Murine infection by the Lyme disease spirochete, B. burgdorferi, results in the generation of pathogen-specific antibody that can provide protection against Lyme disease, but the cells involved in this response are poorly characterized. T cells are not required for generating a protective antibody response to B. burgdorferi infection, but their exact role in providing protection against tissue colonization had not been previously determined. We found that TCRβxδ;-/- mice were susceptible to high pathogen loads and decreased antibody titers, but inhibition of CD40L-dependent interactions resulted in partial protection suggesting that a portion of the help provided by T cells was not dependent on CD40L-CD40 interactions between T and B cells. RAG1-/- mice reconstituted with either un-fractionated or B1-enriched peritoneal cells from previously infected mice generated B. burgdorferi-specific antibody, and upon spirochetal challenge suffered significantly lower levels of pathogen load in the joint and heart. Peritoneal cells from previously infected TCRβxδ-/- mice or B2-enriched or B1-purified peritoneal cells conferred little to only moderate protection, suggesting T cells play an important role in protection against spirochetal infection the joint. Consistent with this, T cells from previously infected donor mice, when transferred with B1 or B2 cells into RAG1-/- mice, generated increased antibody titers and were capable of diminishing bacterial burden in the joint and heart. A previously identified class of protective antibody is directed against the spirochetal surface lipoprotein DbpA, and we found that DbpA is a prominent protein antigen recognized by RAG1-/- mice reconstituted with B1-enriched peritoneal cells. Additionally, we found that mice reconstituted with B1 cells also make antibody directed towards the spirochetal glycolipid antigen, BbGL-IIc, which is recognized by Vα14iNKT cells. Consistent with the idea that T cells are important in providing protection against spirochetal infection, RAG1-/- mice reconstituted with B1 and T cells generated a more robust response against DbpA and BbGL-IIc. These results support the hypothesis that T cells act with B1 cells in a CD40L-independent manner to promote the production of antibodies that play an important role in protection of the joint from spirochetal infection.

Given that Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, an apparent paradox is that the bacterium resides, usually harmlessly, in the nasopharynx of many people. Humoral immunity is thought to be the primary defense against serious pneumococcal infection, and we hypothesized that nasopharyngeal colonization of mice results in the generation of an antibody response that provides long-term protection against lung infection. We found that survival of of C57L/6 mice after intranasal inoculation with wild-type serotype 4 strain TIGR4 pneumococci required B cells but not T cells, suggesting that nasopharyngeal colonization elicited a protective humoral immune response. In fact, intranasal inoculation resulted in detectable pneumococcal-specific antibody responses, and protected mice against a subsequent high-dose S. pneumoniae pulmonary challenge. B cells were required for this response, and transfer of immune sera from i.n. colonized mice, or monoclonal antibodies against phosphorylcholine, a common surface antigen of S. pneumoniae, was sufficient to confer protection. IgA, which is thought to participate in mucosal immunity, contributed to but was not absolutely required for protection from pulmonary challenge. Protection induced by i.n. colonization lasted at least ten weeks. Although it was partially dependent on T cells, depletion of CD4+ T cells at the time of challenge did not alter protection, suggesting that T cells did not provide essential help in activation of conventional memory cells. Peritoneal B1b cells and radiation-resistant, long-lived antibody secreting cells have previously been shown to secrete anti-pneumococcal antibodies and mediate protection against systemic infection following immunization with killed bacteria or capsular polysaccharide [1, 2]. We found that peritoneal cells were not sufficient for colonization-induced protection, but sub-lethally irradiated mice largely survived pulmonary challenge. Thus, our results are consistent with the hypothesis that nasopharyngeal colonization, a common occurrence in humans, is capable of eliciting extended protection against invasive pneumococcal disease by generating long-lived antibody-secreting cells.

Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y. pestis, all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD1. Of these, LcrV from Y. enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL-10. Through expression and purification of recombinant LcrV from Escherichia coliwe show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL-10 and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of F1, a peptide shown to be responsible for 37°C-dependent inhibition of invasion by Y. pestis in vitro, was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y. pestis by environmental cues other than temperature.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea. Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity. In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation. Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.

<p>Systemic fungal infections brought about by <em>Cryptococcus</em> species are associated with some of the highest mortality rates of any infectious disease. Alarmingly these pathogens have overtaken tuberculosis as the second greatest killer among Sub-Saharan AIDS patients and are an emerging disease among immunocompetent populations on the Pacific Coast of North America. This clinical threat has been exacerbated by our inability to discover novel compounds that specifically target fungal cellular architecture at the genus level. To confront this challenge, we have made a concerted effort to biologically prospect the vast chemical potential of Actinomycete bacteria isolated from diverse and underexplored niches around the world. A novel phenotypic screen was developed whereby bacterial small molecule producers were co-cultured on agar plates in an intimate setting with evolutionary distant fungal pathogens <em>Candida albicans</em> and <em>Cryptococcus neoformans</em>. Diffusible small molecules released by the organisms created a signaling environment that stimulated profound phenotypic changes both in the Actinomycetes and the pathogens. We were able to discern a unique relationship whereby the growth of <em>C. neoformans</em> was specifically inhibited by Nigerian soil Actinomycete isolate curated as WAC 2288. Further bioactivity guided purification and chemical analysis lead to the identification of ibomycin, a previously undescribed 34 membered macrolactone decorated with seven sugar moieties. A draft genome of WAC 2288 revealed a 140kb gene cluster containing 12 type I PKS modules and downstream capacity to generate rare sugars are responsible for ibomycin biosynthesis. Purification of ibomycin analogs has revealed that the terminal vancosamine on the molecule is dispensable for bioactivity, establishing a chemical antecedent for target identification through affinity chromatography. Throughout these studies the unprecedented anticryptococcal activity of ibomycin is consistently recapitulated. Future work on the molecule may validate ibomycin as an effective antifungal therapy.</p> / Master of Science (MSc)

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

Sepsis is the leading cause of death among critically ill patients and the 10th most common cause of death overall in the United States. The mortality rates increase with severity of the disease, ranging from 15% for sepsis to 60% for septic shock. Patient with sepsis can present varied clinical symptoms depending on the personal predisposition, causal microorganism, organ system involved, and disease severity. To facilitate sepsis diagnosis, the first sepsis consensus definitions was published in 1991 and then updated in 2001. Early recognition of a sepsis patient followed with timely and appropriate treatment and management strategies have been shown to significantly reduce sepsis-related mortality, and allows care to be provided at lower costs. Despite the rapid progress in the knowledge of pathophysiological mechanisms of sepsis and its treatment in the last two decades, identifying patient with sepsis and therapeutic approaches to sepsis and its complications remains challenging to critical care clinicians. Hence, the objectives of this thesis were to 1) evaluate the test characteristics of the two sepsis consensus definitions and delineate the differences in patient profile among patients meeting or not meeting sepsis definitions; 2) determine the relationship between the changes in several physiological parameters before sepsis onset and sepsis, and to determine whether these parameters could be used to identify sepsis in critically ill adults; 3) evaluate the effect of corticosteroids therapy on patient mortality. Data used in this thesis were prospectively collected from an electronic medical record system for all the adult patients admitted into the seven critical care units (ICUs) in a tertiary medical center. Besides analyzing data at the ICU stay level, we investigated patient information in various time frames, including 24-hour, 12-hour, and 6-hour time windows. In the first study of this thesis, the 1991 sepsis definition was found to have a high sensitivity of 94.6%, but a low specificity of 61.0%. The 2001 sepsis definition had a slightly increased sensitivity but a decreased specificity, which was 96.9% and 58.3%, respectively. The areas under the ROC curve for the two consensus definitions were similar, but less than optimal. The sensitivity and area under the ROC curve of both definitions were lower at the 24-hour time window level than those of the unit stay level, though the specificity increased slightly. At the time window level, the 1991 definitions performed slightly better than the 2001 definition. In the second study, minimum systolic blood pressure performed the best, followed by maximum respiratory rate in discriminating sepsis patients from SIRS patients. Maximum heart rate and maximum respiratory rate can differentiate sepsis patients from non-SIRS patients fairly well. The area under ROC of the combination of five physiological parameters was 0.74 and 0.90 for comparing sepsis to non-infectious SIRS patients and comparing sepsis to non-SIRS patients, respectively. Parameters typically performed better in 24-hour windows compared to 6-hour or 12-hour windows. In the third study, significantly increased hospital mortality and ICU mortality were observed in the group treated with low-dose corticosteroids than the control group based on the propensity score matched comparisons, and multivariate logistic regression analyses after adjustment for propensity score alone, covariates, or propensity score (in deciles) and covariates. This thesis advances the existing knowledge by systemically evaluating the test characteristics for the 1991 and 2001 sepsis consensus definitions, delineating physiological signs and symptoms of deterioration in the preceding 24 hours prior to sepsis onset, assessing the prediction performances of single or combined physiological parameters, and examining the use of corticosteroids treatment and survival among septic shock patients. In addition, this thesis sets an innovative example on how to use data from electronic medical records as these surveillance systems are becoming increasingly popular. The results of these studies suggest that a more parsimonious set of definitional criteria for sepsis diagnosis are needed to improve sepsis case identification. In addition, continuously monitored physiological parameters could help to identify patients who show signs of deterioration prior to developing sepsis. Last but not least, caution should be used when considering a recommendation on the use of low dose corticosteroids in clinical practice guidelines for the management of sepsis.

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.