The Cpx and Rcs responses signal envelope stress through independent mechanisms but are coordinated through the RprA sRNA in the cytoplasm

• Author / Creator

  Guitard, Samantha

• Bacteria have several mechanisms which play a major role in adaptation to environmental stresses. One method involves the use of signal sensing pathways that activate transcription of specific genes encoding proteins to alleviate bacterial stress. Upon sensing an inducing cue, an inner membrane-bound histidine kinase will autophosphorylate on a conserved histidine residue and transfer said phosphate to a conserved aspartate residue of its cognate response regulator in the cytoplasm. This phosphorylated response regulator is then able to control transcription of its regulon members. Notably, the Cpx (conjugative pilus expression) two-component system is important in many Gram-negative bacteria to maintain envelope homeostasis by the upregulation of protein folding and degrading factors, ultimately alleviating stress in the periplasm. In enteric bacteria, the Cpx two-component system has also been found to play a role across many Gram-negative species for proper colonization of hosts and expression of virulence factors. A second phosphorelay, more recently characterized, is the Rcs (regulator of capsule synthesis) phosphorelay, originally discovered for its ability to regulate colanic acid capsular polysaccharide production that protects the cell by promoting biofilm development. The Rcs phosphorelay is an atypical two-component system that has an added level of complexity with an intermediate inner membrane phosphotransfer intermediate, RcsD, that transfers a phosphate from the hisitidine kinase, RcsC, to the response regulator of this system, RcsB. There are also a greater number of auxiliary proteins at the outer membrane and periplasm that generate a signal sensed by RcsC to activate the pathway. The response regulator of the Rcs signal transduction system, RcsB, controls the positive regulation of the small RNA, RprA. Interestingly, the Cpx system has also been recently implicated in regulation of RprA, yet the mechanism behind this interaction is yet to be defined. Several similar inducing signals and toxic molecules have also been shown to activate both the Cpx and Rcs stress responses, such as polymyxin antibiotics and the metal chelator, EDTA. Activation of these responses has also conferred resistance to some of these toxic molecules. The purpose of this study was to test common and potential inducers of each pathway to identify any relationships between these pathways and the regulation of the small RNA, RprA. We identified a link between the microbial response to fluctuations in pH and the regulation of RprA by the noncognate Cpx and Rcs envelope stress responses. Additionally, we discovered that although both stress responses were induced by the same molecules, each pathway was activated independently of members of the noncognate pathways, and by members of each pathway at different cellular locations at both mid- and late-logarithmic growth. Both the Cpx and Rcs signaling pathways have implications in regulating genes that encode both virulence factors and antibiotic resistance mechanisms in Gram-negative pathogens. As the rise of multi-drug resistant bacteria persists, designing novel therapeutics that can efficiently kill bacteria without activating key stress responses involved in transcription of virulence-associated genes or antibiotic resistance genes will be an important feature to prevent further resistance to antimicrobial drugs. To identify whether one stress response implicated in virulence and antibiotic resistance, the Cpx response, can become activated by toxins known to kill Escherichia coli, we tested its ability to sense toxic molecules and bacterial structures that exist in nature but kill E. coli efficiently. These toxins were type B colicins, T4 bacteriophage, and the type VI secretion system of Vibrio cholerae. Each toxin was chosen because of its ability to interact and damage the cell envelope. The Cpx response did not appear to sense exposure to T4 phage or type B colicins, nor did overactivation or repression of the system confer resistance or susceptibility. Overactivation and repression of the Cpx response also did not alter survival during attack by type VI secretion systems of Vibrio cholerae, however it could have potentially activated the Cpx response. Overall, there is evidence that at least type B colicins or T4 bacteriophage could pose as potential antimicrobials that evade at least one important stress response in Gram-negative bacteria while mediating cell killing.

• Subjects / Keywords

  • stress response system
  • Cpx
  • Rcs
  • Escherichia coli

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3GF0NC0G

• License

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