Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

In 2008 the Nobel Prize in Physiology or Medicine was awarded to the co-discoverers of the Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS). This award acknowledged the enormous worldwide impact of the HIV-1/AIDS pandemic and the importance of research aimed at halting its spread. Since the syndrome was first recognized, 25 million people have succumbed to AIDS and over 33 million are currently infected with HIV-1 (www.unaids.org). The most effective strategy for ending the pandemic is the creation of a prophylactic vaccine. Yet, to date, all efforts at HIV-1 vaccine design have met with very limited success. The consistent failures of vaccine candidates stem in large part from the unprecedented diversity of HIV-1. Among the novel theories of vaccine design put forward to address this diversity is the targeted vaccine approach. This proposal is based on the finding that mucosal transmission of HIV-1, the most prevalent form, occurs across a selective bottleneck such that typically only a single (or a few) variants of the viral swarm present in a donor are passed to the recipient. While the mechanisms controlling the selection are largely unknown, the targeted vaccine approach postulates that once they are identified, we can utilize this understanding to design vaccines specifically targeted to the characteristics shared by the rare, mucosally transmissible HIV-1 variants. The studies described in this work were conducted to improve our understanding of the factors influencing viral variant selection during mother-to-child-transmission of HIV-1, a route of mucosal transmission which has globally become the leading cause of child infection. A unique panel was generated, consisting of nearly 300 HIV-1 envelope genes cloned from infected mother-infant pairs. Extensive characterization of the genotypes, phenotypes and phylogeny of these clones was then done to identify attributes differentiating early infant from maternal variants. Low genetic diversity of HIV-1 envelope variants was detected in early infant samples, suggesting a bottleneck and active selection of variants for transmission. Transmitted variants did not differ from non-transmitted variants in CD4 and CCR5 use. Infant isolates replicated poorly in macrophages; a cell subtype hypothesized to be important in the establishment of infection. The sensitivity of infant envelope variants to neutralization by a panel of monoclonal antibodies, heterologous and autologous plasmas and HIV-1 entry inhibitors varied. Most intriguingly, envelopes cloned from infants infected during delivery exhibited a faster entry phenotype than maternal isolates. Together, these findings provide further insight into viral variant selection during mother-to-child transmission. Identification of properties shared by mucosally transmitted viral variants may allow them to be selectively targeted, resulting in improved methods for preventing HIV-1 transmission.

Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

Many virus infections induce a transient state of immune suppression in the infected host. Virus-induced T cell suppression can be caused by T cell activation-induced cell death (AICD), dendritic cell (DC) apoptosis, DC dysfunction, and/or the enhanced expression of immune-suppressive cytokines. It has been previously demonstrated that naïve bystander CD8 T cells derived from hosts experiencing an acute virus-specific T cell response underwent AICD when polyclonally activated by anti-CD3 in vitro (Zarozinski et al., 2000). Susceptibility of naïve bystander T cells to AICD could prevent the development of a new T cell response during an ongoing immune response, and thus render infected hosts immune suppressed. Although immune suppression could result in an enhanced susceptibility to superinfections, virus-infected individuals are more commonly resistant to superinfecting pathogens. Because of these seemingly contradictory conditions, we sought to investigate how acute viral infections impact naïve bystander CD8 T cells in vivo. More specifically, we asked whether bystander CD8 T cells are susceptible to immune suppression or whether they can contribute to the resistance to superinfections. In order to address this, we examined the responses of bystander CD8 T cells activated with cognate antigen during acute viral infections in vivo. We generated several in vivomodels using P14 (LCMV glycoprotein-specific), HY (male antigen-specific), and OT-I (ovalbumin-specific) transgenic CD8 T cells, which we defined as bystander during acute infections with lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), vaccinia virus (VV), and murine cytomegalovirus (MCMV). Consistent with the enhanced susceptibility to cell death noted in vitro, we found that bystander CD8 T cells activated with cognate antigen in vivo during acute viral infections underwent markedly reduced proliferation. Virus-induced transient T cell suppression in vivo was not exclusively mediated by Fas-FasL- or TNF-induced AICD or due to an enhanced susceptibility to apoptosis. Instead, immune suppression in vivowas associated with a delayed onset of division, which we found not to be due to a defect in antigen presentation, but rather due to a T cell intrinsic defect. Despite the suppressed proliferation of TCR-stimulated bystander CD8 T cells in vivo, we found an enhancement of the effector functions exerted by bystander CD8 T cells activated during acute viral infections. During acute viral infections or after stimulation with type 1 IFN (IFN-αβ) inducers, some bystander CD8 T cells were sensitized to immediately exert effector functions such as IFN-γ production and degranulation upon stimulation with high affinity cognate antigen. Sensitization of naïve CD8 T cells required self-MHC I and indirect effects of IFN-αβ, while IL-12, IL-18, and IFN-γ were not individually required. IL-15 was not required for the rapid expression of IFN-γ, but was required for up-regulation of granzyme B (GrzB). P14 and OT-I CD8 T cells, which are capable of homeostatic proliferation, could be sensitized by poly(I:C), but HY CD8 T cells, which are poor at homeostatic proliferation, could not, suggesting that the requirement for MHC I may be to present low affinity cryptically cross-reactive self antigens. Sensitized naive CD8 T cells up-regulated the t-box transcription factor Eomesodermin (Eomes), which can regulate these rapid effector functions. In conclusion, we demonstrate in this thesis that acute viral infections impact naïve bystander CD8 T cells such that their response to cognate antigen is altered. Prior to cognate antigen engagement, bystander CD8 T cells up-regulated Eomes, CD122, and GrzB. Following cognate antigen engagement, bystander CD8 T cells rapidly degranulated and expressed the effector cytokine IFN-γ. The ability of bystander CD8 T cells to rapidly exert effector functions may contribute to the resistance of virus-infected individuals to superinfections. Despite these rapid effector functions, the proliferation of TCR-stimulated bystander CD8 T cells was markedly inhibited. This reduced proliferation was found not to be a defect in antigen presentation, but was a T cell intrinsic defect in initiating division. Thus, bystander CD8 T cells were also susceptible to virus-induced immune suppression. It is also likely that virus-specific CD8 T cells that are not activated until later in the response, so-called latecomer CD8 T cells, may also be susceptible to immune enhancement and suppression. Thus, latecomer CD8 T cells would be able to rapidly exert effector functions at the expense of proliferation. Taken together, we propose that during an immune response, due to spatial and temporal gradients of antigen and inflammation, it is likely that a combination of heterogeneous T cells with different signal strengths and sequences of exposure from cytokines and peptide-MHC constitute the total T cell response to pathogens.

Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRb deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3b sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

Yersinia pestis, the causative agent of plague, specializes in causing dense bacteremia following intradermal deposition of a small number of bacteria by the bite of an infected flea. This robust invasiveness requires the ability to evade containment by the innate immune system. Of the various mechanisms employed by Y. pestis to subvert the innate immune response and to proliferate rapidly in mammalian tissue, only a few are well-characterized. Here, I present two complementary genetic analyses of Y. pestis adaptations to the mammalian environment. In the first, genome-wide fitness profiling for Y. pestis by Tn-seq demonstrates that the bacterium has adapted to overcome limitation of diverse nutrients during mammalian infection. In the second, a series of combinatorial targeted mutations disentangles apparent functional redundancy among the effectors of the Y. pestis type III secretion system, and we report that YpkA, YopT, and YopJ contribute to virulence in mice. We have also begun to investigate a novel relationship between Y. pestis and mammalian platelets, a highly abundant cell type in plasma. I present evidence that Y. pestis has evolved specific mechanisms to interfere with platelet activation, likely in order to evade immune responses and promote maintenance of bacteremia by undermining platelet thrombotic and innate immune functions. The principles guiding this work – systematic genetic analysis of complex systems, coupled with rational modification of in vitro assays to more closely mimic the in vivo environment – are a generalizable approach for increasing the efficiency of discovering new virulence determinants in bacterial pathogens.

The primary objective of this thesis is to differentiate between the risk factors for pre-and post-monsoon cholera epidemics in rural Bangladesh by analyzing the complex interaction between select environmental, cultural/behavioral, and socioeconomic variables over space and time. In rural Bangladesh, cholera epidemics correspond with the annual monsoon: the first, and smallest, occurs between March and June, while the larger cholera peak occurs between September and December. The differences between the spatial and temporal patterns of seasonal cholera are analyzed, and the risk factors are calculated for pre-and post-monsoon cholera epidemics. The theoretical approach that underlies this medical geographical study is disease ecology, which espouses that risk of disease is caused by an interaction between people and their environment. This thesis is structured around a holistic understanding that human-environment interactions are inseparable. In Bangladesh, the monsoon season typically starts between May and June. The 1992 and 1993 cholera peaks occurred just before the monsoon in April and March respectively, while the 1994 cholera peak occurred between April and June. In 1992 and 1993 cholera incidence increased in the post-monsoon period, and peaked in October. The 1994 post-monsoon cholera peak occurred in November. There is a regular temporal pattern to cholera, as the peaks followed a seasonal pattern with the smaller epidemic occurring in the pre-monsoon period and the larger epidemic occurring in the post-monsoon period. This study shows that there are different risks associated with pre-monsoon cholera epidemics and post-monsoon cholera epidemics. The two main risk factors associated with cholera incidence pre-monsoon were bari population (i.e., crowding) and a house located within the flood controlled area. These two variables were even more strongly associated with post-monsoon cholera incidence to a greater degree, along with a number of other variables including water use, sanitation practices, and socioeconomic status.

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI. Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the toxin), with endoribonuclease activity. Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C-terminal domain of MtopI for mutual inhibition. In this study the interaction of Rv1495 with the positively charged C-terminal tail in Mtop I is reported. This new information is useful for rational design and discovery of antibiotics for mycobacteria. Ethacridine, an FDA approved drug has shown activity against MtopI. In this project we studied the mechanisms of resistance associated with this drug as well the use of Ethacridine in combination with Moxifloxacin, to potentiate the bactericidal effect of this current second line drug for TB treatment. Results from sequencing of the genomic DNA isolated from the resistant mutants suggested the involvement of the Holliday-junction Ruv resolvase. Further studies showed that co-treatment with Ethacridine can enhance the moxifloxacin-mediated killing of M. smegmatis. FP-11g, a novel fluoroquinophenoxazine inhibitor of bacterial topoisomerase I, has shown promising activity against M, tuberculosis. We explored the bactericidal activity and resistance mechanisms associated to FP-11g using M. smegmatis as model organism. Additionally, the inhibitory effect of FP-11g was also evaluated on M. abscessus. FP-11g at concentration 4X MIC showed complete bactericidal activity against M. smegmatis after 24 hours. Inhibitory activity against M. abscessus was also observed. Results from sequencing of the genomic DNA isolated from the M. smegmatis resistant mutants revealed mutations in genes associated with general drug resistance.