No description available.

No description available.

No description available.

Salmonella enterica serovar Typhimurium (Salmonella) is a Gram-negative bacterial pathogen capable of causing both gastroenteritis and bacteraemia in human hosts. During infection, Salmonella invokes a complex network of virulence factors, regulatory systems, and metabolic pathways to promote immune evasion, sometimes demanding antibiotic treatment for resolution. Unfortunately, antibiotic resistance has reached critical levels in this and other pathogens, necessitating the discovery of new anti-infective targets and treatment options. Herein, we have sought to exploit the dynamic interactions between Salmonella and the host immune system to identify new, conditionally active anti-Salmonella therapies. In chapter 2, we aim to identify chemical compounds that are selectively antimicrobial against intracellular Salmonella, and discover that the anxiolytic drug metergoline inhibits Salmonella survival in cultured macrophages and systemically infected mice. In chapter 3, we screen for anti-virulence compounds that target regulatory signaling in Salmonella, and characterize the inhibitory activity of methyl-3,4-dephostatin, which perturbs SsrA/B and PmrB/A signaling and enhances sensitivity to colistin in vitro and in vivo. In chapter 4, we identify several host-directed compounds that modulate macrophage immunity and investigate their ability to attenuate a multidrug resistant Salmonella infection. Together, the work presented in this thesis demonstrates the potential for drug screening in infection-relevant conditions to identify new anti-infectives with non-traditional targets. / Thesis / Doctor of Philosophy (PhD)

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In the U.S., soybean mosaic virus (SMV) is classified into seven strain groups, designated G1 to G7, based on their different responses on resistant soybean [Glycine max (L.) Merr.] cultivars. These responses are: symptomless or resistant (R), necrotic (N), and mosaic or susceptible (S). The gene-for-gene model has been proposed for SMV-soybean interactions. In the majority of cultivars, a single dominant gene, Rsv1, confers both the R and N responses. In the first part of this study, the coat protein (CP) genes of two SMV strains, G1 and G6 were isolated, cloned, and sequenced. Gene isolation was done by reverse transcription-polymerase chain reaction (RT-PCR) on partially purified virus preparation without prior RNA extraction. Amplified products were blunt-end ligated into pNoTA/T7 vector and transformed into competent cells. Sequencing was performed in both directions on heat-denatured double-stranded plasmids. The predicted 265 amino acid sequence of the CP of G1 and G6 strains were 98.9% identical, with only two amino acid differences. Correlating the CP sequences of G1, G2, G6, and G7, with their virulence on resistant soybean cultivars indicated that the CP is not likely to be the R- and/or N-determinant in the SMV-soybean system. The second part of the study involved studying the pathogenesis of G1, G6, and G7 strains on inoculated leaves of R, N, and S soybean cultivars by leaf imprint immunoassay. Results indicated four types of reactions: i) susceptible, showing unrestricted replication and spread; ii) immune, where no virus was detected; iii) systemic spread, showing unrestricted replication but limited spread along the veins; and iv) restricted replication and spread, where infection was restricted to few foci along the veins. Results of this study indicated that Rsv1-mediated resistance is a multicomponent type of resistance that involves both inhibition of virus replication as well as cell-to-cell movement. The third part of the study aimed at investigating Rsv1-mediated resistance at the cellular level. For this purpose, an SMV-soybean protoplast system was developed. Protoplast isolation was based on a combined cellulase-pectolyase Y-23 digestion and metrizamide-sorbitol gradient purification protocol. Virus inoculation of protoplasts was facilitated by either polyethelene glycol (PEG) or poly-L-ornithine (PLO), and method of detection was by Western blotting using antiserum to whole virus. Inoculation by PEG was successful, but results were irreproducible because of the adverse effect of PEG on protoplast viability. Inoculation by PLO was inconclusive because of the high background from residual inoculum. Additional research is needed before a protoplast system can be used to study the mechanism of Rsv1 resistance to SMV at the cellular level. / Ph. D.

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Salmonella enterica is an enteric pathogen with a broad host tropism that can cause disease ranging from self-limited gastroenteritis to enteric fever. The evolution of S. enterica as a pathogen is driven by the horizontal acquisition of genes that promote virulence and survival within host immune cells, as well as the coordinated regulation of these and ancestral genes by two-component systems (TCS). TCS integrate environmental cues with the transcriptional reprogramming of bacteria, and in the case of Salmonella, result in niche-specific gene expression in response to anti-bacterial cues produced by the host. The TCS SsrA-SsrB in S. enterica is considered the master regulator for intracellular virulence, where SsrA is a sensor kinase that triggers the activation of the DNA binding protein SsrB. The full suite of genes regulated by SsrB in S. enterica, as well as the cues that activate this TCS, have not been fully characterized. Here, we demonstrated that horizontally acquired and ancestral genes in the S. enterica genome have evolved to be regulated by SsrB, and the repression of a set of ancestral genes involved in flagellar motility promotes evasion of the host immune system. Additionally, we identified the production of reactive oxygen species (ROS) by host immune cells as a signal that can activate a cluster of genes regulated by the SsrA-SsrB TCS, likely mediated by SsrA sensing of these ROS. Together, these results expand our understanding of the complex interplay between the pathogen S. enterica and the host that results in bacterial infections. / Thesis / Doctor of Philosophy (PhD) / Salmonella enterica (S. enterica) is a species of bacteria that can cause food poisoning in various animals, including humans, through consumption of contaminated food and water. During an infection, host cells activate numerous defense mechanisms to prevent disease. S. enterica has evolved to turn specific genes on or off in response, resulting in modifications to bacterial and host cell behaviour that promote infection. The timing of these genetic changes is controlled by proteins that can sense specific environmental signals and adjust gene expression accordingly. The specific signals sensed by S. enterica that allow for adaptive gene expression within the host, and the types of genes that are regulated to promote survival, have not been fully identified. Here, we show that S. enterica evolved to repress genes involved in flagellar motility to hide from the host immune response. We further demonstrate that S. enterica can sense anti-bacterial molecules produced by the host, called reactive oxygen species, to trigger specific changes in gene expression. Together, this work reveals novel aspects for the molecular basis of Salmonella enterica pathogenesis.

No description available.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy