Graduate Thesis Or Dissertation
 

The role of Salmonella typhimurium type three secretion system (T3SS) and effector proteins in chromatophore cell response.

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  • Bacterial contamination of food poses a great risk to human health worldwide. A chromatophore cell-based biosensor, utilizing B. splendens erythrophore cells, is an emerging technology that has shown potential to detect bacterial toxicity based on function-dependent mechanisms. Previous studies have investigated the response of erythrophore cells to foodborne pathogens, pesticides, and environmental toxicants. Numerous studies reveal that the chromatophore cell type, erythrophore, can rapidly detect, by aggregating their pigment organelles, several food associated bacterial pathogens, including Salmonella typhimurium. This study describes the Betta splendens erythrophore cells response to Salmonella typhimurium, including an analysis of the bacterial mechanism(s) that may be involved in this response. Previous studies using mutagenesis, identified genes of S. typhimurium that contributed to a response in erythrophore cells. The results indicated a potential role for the Type Three Secretion System (T3SS) apparatus coded for by the prgHIJK operon. In the current study, a mutation was identified in a region different from the mutation that disrupted the prgHIJK operon. However, both mutantions induced a similar but not identical response in erythrophore cells. The first objective of this study was to test the hypothesis that this second S. typhimurium (ST10) mutant has a mutation residing in either the gene(s) coding for the process regulating expression of the prgHIJIK operon or the gene(s) coding for the effector proteins secreted by the PrgHIJK apparatus. The S. typhimurium (ST10) mutant contained an insertional mutation in the hilD gene, a regulator involved in regulating the expression of the prgHIJK operon. Failure to express hilD in S. typhimurium impacts pigment organelle movement in erythrophore cells. The second objective of this study was to test the hypothesis that the effector proteins of T3SS (SipA, SopB, SopE2, SptP) are the components of S. typhimurium’s invading mechanism directly involved in inducing pigment organelles to undergo an aggregative response in erythrophore cells. Mutations in sopE2, sipA, or sptP did not change the erythrophore cell response to S. typhimurium as the magnitude and kinetics of the response was similar to that observed with wildtype S. typhimurium. A mutation in the sopB gene that encodes the SopB effector protein, which is involved in actin filament rearrangement, did change erythrophore cell response to S. typhimurium as the magnitude and kinetics of the response was different to that observed for the wildtype S. typhimurium. This is the first evidence in support of a direct role for the S. typhimurium SopB effector protein in pigment organelle aggregation in erythrophore cells. Results from this research effort suggest that hilD and sopB genes of S. typhimurium are involved in pigment organelle movement in a neuron-like pigment cell, an erythrophore cell of the chromatophore cell classification, isolated from the fish species, Betta splendens. The outcome to this research effort is to increase understanding of the biological basis of the erythrophore cell response to bacterial pathogens. Increased understanding will lead to better utility of erythrophore cells as biosensors for bacterial toxicity testing.
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