Excision mechanisms of pathogenicity islands and phages among vibrio pathogens
Date
2016
Authors
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Publisher
University of Delaware
Abstract
Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. The work in this dissertation examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island-1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1, and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1 and VPI-2 encoded integrases. We demonstrated that the RDFs Vibrio excision factor (Vef) A and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross-talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. In chapter 3 we identify several islands with novel cargo genes and variant combinations of the VPI recombination modules described in chapter2. An island we named VPI-3 in V. cholerae NRT36S contains a type three secretion system (T3SS) and an island we named VPI-6 in V. cholerae RC385 contains genes encoding for a CRISPR-Cas system and type VI secretion system (T6SS) were examined in this study. We showed that both VPI-3 and VPI-6 can excise from the bacterial chromosome. Evolutionary analysis of these island regions reveals a modular structure indicating that parts of these islands were likely acquired separately. These data demonstrate that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes. In chapter 4 we examine the role of host encoded factors on excision of a filamentous phage named f237, found in the human pathogen Vibrio parahaemolyticus. Here we demonstrate that the quorum sensing regulator luxO is involved in regulating f237 excision and transcription of f237 encoded genes. Specifically, we present evidence for the direct regulation of phage f237 by the high cell density quorum sensing regulator OpaR. In a luxO mutant, cells are locked in a high cell density state and OpaR is constitutively expressed; in this mutant we showed that several f237 genes are more highly expressed, ranging from 3-fold to over 60-fold, relative to wild-type. We found that this increase in expression also correlated with a greater than 20-fold increase in production of f237 phage circular intermediates. Additionally, we found an increase in the amount of the attB excision product in a luxO mutant relative to wild-type. The function of this excision remains unknown and its effects on the physiology of the cell, if any. Given that increased f237 expression and circular intermediate (CI) production were observed in a luxO mutant, we determined whether OpaR may be responsible for these phenotypes. We confirmed that OpaR could bind to these DNA regions using EMSA assays. All these data support the model that OpaR is directly and positively regulating the transcription of f237 genes as well as production of f237 circular intermediates.