Role of the RpoE sigma factor and two-component Cpx systems, in biofilm formation of Salmonella enterica subspecies enterica serovar Enteritidis
Date
2019-03-28
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0003-3907-1108
Type
Thesis
Degree Level
Masters
Abstract
The cell envelope of the Gram negative bacteria is a complex multilayered structure that functions to protect bacteria from various stressors. The cell envelope is involved in maintenance of cellular shape and rigidity, transport of nutrients and waste, cell division as well as in growth and metabolism. Thus, it is crucial for bacteria to ensure the integrity of the cell envelope in response to environmental assaults and maintain a continuous, semi-permeable barrier provided by the cell envelope. Bacteria have evolved specialized response systems, called extracytoplasmic stress responses systems (ESRs), that function to sense the damage of the cell envelope and accordingly regulate the genes essential to combat the stress, thus restoring envelope and intracellular homeostasis. Two of these ESR systems, the RpoE sigma factor and the Cpx two-component system, are involved in regulating genes in response to the envelope stresses. These regulatory systems have been linked to pathogenesis and biofilm formation in several Gram negative pathogens, although the complex network mediated by these systems, along with their interaction, remain unclear. Accordingly, a first goal of this thesis was to investigate the individual and synergistic role of the RpoE and Cpx (CpxA and CpxR) regulatory systems in their abilities to affect biofilm formation in Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis). For this purpose, I generated in-frame deleted rpoE, cpxR and double-deleted rpoE/cpxR strains. These mutant strains were assessed for underlining characteristic differences in biofilm formation compared with wild-type (WT) S. Enteritidis. It was determined that cpxR (a response regulator of Cpx system) and rpoE/cpxR knock-out mutant strains of S. Enteritidis resulted in decreased biomass in biofilms compared to that of the parental WT strain. The cells in the cpxR knock-out mutant biofilm additionally exhibited an unusual filamentous phenotype. In contrast, the rpoE knock-out mutant strains exhibited architecture and the cellular morphology similar to WT, which formed benchmark Salmonella biofilms with decreasing porosity and increased biomass overtime. Based on the above results on biofilm formation, I report that RpoE by itself did not significantly influence the biofilm formation in S. Enteritidis. However, in conjunction with CpxR, the RpoE sigma factor might co-regulate essential genes required for biofilm formation indicating an integrated role of these two ESRs in biofilm formation. The above findings revealed that CpxR individually, as well as in combination with RpoE sigma factor, played an important role in biofilm formation of S. Enteritidis. Accordingly, the second goal of this thesis was to characterize the expression of genes under control of the Cpx regulon in S. Enteritidis in response to biofilm formation. For this purpose, comparative transcriptome profiling between WT and cpxR mutant strains were performed to identify CpxR-regulated genes involved in biofilm formation. My results suggest that CpxR is involved in regulation of a number of regulatory pathways linked to adhesion, motility, O-antigen biosynthesis and virulence. Some of the operons that were transcriptionally-repressed by deletion of cpxR includes, the peg and saf operon involved in adherence; the rfb and gtr operon involved in O-antigen synthesis; and the hsd operon involved in virulence. Together these results suggest that Cpx controls regulatory pathways affecting adhesion, motility and O-antigen biosynthesis which, together or individually, could impact biofilm formation by S. Enteritidis.
Overall, my work has revealed an essential role of CpxR regulatory protein in biofilm formation and highlighted several genes that are directly regulated by CpxR. It offers new insights on the CpxR-regulon required for successful surface adaptation, and helps to elucidate the complex stress-response metabolic pathway utilized by S. Enteritidis. To my knowledge, this is the first study providing a transcriptomic analysis of the Cpx regulon in Salmonella.
Description
Keywords
Salmonella, Biofilm, Extra-cytoplasmic stress-response system, transcriptomic study.
Citation
Degree
Master of Science (M.Sc.)
Department
Food and Bioproduct Sciences
Program
Applied Microbiology