lipid metabolism-related proteins, Rab GTPases, chaperones and cytoskeleton proteins). The expression levels of a subset of 205 proteins (2 biological replicates, 6 runs total, relative standard deviation less than 50%, for LD-uninfected, LD-20 hpi and LD-40 hpi) were calculated, allowing an assessment of changes in the LD proteome in response to infection. PLIN3 (Tip47) was the most abundant LD protein, representing by itself ~ 11% of the total mass. Perilipins, lipid metabolism proteins, chaperones and cytoskeleton proteins were dominant, representing ~ 61% of the total mass. In response to infection, lipid-metabolism proteins were found to be increased in LDs whereas a large number of ER chaperones, MHC class I and PDI were decreased. We propose that CT targets LD to co-opt lipid transport. In addition, the association of immune-related molecules with LDs indicated a possible intersection between these organelles and innate immune responses. Because interferon (IFN) responses are important in chlamydial containment, we tested how the LD proteome changed upon IFN-gamma treatment in MEF (mouse embryo fibroblast) cells, where Chlamydia induces a strong IFN response. Surprisingly, a number of p47 GTPases and other IFN-induced proteins were highly enriched in LDs upon IFN-gamma treatment. Because these proteins are important to contain Chlamydia and other intracellular pathogens it is possible that LDs may participate in the cell autonomous immune response against intracellular pathogens. As such, the translocation of LDs into the Chlamydia parasitopherous vacuole may prevent the proper function of effector proteins of the interferon response and represent a mechanism of immune evasion by this pathogen.
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2009 Bacteriology Meritorious Research Travel Award Recipients
| Name: | Ine Jorgensen, Ph.D. |
| Lab: | Valdivia |
| Conference: | 2009 FASEB Summer Research Conference – Microbial Pathogenesis: Mechanisms of Infectious Disease |
| Date: | July 19-24, 2009 |
| Location: | Snowmass, Colorado |
| Purpose: | To present talk and poster |
Abstract:
The Chlamydial protease CPAF targets secreted effector proteins
The intracellular bacterial pathogen Chlamydia trachomatis modulates a range of host cellular functions by delivering effector proteins across the membrane of the pathogen-containing vacuole (“inclusion”)1. The secreted Chlamydial Protease-Like Activity Factor (CPAF) targets multiple mammalian proteins for degradation and is involved in downregulating MHC expression2, inhibiting apoptosis3 and re-arranging of the host cytoskeleton4. Given the broad substrate specificity of CPAF, we hypothesized that this protease may also target chlamydial proteins during infection. We screened 325 GFP-tagged chlamydial proteins expressed in yeast for sensitivity to proteolysis. This screen identified 29 ORFs that were either processed or degraded after incubation with crude lysate from infected cells. At least five ORFs were confirmed as substrates of CPAF – Ct005, Ct115, Ct116, Ct233, and Ct813. Purified versions of these proteins were processed into distinct cleavage fragments or completely degraded after incubation with recombinant CPAF in a cell free assay. Interestingly, Ct005, Ct115 (IncD), Ct116 (IncE) and Ct233 (IncC) contain a hydrophobic domain common to chlamydial proteins that associate with the inclusion membrane (Incs). Additionally, all five CPAF substrates belong to a subclass of incs that are expressed during the very early stages of infection, and were either confirmed or predicted to be secreted by the type III secretion apparatus. We generated antibodies against Ct005, IncD and IncC and determined that they are expressed during early stages of infection and localize to the inclusion membrane. These findings suggest a novel role for CPAF in regulating secreted bacterial proteins in the host cytosol. C. trachomatis actively down-regulates the immune response and inhibits apoptosis in infected cells. We propose that CPAF degradation of secreted proteins precludes MHC antigens presentation of peptides derived from these proteins, and prevents the accumulation of toxic effectors that could initiate apoptosis in infected cells. Alternatively, CPAF may prevent infection and development of a second inclusion in previously infected cells, by degrading effectors important to the initial development of the inclusion once these proteins are no longer needed.
| Name: | Amanda Sheets, Ph.D. |
| Lab: | St. Geme |
| Conference: | Microbial Pathogenesis and host Response Meeting |
| Date: | September 8-12, 2009 |
| Location: | Cold Spring Harbor, New York |
| Purpose: | To present talk and poster |
Abstract:
Phase-variable adhesive activity of the Haemophilus cryptic genospecies trimeric autotransporter Cha
Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. In recent work, we identified a Haemophilus cryptic genospecies protein called Cha, which mediates efficient adherence to genital and respiratory epithelium. The Cha adhesin belongs to the trimeric autotransporter family and contains an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal beta barrel domain that trimerizes and anchors the protein in the bacterial outer membrane. Similar to a number of other bacterial adhesins, the passenger domain in Cha contains scattered clusters of YadA-like head domains and 4 non-contiguous HIM motifs. In addition, the passenger domain contains a series of tandem 28-amino acid repeats. In the current study, we report that Cha-mediated adherence is phase-variable and is linked to spontaneous changes in the number of 28-amino acid repeats. Variation in the number of repeats has a direct effect on Cha adhesive activity, independent of an impact on transcription of the cha locus or surface localization of Cha protein. Based on experiments evaluating the adherence capacity of H. influenzae expressing Cha protein with internal deletions, we independently established that the N-terminal 473 residues of Cha harbor the binding domain responsible for Cha-mediated adherence to epithelial cells. Additionally, this same region appears to facilitate bacterial aggregation through intermolecular Cha-Cha binding which is inhibited by expansion of the repeat domain. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats influences the conformation of Cha thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface which have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells.
| Name: | Varsha Singh, Ph.D. |
| Lab: | Aballay |
| Conference: | 17th International C. elegans Meeting |
| Date: | June 24-28, 2009 |
| Location: | Los Angeles, California |
| Purpose: | To present talk and poster |
Abstract:
Over-activation of FOXO transcription factor, DAF-16, causes pathogen susceptibility in Caenorhabditis elegans
DAF-16 is a FOXO transcription factor involved in regulation of metabolism and stress response in C. elegans. Previous studies have shown that overexpression of DAF-16 increases lifespan and resistance to pathogens. Here, we report that hyper-activation of DAF-16 may lead to deleterious consequences during infection with bacterial pathogens. We also provide evidence that a stress-inducible Heat Shock Factor (HSF)-1 is utilized to control DAF-16 activitation by promoting its export from the nucleus. The effect of HSF-1 appears to be mediated through Hsp70 which is also induced and translocated to the nucleus during stress. Aquaporin upregulation is partly responsible for enhanced susceptibility to pathogens. Susceptibility due to DAF-16 activation could also be rescued by increasing the osmolarilty of the exogenous medium indicating that osmotic balance is an important aspect of infection. In summary, while overexpression of DAF-16 is beneficial, its sustained activation leads to enhanced susceptibility to pathogens. C. elegans utilizes chaperones to promote DAF-16 nuclear export in a timely manner and prevent susceptibility.
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2008 Bacteriology Meritorious Research Travel Award Recipients
| Name: | Jordan Cocchiaro, Ph.D. |
| Lab: | Valdivia |
| Conference: | 108th General Meeting of the American Society for Microbiology |
| Date: | June 1-5, 2008 |
| Location: | Boston, Massachusetts |
| Purpose: | To present talk and poster |
Abstract:
Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia inclusion
Chlamydia trachomatis is an obligate, intracellular gram-negative bacterium that causes many diseases in humans, most notably blinding trachoma and sexually transmitted infections. Chlamydia replicates within a membrane-bound vacuole, termed an inclusion, from which it acquires host-derived lipids essential for bacterial growth and survival. Some of these lipids are acquired through re-routing of vesicular traffic from the Golgi and endosomal multivesicular bodies (MVBs) to the inclusion; however, non-vesicle mediated pathways have also been implicated in this process. We recently determined that Chlamydia infection disrupts host neutral-lipid homeostasis, and cytoplasmic Lipid Droplets (LDs) – eukaryotic, neutral-lipid storage organelles – accumulate at the inclusion periphery. We hypothesized that LDs may represent an alternate source of lipids for Chlamydia. In this study, we show by live cell, time-lapse fluorescence microscopy and thin-section electron microscopy that intact LDs are translocated from the host cytoplasm into the inclusion lumen. LDs dock at the surface of the inclusion, penetrate the inclusion membrane, and intimately associate with Reticulate Bodies – the replicative form of Chlamydia. Furthermore, the bacterial inclusion membrane protein IncA, unlike other Inc proteins, co-fractionates biochemically with LDs, and IncA-positive structures accumulate in the inclusion lumen. Therefore, we postulate that the translocation of LDs requires the involution of IncAenriched sub-domains of the inclusion membrane. Finally, we provide evidence that the chlamydial protein Lda3 participates in the co-option of these organelles by linking cytoplasmic LDs to inclusion membranes and by displacing the protective coat protein adipocyte differentiation related protein (ADRP) from LDs, presumably to initiate lipolysis. The wholesale transport of LDs into the lumen of a parasitophorous vacuole represents a unique mechanism of organelle sequestration and subversion by a bacterial pathogen.
| Name: | Kris Spaeth, Ph.D. |
| Lab: | Valdivia |
| Conference: | 108th General Meeting of the American Society for Microbiology |
| Date: | June 1-5, 2008 |
| Location: | Boston, Massachusetts |
| Purpose: | To present talk and poster |
Abstract:
The protein-protein interaction map of the Chlamydia trachomatis Type III secretion system
The obligate intracellular pathogen Chlamydia trachomatis uses a Type III Secretion (TTS) system to deliver effector proteins into the host cytoplasm and the membrane of the pathogen-containing vacuole. A model of the chlamydial TTS apparatus has been proposed based on homologies to enteric TTS systems since there is a lack of genetic tools in Chlamydiae to directly define these components. However, because not all TTS components are conserved in C. trachomatis and several open reading frames (ORFs) of unknown function are co-transcribed with the core components, we predicted that these divergent proteins perform functions important to the assembly and function of the TTS apparatus. In this study, we generated a basic physical map of the C. trachomatis TTS system based on protein-protein interactions identified by yeast two-hybrid analysis. All ORFs associated with the three chromosomal loci encoding putative core TTS components were fused to the yeast Gal4 DNA binding domains (DBD) and Activator Domains (AD) and co-expressed in yeast cells. Positive chlamydial protein-protein interaction was assessed by expression of reporter genes driven by Gal4 promoters. Overall, our results suggest that predicted interactions among TTS core components are evolutionarily conserved and many ORFs of unknown function co-transcribed with TTS components interact with the secretion apparatus. Next, we expanded our search for TTS-interacting proteins to all Chlamydia-specific ORFs and putative effector proteins. This analysis revealed additional proteins that interacted with the basal ring of the TTS apparatus, with two of these proteins, CT260 and CT700, acting as central nodes of protein-protein interaction clusters. Because proteins within these clusters include known TTS effectors, we postulate that these central node proteins may be TTS secretion chaperones or escorts. Consistent with this, bioinformatic analysis indicates that of these proteins Ct260 shares structural features with known TTS chaperones. Our findings provide a framework for the biochemical analysis of TTS assembly and regulation in this clinically important pathogen.
| Name: | Roma Kenjale, Ph.D. |
| Lab: | St. Geme |
| Conference: | 108th General Meeting of the American Society for Microbiology |
| Date: | June 1-5, 2008 |
| Location: | Boston, Massachusetts |
| Purpose: | To present talk and poster |
Abstract:
Structural determinants of autoproteolysis by the Haemophilus influenzae Hap autotransporter
Non-typeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The process of colonization involves both pilus and non-pilus adhesins. Hap is a non-pilus adhesin that belongs to the autotransporter family and mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells. Hap also contains serine protease activity associated with autoproteolysis, resulting in extracellular release of the Haps passenger domain and leaving the Hapβ beta domain embedded in the outer membrane. Autoproteolysis is mediated by a catalytic triad that includes residues H98, D140 and S243. The primary site of cleavage is the LN 1036-37 bond, and alternative sites include the LT1046-7, FA1077-8, and FS1067-8 bonds. In this study, we set out to characterize the structural determinants of Hap autoproteolysis. We utilized site-directed mutagenesis to study the P1-P4 residues at the Hap cleavage sites. In addition, we used the crystal structures of the E. coli Hbp autotransporter, alpha-chymotrypsin and trypsin to model the Hap protease domain and then exploited this model to mutate residues that correspond to the predicted S1, S2, and S4 subsites. We found that non-conservative mutations at the P1 and P2 positions but not at the P3 and P4 positions disrupted proteolysis, suggesting that the P1 and P2 positions are important determinants of Hap substrate specificity. In addition, we observed that mutations at the predicted S2 and S4 subsites eliminated proteolysis, arguing that the model of the Hap protease domain is an accurate representation of the Hap substrate groove. This information may be helpful in identifying potential host targets for Hap proteolysis and in developing novel inhibitors of Hap activity and NTHi disease.
| Name: | Jeongmin Song, Ph.D. |
| Lab: | Abraham |
| Conference: | The American Society for Cell Biology (ASCB) Meeting |
| Date: | December 13-17, 2008 |
| Location: | San Francisco, CA |
| Purpose: | To present talk and poster |
Abstract:
TLR4-Mediated Expulsion of E. coli from infected bladder epithelial cells
Uropathogenic E. coli invade bladder epithelial cells (BECs) by direct entry into specialized cAMP regulated exocytic compartments. Remarkably, a significant number of these intracellular bacteria are subsequently expelled in piecemeal fashion by infected BECs. Here, we report that expulsion of intracellular E. coli by infected BECs is initiated by the pattern recognition receptor, Toll-Like Receptor (TLR) 4, following activation by LPS. Additionally, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. This capacity of TLR4 to mediate the expulsion of intracellular bacteria from infected cells represents a previously unrecognized function for this innate immune receptor.