Characterization of the Vibrio cholerae Phage Shock Protein Response

Abstract

The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Keywords: Psp; Vibrio cholerae; cholera; stress response.

FIG 1

Genetic organization of Psp systems and PspA sequence similarity. (A) Comparison of the genetic organization of the psp genes in V. cholerae with that of three well-studied Gram-negative species, E. coli, S. enterica, and Y. enterocolitica. (B) Alignment of the amino acid sequences of PspA for the four species. Multiple sequence alignment created with ExPASy 3.21 BOXSHADE server (https://sourceforge.net/projects/boxshade/). Black shading or *, fully conserved residues; gray shading or dot, semiconserved residues; white, no conserved residues.

FIG 2

The T2SS secretin, GspD, induces the Psp response in V. cholerae. The four identified secretins of V. cholerae (GspD, TcpC, MshL, and PilQ) were overexpressed from pTTQ18, containing C-terminal 6×His epitopes for detection. (A) Activity of the pspA promoter as detected by β-galactosidase production from a lacZΩPpspA reporter. (B) Chromosomal expression of PspA in response to overexpressed secretins. ***, P < 0.001 by Student's t test. Error bars represent ± standard deviations.

FIG 3

GspD forms heat-resistant multimers that mislocalize to the inner membrane, where the other V. cholerae secretins do not. (A) Cultures were grown to mid-log phase when protein production was induced by the addition of arabinose for 1 h. Samples were resuspended in sample buffer containing SDS and boiled for 5 min before separation by electrophoresis. (B) Induced cultures were fractionated into total membrane (M), inner membrane (IM), and outer membrane (OM) fractions, and GspD multimer and monomers were detected using anti-His antibody. OmpT-FLAG and ToxR were used as outer membrane and inner membrane controls, respectively. Figure is representative of 3 experiments.

FIG 4

GspD overexpression increases psp transcript levels and leads to PspA membrane association. Cultures were grown to mid-log phase when protein production was induced by the addition of arabinose for 1 h. (A) RNA was harvested and reverse transcribed. Transcript levels were normalized to the housekeeping gene recA. (B) Induced and uninduced cultures were fractionated into soluble and membrane fractions. Crp-FLAG and ToxR were used as cytoplasmic and membrane controls, respectively. Figure is representative of 3 experiments.

FIG 5

PspA is a negative regulator of the Psp response, and PspB, -C, and -F are positive regulators. Cultures were grown to mid-log phase (4 h). β-Galactosidase activity was produced from a chromosomal promoter fusion of lacZΩPpspA. (A) Loss of pspA leads to a large increase in PpspA activity, which can be reduced by complementation. (B) Loss of pspB, pspC, or pspF results in the inability to induce the Psp response. ***, P < 0.001 by Student’s t test. Error bars represent ± standard deviations.

FIG 6

The Psp response can be induced by specific environmental conditions. (A) Cultures were grown for 24 h, and 1-ml aliquots were removed at the indicated time points. Cultures were normalized by OD₆₀₀, and chromosomal PspA-6×His expression was detected by immunoblotting. (B) The indicated stressors were added after 3 h of growth. After 1 h (or the indicated time), cultures were normalized by OD₆₀₀, and total protein concentration and chromosomal PspA-6×His expression were detected by immunoblotting. Loading control is a cross-reactive protein to ToxR antisera (59). Both panels are representative of three experiments.

FIG 7

The psp null strain shows reduced colonization in the zebrafish model of cholera infection but no defect in infant mice. (A) Infant mice were orally inoculated with a 1:1 ratio of 10⁶ O395ΔlacZ and O395ΔpspFABC. (B) Zebrafish were incubated in water containing either 10⁸ O395ΔlacZ or O395ΔpspFABC. After overnight infections, intestines were harvested and bacteria were plated for enumeration. Each data point represents data from one fish or mouse. (C) The water was tested for mucin concentration postinfection as a measure of fish diarrhea. The bar diagrams show the mucin level in excreted water after 24 h. (D) Bacterial numbers in the water postinfection were quantified to determine the levels of excreted bacteria. ***, P < 0.001. Error bars represent ± standard error of the means in panel B and standard deviations in panels C and D.

FIG 8

Model of Psp response in V. cholerae. In the absence of stress, the transcriptional activator PspF is bound by PspA, inhibiting transcription of psp genes. In the presence of stressors, such as mislocalized secretins from the type II secretion system (T2SS), PspA is sequestered to the inner membrane and PspF is free to initiate transcription of the psp genes. The inner membrane proteins PspB and PspC are predicted to aid in sensing damage and binding PspA to the inner membrane.