Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.

Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.

Emerging infectious diseases, pollution and climate change are associated with amphibian extinction events worldwide; however, direct causation is often obscured by the interactions of these stressors. Elucidating the possible synergies between metal contamination and disease is, therefore, critical in advancing our knowledge of the co-evolutionary mechanisms in host-pathogen systems and helping with the ability to better forecast the spread of diseases in metal-stressed environments. Additionally, increasing ecotoxicological research has improved our understanding of the complex influence trace metals may have on the physico-chemical nature of aquatic systems; however, the discrepancy in concentration-response within the toxicological literature makes it difficult to accurately define the range of toxicity, often due to the variability in media used in experimentation. The first chapter of this thesis reports an evaluation of copper, nickel and copper/nickel concentrations on several Northern Leopard Frog (Lithobates [Rana] pipiens) larvae life history traits within field collected smelting effluent water. Overall, results indicated that copper had a stronger negative impact on survival than nickel. However, tadpoles exposed to copper displayed increased growth and developmental patterns while those exposed to nickel demonstrated opposing life history traits. These results indicate that tadpoles are displaying different fitness strategies, in terms of survival and life history, in the presence of increased copper and/or nickel stress.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

Infectious diseases from novel viruses have become a major public health concern. Rapid identification of virus–host interactions can reveal mechanistic insights into infectious diseases and shed light on potential treatments. Current computational prediction methods for novel viruses are based mainly on protein sequences. However, it is not clear to what extent other important features, such as the symptoms caused by the viruses, could contribute to a predictor. Disease phenotypes (i.e., signs and symptoms) are readily accessible from clinical diagnosis and we hypothesize that they may act as a potential proxy and an additional source of information for the underlying molecular interactions between the pathogens and hosts. We developed DeepViral, a deep learning based method that predicts protein– protein interactions (PPI) between humans and viruses. Motivated by the potential utility of infectious disease phenotypes, we first embedded human proteins and viruses in a shared space using their associated phenotypes and functions, supported by formalized background knowledge from biomedical ontologies. By jointly learning from protein sequences and phenotype features, DeepViral significantly improves over existing sequence-based methods for intra- and inter-species PPI prediction. Lastly, we propose a novel experimental setup to realistically evaluate prediction methods for novel viruses.

No description available.

Bacterial pathogens are a major cause of diseases in human, agricultural plants and farm animals. Even after decades of research they remain a challenge to health care as they are known to rapidly evolve and develop resistance to the existing drugs. Systems biology is an emerging area of research where all of the components of the system, their interactions, and the dynamics can be studied in a comprehensive, quantitative, and integrative fashion to generate predictive models. When applied to bacterial pathogenesis, systems biology approaches will help identify potential novel molecular targets for drug discovery. A pre-requisite for conducting systems analysis is the identification of the building blocks of the system i.e. individual components of the system (structural annotation), identification of their functions (functional annotation) and identification of the interactions among the individual components (interaction prediction). In the context of bacterial pathogenesis, it is necessary to identify the host-pathogen interactions. This dissertation work describes computational resources that enable comprehensive systems level study of host pathogen system to enhance our understanding of bacterial pathogenesis. It specifically focuses on improving the structural and functional annotation of pathogen genomes as well as identifying host-pathogen interactions at a genome scale. The novel contributions of this dissertation towards systems biology of bacterial pathogens include three computational tools/resources. “TAAPP” (Tiling array analysis pipeline for prokaryotes) is a web based tool for the analysis of whole genome tiling array data for bacterial pathogens. TAAPP helps improve the structural annotation of bacterial genomes. “ISO-IEA” (Inferred from sequence orthology - Inferred from electronic annotation) is a tool that can be used for the functional annotation of any sequenced genome. “HPIDB” (Host pathogen interaction database) is developed with data a mining capability that includes host-pathogen interaction prediction. The new knowledge gained due to the implementation of these tools is the description of the non coding RNA as well as a computationally predicted host-pathogen interaction network for the human respiratory pathogen Streptococcus pneumoniae. In summary, the computation tools and resources developed in this dissertation study will enable building systems biology models of bacterial pathogens.

Bacteria inhabit diverse environmental niches and consequently, must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as regulating bacterial interactions with its host. This thesis focuses on the coordination of virulence gene expression and evasion of host immunity by (p)ppGpp in Salmonella enterica serovar Typhimurium. In the first data chapter, I describe the role of (p)ppGpp in mediating bacterial resistance to killing by the human complement system. I identified that (p)ppGpp activates ppnN, a nucleotide metabolism associated enzyme, and the biosynthesis of lipopolysaccharide O-antigen to protect Salmonella from cell lysis by complement. The second data chapter compares and contrasts the stringent response of an invasive clinical isolate of Salmonella Typhimurium to a strain of Salmonella Typhimurium that causes acute gastroenteritis using RNA-sequencing. Critical analysis of our transcriptomics dataset showed that flagellar-based motility is differentially regulated by (p)ppGpp in the two strains of Salmonella. Together, these findings demonstrate that (p)ppGpp has significant functional roles beyond mediating adaptation to nutrient limitation. / Thesis / Doctor of Philosophy (PhD)

Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. / Thesis / Master of Science (MSc) / Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

Catfish, the "king" of the U.S. aquaculture, is threatened by a severe, systemic bacterial disease known as enteric septicemia of catfish (ESC). This disease causes high mortality and massive economic losses in cultured channel catfish (Ictalurus punctatus) in the United States. E. ictaluri penetrates catfish intestinal epithelia quickly and establishes a systemic infection rapidly. However, our knowledge on catfish intestine and E. ictaluri interaction is very limited. In Particular, catfish intestinal immune responses and virulence genes needed by E. ictaluri to evade host defenses are not well understood. Hence, our long-term goal is to identify the molecular mechanisms of E. ictaluri-host interactions. The overall objectives of this study were to understand catfish immune responses to E. ictaluri infection and determine essential genes of E. ictaluri during the intestinal invasion. To accomplish the overall objectives of this research, intestinal ligated loops were constructed surgically in live catfish and loops were injected with wild-type E. ictaluri and two live attenuated E. ictaluri vaccine strains developed recently by our research group. We first determined catfish intestinal immune responses against E. ictaluri wild-type and live attenuated vaccine strains. Then, we analyzed the global gene expression patterns of wild-type E. ictaluri and vaccine strains during catfish intestinal invasion using high throughput RNA-Seq technology. Results showed a moderate level of neutrophil and B cell infiltration correlated with significantly lower expression of TNF-α, CD4-1, and CD8-α in the vaccine injected intestinal tissue compared to that of wild-type injected intestinal tissue. Further, RNA-Seq data analysis showed the prominent expression of genes related to bacterial secretion systems, ATP production processes, and multidrug resistance (MDR) efflux pumps in wild-type E. ictaluri. In contrast, the prominently expressed genes in vaccine strains were related to the phosphotransferase system and sugar metabolism processes. All these data suggest that our live attenuated vaccines are capable of triggering effective immune responses in catfish without causing damage to the host.