The role of quorum sensing regulators in Vibrio parahaemolyticus
Date
2017
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne
gastroenteritis worldwide as well as a significant pathogen of shellfish and fish. In
addition, the geographic range of this species and others within the genus is spreading
globally due to climate change. Our knowledge of how this pathogen colonizes the
human intestine, the first step in its pathogenesis, is limited. Some of the most
influential factors for survival and pathogenesis of bacteria in changing environments
include stress response mechanisms, motility, production of virulence factors and
formation of a biofilm. Bacteria possess a communication mechanism, where they
synchronize actions with other bacteria in the population by regulating the expression
of genes that control these actions. Bacteria do this by secreting molecules called
autoinducers that act as extracellular signals that other bacteria in the environment can
respond to. This process of bacterial cell to cell communication is called quorum
sensing. The work in this dissertation examined the role of the downstream regulators
of the quorum sensing pathway in V. parahaemolyticus colonization and fitness. By
examining single and double deletion mutants of the three downstream regulators of
the quorum sensing pathway, the response regulator luxO and the two quorum sensing
master regulators opaR and aphA, we determined that the low cell density master
regulator AphA is important for in vivo fitness and that over-expression of the high
cell density master regulator opaR is detrimental to in vivo fitness. We then performed
RNASeq transcriptome analysis on the wild-type and the luxO mutant, which was
found to be defective in vivo and over-expressed the high cell density regulator opaR.
The transcriptome analysis revealed that 60% of genes downregulated in the luxO
mutant were involved in metabolism and transport, suggesting that the mutant could
have a metabolic disadvantage compared to wild-type. This was indeed confirmed by
carbon phenotype microarrays, which revealed that the luxO mutant was significantly
defective in growth compared to the wild-type when grown in a number of carbon
sources. Additionally, the aphA mutant, which was also defective in vivo, exhibited
similar growth defects compared to wild-type. Both luxO and aphA mutant strains
were defective in 25 carbon sources compared to wild-type. By bioinformatics and
biochemical analysis, we further showed that OpaR binds directly to the regulatory
regions of genes and operons involved in carbon transport and metabolism thereby
indicating a role for this regulator in metabolism in the bacteria. ☐ In Chapter 3, we perform comparative transcriptome analysis of wild-type
versus the super-colonizer rpoN mutant. RpoN is a sigma factor that is involved in the
transcription of 100s of genes but requires an activator protein to be functional. We
examined this regulator since one of its activators is LuxO and together they regulate
the expression of the quorum sensing master regulators OpaR and AphA. We found a
number of genes were differentially regulated between the wild-type and the rpoN
mutant including a number of genes encoding for ribosomal proteins and tRNAs that
were upregulated in the rpoN mutant. This was in keeping with the fact that we had
previously found the mutant to be metabolically more fit than the wild-type.
Expression of the QS master regulators in the rpoN mutant did not match the
expression pattern seen in the luxO mutant. We speculate that these differences in
expression between the luxO and rpoN mutants might be due to increased expression
of one of the quorum regulatory small RNA, qrr2 in the rpoN mutant. ☐ Other work in this dissertation focuses on the acid and osmotic stress response
mechanisms in V. parahaemolyticus. As a marine bacteria and a gastrointestinal
pathogen, V. parahaemolyticus constantly encounters various salt gradients and is
subjected to acid stresses within the gastrointestinal tract. We examined the effect of
lethal acid stress on the bacteria under varying degrees of salt concentration. We found
that preadaptation of the bacteria to high salt is beneficial for the bacteria as it leads to
improved survival under lethal acid stress and other lethal environmental stresses. ☐ Finally, we performed whole genome sequencing on an environmental isolate
of V. parahaemolyticus, strain UCM-V493. UCM-V493 was isolated from a sediment
sample in Spain in 2002. We were interested in determining differences between
pathogenic and non-pathogenic strains. We sequenced this strain to completion and
closed both circular chromosomes with a combination of Illumina and Pacbio
sequencing. Comparative genomic analysis uncovered many regions of difference
between UCM-V493 and RIMD2210633, the canonical clinical isolate.