Bacteriophage-Encoded Molecules Regulate STEC Virulence
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Shiga toxin (Stx)-encoding Escherichia coli (STEC)  are a type of pathogenic E. coli that cause human disease with symptoms such as severe stomach cramps, diarrhea (often bloody), fever, vomiting, and, rarely, hemolytic uremic syndrome, kidney failure, and death. Most of what we know about STEC is from studies of a STEC O157:H7 strain, which was first identified as a pathogen in 1982. Less is known about non-O157 STEC strains, due to diagnostic limitations and inadequate surveillance. STEC O157 strains cause 73,000 illnesses annually in the United States and non-O157 STEC strains cause at least 37,000. STEC get their name from the exotoxin they produce, Stx, which is encoded on lambdoid prophages that they harbor. Lambdoid prophages are temperate, meaning they can choose between two developmental fates- lysis or lysogeny. In the lytic pathway, phage-mediated bacterial lysis leads to death of the infected cell and, subsequently, Stx production and release. In the lysogenic pathway, phages integrate their own DNA within the host chromosome and remains dormant. The prophage can also switch from the lytic to the lysogenic pathway in a process called induction. Since antibiotics cause prophages to induce, there is no current treatment for STEC-mediated disease. The population structure of E. coli O157:H7 lineage is highly homogenous, but the identities and gene content of Stx-encoding phages in these strains are highly variable. Moreover, STEC-mediated disease severity varies from outbreak to outbreak. Since Stx production, and thus disease, depends on phage lytic growth, our central hypothesis is that the severity of STEC infections depends on the rate of prophage induction, which is primarily regulated by phage-encoded molecules. This dissertation examines the roles that two phage proteins, namely the phage-encoded repressor and a phage-encoded methylase, play in modulating phage induction and thereby regulating STEC virulence. Since induction requires disabling the phage-encoded repressor’s DNA binding abilities, part of this dissertation focuses on determining how the repressor regulates induction and thereby STEC-mediated virulence. DNA binding studies, together with quantitative RT-PCR and dotblots were used to characterize the phage-encoded repressor in BAA2326, an Stx-encoding phage harbored in STEC O104:H4 and implicated in one the most virulent STEC outbreaks. The results of the DNA binding studies show that the BAA2326 repressor uses alternate binding that leads to lowered intracellular levels of transcript and protein. This ultimately leads to a higher susceptibility to induction, which results in increased virulence. We have also identified a ‘novel’ but widely prevalent methylase that is responsible for increased virulence in strains bearing two Stx-encoding phages PA2 and PA8 (originally found in STEC O157:H7 strains). A trypan blue exclusion test that was used to assess cell viability in the bacterial predator Acanthamoeba castellanii showed that the methylation activity of the methylase is required to exhibit the high killing potential of methylase+ lysogens. Quantitative PCR showed that the methylase positively regulates the number of phages being released as a function of time post induction. Quantitative dotblots showed that the methylase affects induction by downregulating repressor levels in cells. We compared our results with other well-characterized lambdoid phages namely, Stx-encoding phage 933W and non Stx-encoding phages, 434 and λ. We find that Stx-encoding phages, BAA2326, 933W, PA2, and PA8 use more than one mechanism to have a higher frequency of spontaneous induction and exhibit ‘hair-trigger’ induction. The increased virulence of strains bearing these phage is regardless of the host genotype. Hence, we conclude that Stx-encoding phages are incorporating subtle changes to evolve and become more potent in toxin production and release. This has a direct impact on the severity of STEC-mediated disease. Now that we know two phage-encoded molecules that regulate induction, and subsequently, affect disease severity, targeted treatment options can be developed.