Several types of cell death including apoptosis, necroptosis and autophagic cell death play diverse roles in different biological processes. In addition to its essential roles in development and metabolism, programmed cell death is indispensable for host immunity. Interestingly, current research shows that these processes are connected but the nature and extent of the crosstalk between host defense and programmed cell death still remains an area of great interest. The NFkB factor Relish is best characterized as a crucial component of Drosophila Imd pathway, which generates immune responses by producing antimicrobial peptides following Gram-negative bacterial infection. In this dissertation, I demonstrate a novel role of Relish in developmentally programmed cell death. During metamorphosis, Drosophila salivary glands are degraded by the collective actions of caspase-dependent and autophagic cell death. Here I show that Relish mutants displayed improper salivary gland degradation and the persistence of salivary gland cell fragments. Expression of Relish in salivary glands rescued this phenomenon. Among the upstream components of the Imd pathway, mutants in the bacterial peptidoglycan receptors, PGRP-LC and-LE also exhibited similar defects in gland degradation, but surprisingly none of the other Imd pathway components examined had any such effect. As both Relish and PGRPs are critical for host defense against bacterial infection, our next concern was the role of host microflora in salivary gland degradation. However, observation of normal salivary gland cell death in axenic flies ruled out the possible involvement of microbiota. Robust genetic analyses proved that Relish-mediated cell death occurs in caspase-independent but autophagy-dependent manner. Moreover, expressions of either active version of Relish or PGRP-LC resulted in the premature gland degradation and induction of autophagy. Finally, I show that Relish controls autophagy by regulating the expression of Atg1, a core component of the autophagy pathway. Together these findings suggest the existence of a novel pathway, which connects immune response factors to developmentally programmed cell death.

The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.

CD4 T cells differentiate into multiple effector subsets that mediate pathogen clearance. ThCTL are anti-viral effectors with MHC-II restricted cytotoxicity. The factors regulating ThCTL generation are unclear, in part due to a lack of a signature marker. I show here that in mice, NKG2C/E identifies ThCTL that develop in the lung during influenza A virus (IAV) infection. ThCTL phenotype indicates they are highly activated effectors with high levels of binding to P-selectin, T-bet, IFNγ production, and degranulation. ThCTL express increased levels of granzymes and perforin and lower levels of genes associated with memory and recirculation compared to non-ThCTL lung effectors. ThCTL are also restricted to the site of infection, the lung in IAV and systemically in LCMV. ThCTL require Blimp-1 for their differentiation, suggesting a unique effector CD4 population. As ThCTL are highly activated, they also require antigen signaling post priming during IAV infection. Late antigen was necessary and sufficient for the differentiation of ThCTL. In the context of late antigen encounter, ThCTL surprisingly do not require CD80 and CD86 costimulation for their differentiation. Additionally ThCTL do not require late IL-2 for their differentiation and instead require late IL-15 signals for their efficient generation. Thus these data suggest ThCTL are marked by the expression of NKG2C/E and represent a unique CD4 effector population specialized for cytotoxicity.

Mycobacterium tuberculosis stably adapts to respiratory limited environments by entering into a nongrowing but metabolically active state termed quiescence. This state is inherently tolerant to antibiotics due to a reduction in growth and activity of associated biosynthetic pathways. Understanding the physiology of the quiescent state, therefore, may be useful in developing new strategies to improve drug efficiency. Here, we used an established in vitro model of respiratory stress, hypoxia, to induce quiescence. We utilized metabolomic and genetic approaches to identify essential and active pathways associated with nongrowth. Our metabolomic profile of hypoxic M. tuberculosis revealed an increase in several free fatty acids, metabolite intermediates in the oxidative pathway of the tricarboxylic acid (TCA) cycle, as well as, the important chemical messenger, cAMP. In tandem, a high-throughput transposon mutant library screen (TnSeq) revealed that a cAMP-regulated protein acetyltransferase, MtPat, was conditionally essential for survival in the hypoxic state. Via 13C-carbon flux tracing we show an MtPat mutant is deficient in re-routing hypoxic metabolism away from the oxidative TCA cycle and that MtPat is involved in inhibiting fatty-acid catabolism in hypoxia. Additionally, we show that reductive TCA metabolism is required for survival of hypoxia by depletion of an essential TCA enzyme, malate dehydrogenase (Mdh) both in in vitro hypoxia and in vivo mouse infection. Inhibition of Mdh with a novel compound resulted in a significantly greater killing efficiency than the first-line anti-M. tuberculosis drug isoniazid (INH). In conclusion, we show that understanding the physiology of the quiescent state can lead to new drug targets for M. tuberculosis.

Presentation of antigenic peptides on MHC-II molecules is essential for induction of tolerance to self and for effective immunity against foreign pathogens. The non- classical MHC-II molecule DO (HLA-DO in humans, H2-O in mice) functions in selection of MHC-II epitopes by competitively inhibiting the peptide exchange factor DM. Previous studies have suggested a role for DO in development of autoimmunity and in the immune response to retroviral infection, presumably via modulation of the MHC-II peptidome, but the precise effect of DO has been difficult to discern. Through characterization of the full spectrum of peptides from DO-sufficient and DO-deficient cells, we demonstrate that DO functions to broaden the diversity of peptide species presented on MHC-II. DO is regulated differently from other components of the MHC-II processing machinery, with expression limited to B cell and dendritic cell subsets, as well as thymic epithelial cells, suggesting a role for DO in mediating central tolerance. In a mouse model lacking DO, we show that selection of T regulatory cells (Tregs) is increased and that DO- deficient Tregs are more activated and exert greater suppressive capacity. Despite augmented Treg function, mice lacking DO display enhanced susceptibility to autoimmunity, with altered germinal center (GC) Tregs and B cells indicative of an aberrant GC reaction. These data suggest that DO expression serves to fine-tune the immunopeptidome in order to promote self-tolerance to a wide spectrum of epitopes and to select a Treg population with appropriate specificity for self- antigens.

Transcriptional regulation of CD8+ T cell differentiation during acute and chronic viral infections is an intricate web made up of many of transcription factors. While several transcription factors have been elucidated in this process, there are still many more that remain elusive. In this work, we look into the role of two transcription factors, IRF4 and Runx2, and their role in CD8+ T cell terminal effector cells and memory precursor cells during acute LCMV-Armstrong infection. We found that IRF4 expression was regulated by TCR signal strength during infection, and that IRF4 expression levels directly correlated with the magnitude of the effector cell response. IRF4 was also shown to regulate T-bet and Eomes, two transcription factors critical for CD8+ T cell differentiation into effector and memory cells. From these results, we were interested in the potential role of IRF4 during chronic LCMV-clone 13 infection, where ratios of T-bet and Eomes are critical for viral clearance. We found that haplodeficiency of IRF4 in the T cell compartment lead to an increase in the ratio of Eomes to T-bet in T cells, which in turn affected the proportion of Eomeshi versus T-bethi cells and resulted in a loss in ability to clear viral infection. Irf4+/-Eomes+/- compound heterozygous mice were generated to test if decreasing Eomes expression would rescue the Irf4+/- phenotype. Irf4+/-Eomes+/- mice were phenotypically similar to WT mice in terms of Eomes to T-bet ratios, and were able to clear viral infection, demonstrating a critical role of IRF4 in regulating T-bet and Eomes during chronic viral infection. Next we looked into the role of Runx2 during acute LCMV-Armstrong infection and found that Runx2-deficient pathogen-specific CD8+ T cells had a defect in the total number of memory precursor cells compared to WT controls. We further showed that Runx2 was inversely correlated with TCR signal strength, and that Runx2 expression was repressed by IRF4. From these work, we have introduced two more transcription factors that are critical for CD8+ T cells differentiation during acute and chronic viral infection. Given the sheer number of transcription factors known to regulate these processes, having a full understanding of the transcriptional network will allow us to find the best targets for therapeutic intervention for treatments ranging from vaccine development and autoimmunity to cancer immunotherapy and treatment of chronic viral infections.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Dengue virus (DENV) causes over 500,000 infections annually with a spectrum of clinical diseases ranging from subclinical infection to dengue, a mild febrile illness, to life-threatening severe dengue. Vascular leakage without endothelial cell damage is the hallmark symptom of severe dengue illness and is proposed to be directly mediated by soluble inflammatory mediators IL-8 and TNFα. IL-8 production occurs in response to DENV infection, is elevated during severe dengue, is proposed to inhibit interferon, and could potentially recruit target cells to sites of infection. We previously showed that expression of DENV NS5 activates the IL-8 promoter, induces IL-8 transcription, and induces IL-8 protein production in HepG2 and HEK293A cell lines. As multiple DENV proteins are reported to interact with important signaling pathways, we hypothesized that other DENV proteins could contribute to the activation of IL-8. We found that plasmids expressing prM-E together, the GPI-linked variant of NS1 (NS1G), the carboxyl-terminal 112 amino acids of NS4B, as well as NS5 each induced expression from an IL-8 promoter-driven reporter plasmid. Expression of NS5 also induced activation of a RANTES promoter construct and TNFα mRNA expression. Further, we found that the carboxyl-terminal polymerase domain of NS5 was sufficient to induce IL-8 secretion but polymerase function was not required. Like NS5, prM-E and NS1G induced luciferase expression from an AP-1-driven reporter plasmid. We further tested whether activation of the IL-8 promoter depended on any single transcription factor within IL-8 using IL-8 promoter-driven plasmids mutated at the AP-1, C/EBP or NF-κB binding sites. We found that activation of the IL-8 promoter by prM-E, NS1G and NS4B did not depend on activation of any single transcription factor. Our data suggested that AP-1 may be both positively and negatively inducing transcription, fitting with previous theories that DENV regulates IL-8 induction. However, we did not observe any differences in activation of AP-1 subunit c-Jun, or the inhibitory subunits Fra-1 or Fra-2 between DENV and mock-infected cells. These data support a model in which multiple DENV proteins activate the IL-8 promoter, provide a potential basis of IL-8 induction by DENV in multiple cell types, and further supports a mechanism by which DENV contributes to severe dengue illness.

When an outbreak of an infectious disease occurs, public health officials need to understand the dynamics of disease transmission in order to launch an effective response. Two quantities that are often used to describe transmission are the basic reproductive number and the distribution of the serial interval. The basic reproductive number, R0, is the average number of secondary cases a primary case will infect, assuming a completely susceptible population. The serial interval (SI) provides a measure of temporality, and is defined as the time between symptom onset between a primary case and its secondary case. Investigators typically collect outbreak data in the form of an epidemic curve that displays the number of cases by each day (or other time scale) of the outbreak. Occasionally the epidemic curve data is more expansive and includes demographic or other information. A contact trace sample may also be collected, which is based on a sample of the cases that have their contact patterns traced to determine the timing and sequence of transmission. In addition, numerous large scale social mixing surveys have been administered in recent years to collect information about contact patterns and infection rates among different age groups. These are readily available and are sometimes used to account for population heterogeneity. In this dissertation, we modify the methods presented in White and Pagano (2008) to account for additional data beyond the epidemic curve to estimate R0 and SI. We present two approaches that incorporate these data through the use of a Bayesian framework. First, we consider informing the prior distribution of the SI with contact trace data and examine implications of combining data that are in conflict. The second approach extends the first approach to account for heterogeneity in the estimation of R0. We derive a modification to the White and Pagano likelihood function and utilize social mixing surveys to inform the prior distributions of R0. Both approaches are assessed through a simulation study and are compared to alternative approaches, and are applied to real outbreak data from the 2003 SARS outbreak in Hong Kong and Singapore, and the influenza A(H1N1)2009pdm outbreak in South Africa.

The close proximity of the United States to the Mexican border poses a concern for communicable diseases because of the high flow of population movement. The purpose of this retrospective, quantitative study was to identify risks associated with respiratory diseases using an analysis of archived data from the Severe Acute Respiratory Illness (SARI) surveillance program. Based on the epidemiologic triangle theory, demographic and etiologic factors were analyzed to examine any associations with SARI in this population. Between 2010 and 2012, 798 subjects enrolled in this program, with 336 (42.1%) testing positive for respiratory pathogens. Chi square analysis determined that age (X2 (4, N = 786) = 255.361, p < 0.001), clinic location (X2 (3, N = 780) = 290.841, p < 0.001), and race/ethnicity (X2 (4, N = 762) = 1456.701, p < 0.001) showed significant associations with SARI in the population. The logistic regression model showed that the youngest age group (0-4) had the highest risk of developing SARI compared to other age groups (5-24 OR = 0.521, 95% CI [0.311-0.871]; 25-49 OR = 0.377, 95% CI [0.224-0.636]; 50-64 OR = 0.211, 95% CI [0.118-0.376]; >65 OR = 0.225, 95% CI [0.143-0.356]. African Americans were also at higher risk of developing SARI compared to Hispanic Americans (OR = 3.997, 95% CI [1.272-12.558]. This study promotes positive social change by informing efforts to increase vaccination and health literacy, improve the accessibility and availability of preventive health care in low socioeconomic communities, and promote healthy lifestyles among at-risk groups. These steps will improve the overall health of the communities along the U.S.-Mexico border region.