Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact

Abstract

The extracytoplasmic-stress-responsive CpxRA two-component signal transduction pathway allows bacteria to adapt to growth in extreme environments. It controls the production of periplasmic protein folding and degradation factors, which aids in the biogenesis of multicomponent virulence determinants that span the bacterial envelope. This is true of the Yersinia pseudotuberculosis Ysc-Yop type III secretion system. However, despite using a second-site suppressor mutation to restore Yop effector secretion by yersiniae defective in the CpxA sensor kinase, these bacteria poorly translocated Yops into target eukaryotic cells. Investigation of this phenotype herein revealed that the expression of genes which encode several surface-located adhesins is also influenced by the Cpx pathway. In particular, the expression and surface localization of invasin, an adhesin that engages beta1-integrins on the eukaryotic cell surface, are severely restricted by the removal of CpxA. This reduces bacterial association with eukaryotic cells, which could be suppressed by the ectopic production of CpxA, invasin, or RovA, a positive activator of inv expression. In turn, these infected eukaryotic cells then became susceptible to intoxication by translocated Yop effectors. In contrast, bacteria harboring an in-frame deletion of cpxR, which encodes the cognate response regulator, displayed an enhanced ability to interact with cell monolayers, as well as elevated inv and rovA transcription. This phenotype could be drastically suppressed by providing a wild-type copy of cpxR in trans. We propose a mechanism of inv regulation influenced by the direct negative effects of phosphorylated CpxR on inv and rovA transcription. In this fashion, sensing of extracytoplasmic stress by CpxAR contributes to productive Yersinia sp.-eukaryotic cell interactions.

FIG. 1.

HeLa cell association by Y. pseudotuberculosis. Strains were allowed to infect a monolayer of growing HeLa cells at either 26°C (A) or 37°C (B). Unattached and loosely attached bacteria were removed, and viable-cell counts were performed on the remaining tightly cell-associated bacteria. Shown is the mean percentage ± the standard error of the mean from at least four independent experiments of infecting bacteria that remained tightly associated with cells. Strains: parent, YPIII/pIB102; ΔyadA inv::kan double mutant, SF104/pYH7; ΔcpxA null mutant, YPIII07/pIB102; ΔcpxA mutant complemented with pcpxA⁺, YPIII07/pIB102/pMF581. Expression of cpxA from pMF581 was induced by IPTG.

FIG. 2.

RT-PCR of mRNA isolated from Y. pseudotuberculosis. RNA was isolated from logarithmic-phase bacterial cultures grown at 26°C or 37°C in LB medium. Samples were subjected to RT-PCR with primers specific for ail, ail-like, ail2, ompX, psaA, and inv. Amplification of rpoA was used as an internal standard. The ppiA, degP, and cpxP alleles are representative members of the Cpx regulon that served as controls to monitor the regulatory influence of Cpx pathway activation. Where indicated, IPTG was added to induce ectopic expression of cpxA. Lanes: parent, YPIII/pIB102; ΔcpxA null mutant, YPIII07/pIB102; complemented ΔcpxA/pcpxA⁺ mutant, YPIII07/pIB102/pMF581. All images where first acquired with a Fluor-S MultiImager (Bio-Rad). After image inversion, the intensity of each band was quantified with the Quantity One quantitation software version 4.2.3 (Bio-Rad) and is given below each image as a value relative to the gene expression in parental bacteria grown at the lower temperature.

FIG. 3.

Analysis of invasin, RovA, and H-NS production by Y. pseudotuberculosis. Protein was isolated from stationary-phase bacteria grown in LB medium at either 26°C or 37°C, separated by SDS-PAGE, and then identified by immunoblot analysis with polyclonal rabbit antiserum raised against invasin, RovA, or E. coli H-NS. Where indicated, IPTG (final concentration of 0.4 mM) or arabinose (0.02%) was added. Lanes: parent, YPIII/pIB102; ΔyadA, inv::kan double mutant, SF104/pYH7; ΔcpxA null mutant, YPIII07/pIB102; ΔcpxA mutant complemented with pcpxA⁺, YPIII07/pIB102/pMF581; ΔcpxA mutant suppressed with pinv⁺, YPIII07/pIB102/pIRR1; ΔcpxA mutant suppressed with provA⁺, YPIII07/pIB102/pGN37; ΔcpxR null mutant, YPIII08/pIB102; ΔcpxR mutant with vector control, YPIII08/pIB102/pMMB208; ΔcpxR mutant producing wild-type CpxR in trans, YPIII08/pIB102/pKEC021; ΔcpxR mutant producing nonphosphorylatable CpxR_D51A in trans, YPIII08/pIB102/pJF015. Molecular masses in parentheses were deduced from the primary sequence.

FIG. 4.

Y. pseudotuberculosis-HeLa cell association and uptake efficiency. Strains were allowed to infect monolayers of growing HeLa cells at 26°C (black bars) and at 37°C (gray bars). (A) The percentage of infecting bacteria that remained tightly associated with cells was determined as outlined in the legend to Fig. 1. (B) The remaining duplicates were subjected to gentamicin to recover internalized bacteria protected from antibiotic exposure. Their number is expressed as a mean percentage ± the standard error of the mean from at least four independent experiments of those bacteria tightly associated with cells. Where indicated, ectopic expression of cpxA and the cpxR allelic variants was induced with IPTG. rovA expression was induced with arabinose. Strains: parent, YPIII/pIB102; ΔcpxA null mutant alone, YPIII07/pIB102; ΔcpxA mutant complemented with pcpxA⁺, YPIII07/pIB102/pMF581; ΔcpxA mutant suppressed with pinv⁺, YPIII07/pIB102/pIRR1; ΔcpxA mutant suppressed with provA⁺, YPIII07/pIB102/pGN37; ΔcpxR null mutant alone, YPIII08/pIB102; ΔcpxR mutant producing wild-type CpxR in trans, YPIII08/pIB102/pKEC021; ΔcpxR mutant producing nonphosphorylatable CpxR_D51A in trans, YPIII08/pIB102/pJF015; ΔcpxR mutant with vector control, YPIII08/pIB102/pMMB208.

FIG. 5.

Infection of HeLa cells by Y. pseudotuberculosis. Strains were allowed to infect a monolayer of growing HeLa cells. At ∼1 h postinfection, the effect of the bacteria on the HeLa cells was recorded by phase-contrast microscopy. Infection with bacteria capable of translocating the YopE cytotoxin caused extensive rounding of the HeLa cells. In the absence of translocated YopE, infected cells maintained a typically elongated morphology. Ectopic expression of cpxA and rovA was induced with IPTG and arabinose, respectively. Shown are phase-contrast images of parental strain YPIII/pIB102 (A); ΔyadA inv::kan double-mutant strain SF104/pYH7 (B); ΔcpxA null mutant strain YPIII07/pIB102 (C); a ΔcpxA mutant strain complemented with pcpxA⁺, YPIII07/pIB102/pMF581 (D); a ΔcpxA mutant strain suppressed with pinv⁺, YPIII07/pIB102/pIRR1 (E); a ΔcpxA mutant strain suppressed with provA⁺, YPIII07/pIB102/pGN37 (F); a ΔcpxA mutant strain with vector control, YPIII07/pIB102/pMF200 (G); ΔcpxA yscU double-mutant strain YPIII07/pIB75 (H); a ΔcpxA yscU mutant strain producing CpxA in trans, YPIII07/pIB75/pMF581 (I); a ΔcpxA yscU mutant strain producing invasin in trans, YPIII07/pIB75/pIRR1 (J); and a ΔcpxA yscU mutant strain producing RovA in trans, YPIII07/pIB75/pGN37 (K).

FIG. 6.

RT-PCR of mRNA isolated from Y. pseudotuberculosis. RNA was isolated from stationary-phase bacterial cultures grown at 26°C or 37°C in LB medium. Samples were subjected to RT-PCR with primers specific for rovA, hns, inv, and cpxR. Amplification of rpoA was used as an internal standard. Cpx regulon members ppiA, degP, and cpxP served as controls to monitor the regulatory influence of Cpx pathway activation. Lanes: parent strain YPIII/pIB102; ΔcpxA null mutant strain YPIII07/pIB102; complemented ΔcpxA/pcpxA⁺ mutant strain YPIII07/pIB102/pMF581; ΔcpxR null mutant strain YPIII08/pIB102; a ΔcpxR mutant strain producing wild-type CpxR in trans, YPIII08/pIB102/pKEC021; and a ΔcpxR mutant strain producing nonphosphorylatable CpxR_D51A in trans, YPIII08/pIB102/pJF015. All images were processed as described in the legend to Fig. 2.

FIG. 7.

Y. pseudotuberculosis CpxR-His₆ binds to rovA and inv control regions. Mobility shift assays were performed with purified CpxR-His₆ (26 μg/ml) and agarose gel-extracted PCR fragments harboring the regulatory regions of cpxR/cpxP, degP, rovA, and inv (40 to 80 μg/ml, depending on the fragment). Where indicated, CpxR-His₆ was phosphorylated with acetyl phosphate (AP). An internal fragment of CpxR was used as a negative control. Constituents of each lane are indicated by plus signs. The approximate size of each amplified PCR fragment is given in parentheses. The electrophoretic mobility of these DNA fragments in the absence of protein is indicated by asterisks, while DNA-CpxR-P complexes are indicated by arrowheads.

FIG. 8.

Model of the effect of CpxR-P on rovA and inv expression. Activated CpxA acts as a kinase phosphorylating CpxR (+P). CpxR-P can then bind directly to rovA and inv control regions to ultimately repress transcription (line with a horizontal bar). When homeostasis is achieved, CpxR-P is quenched by the intrinsic phosphatase activity of CpxA (−P). This relieves the repression, allowing the production of an elevated level of RovA that, in turn, activates its own synthesis, as well as that of invasin (line with arrows). However, in the absence of CpxA, CpxR-P levels will remain high, forcing continued repression of inv and rovA transcription. This may even be further amplified by the CpxA-independent phosphorylation of CpxR via second-messenger phosphodonors such as acetyl phosphate (acetyl∼P) (indicated by a question mark). Cpx system activation also diminishes efficient T3S by yersiniae by an undisclosed mechanism (dotted line) (6). Additionally, the production of surface-located organelles can affect the activity of other similarly located virulence determinants, but such a connection between invasin and Ysc-Yop T3S remains unexplored (also indicated by a question mark).