HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment

Abstract

Yersinia pestis biofilm formation, controlled by intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP), is important for blockage-dependent plague transmission from fleas to mammals. HmsCDE is a tripartite signaling system that modulates intracellular c-di-GMP levels to regulate biofilm formation in Y. pestis. Previously, we found that Y. pestis biofilm formation is stimulated in reducing environments in an hmsCDE-dependent manner. However, the mechanism by which HmsCDE senses the redox state remains elusive. Using a dsbA mutant and the addition of Cu²⁺ to simulate reducing and oxidizing periplasmic environments, we found that HmsC protein levels are decreased and the HmsC-HmsD protein-protein interaction is weakened in a reducing environment. In addition, we revealed that intraprotein disulphide bonds are critical for HmsC since breakage lowers protein stability and diminishes the interaction with HmsD. Our results suggest that HmsC might play a major role in sensing the environmental changes.

Keywords: HmsC; HmsD; Yersinia pestis; biofilm formation; c-di-GMP.

Figure 1

HmsC is involved in sensing the redox environment. (A) Relative amount of adhered biofilm formed by the Yersinia pestis KIM6+ parent strain and its isogenic derivatives with (gray) or without (white) CuSO₄. (B) Biofilm formation by Y. pestis KIM6+, ΔdsbA, ΔdsbAΔhmsD, ΔdsbAΔhmsT, and ΔdsbAΔhmsE mutant strains after transformation with the empty pUC19 vector or plasmids containing hmsC (pYC212), hmsD (pYC211) or dsbA (pYC257). ^**P < 0.01, ns = not significant. Results are means and standard deviations of four independent experiments.

Figure 2

Intracellular c-di-GMP levels in Y. pestis and its derivatives. Intracellular c-di-GMP was extracted and measured as described in the experimental procedures. ^**P < 0.01. Results are means and standard deviations of three independent experiments.

Figure 3

Effect of the dsbA mutant on HmsC, HmsD, and HmsE protein levels in Y. pestis. (A) HmsC protein levels detected by western blotting. Whole cells were extracted from Y. pestis SY1540 (hmsC-Flag) and Y. pestis SY1542 (ΔdsbA, hmsC-Flag) containing plasmid pYC257 (p-dsbA) or pYC212 (p-hmsC), and from cells grown in LB medium supplemented with CuSO₄. (B) HmsD and HmsE protein levels detected by western blotting. Whole cells were extracted from Y. pestis SY1562 (hmsD-Myc), SY2046 (hmsE-HA), Y. pestis SY1564 (ΔdsbA, hmsD-Myc), and Y. pestis SY2994 (ΔdsbA, hmsE-HA) containing plasmid pYC257 (p-dsbA), and from cells supplemented with CuSO₄. HmsC, HmsD, and HmsE protein levels were quantified using Image J. Numbers below blots indicate the ratio of proteins in the indicated sample compared with samples collected at mid-stationary phase (Set as 1) based on at least two independent experiments.

Figure 4

Disulphide bond formation affects the interaction between HmsC and HmsD. HmsC, and HmsC mutants were co-purified with the periplasmic domain of HmsD in vivo. P-MBP-HmsD^N49−155 was co-expressed with HmsC-His₈ in the wild-type strain (lane 1) or the dsbA mutant (lane 2). P-MBP-HmsD^N49−155 was co-expressed with HmsC^{C87AC126AC161AC168A}-Flag-His₈(C1234A) (lane 3), purified using Ni-NTA resin, and detected with anti-MBP tag and anti-Flag antibodies.

Figure 5

Disulphide bonds are important for HmsC function. (A) Relative amount of adhered biofilm from the hmsC mutant strain and hmsC mutant strains transformed with the pUC19 vector or the plasmids containing hmsC (pYC212), hmsC^C87A (pYC316, C1A), hmsC^C126A (pYC317, C2A), hmsC^C161A (pYC318, C3A), hmsC^C168A (pYC317, C4A), hmsC^C87AC126A (pYC320, C1AC2A), hmsC^C87AC161A (pYC321, C1AC3A), hmsC^C87AC168A (pYC322, C1AC4A), hmsC^C126AC161A (pYC323, C2AC3A), hmsC^C126AC168A (pYC324, C2AC4A), hmsC^C161AC168A (pYC325, C3AC4A), hmsC^{C87AC126AC161A} (pYC326, C1AC2AC3A), hmsC^{C87AC126AC168A} (pYC327, C1AC2AC4A) or hmsC^{C87AC126AC161AC168A} (pYC333, C1AC2AC3AC4A). (B) Protein levels in hmsC mutant strains transformed with the empty pUC19 vector, or plasmids containing hmsC (pYC212) or hmsC conserved cysteine mutants. ^**P < 0.01. Results are means and standard deviations of four independent experiments. HmsC protein levels were quantified using Image J. Numbers below blots indicate the ratio of proteins in the indicated samples compared with samples collected at mid-stationary phase (Set as 1) based on at least two independent experiments.

Figure 6

Regulation of Y. pestis biofilm formation by DsbA and HmsC. The periplasmic protein HmsC possesses four conserved cysteines that likely form disulphide bonds that are modulated by DsbA. The formation of disulphide bonds controls HmsC protein degradation and protein conformation, and determines whether HmsC binds to or disassociates from the periplasmic sensor domain of HmsD, which controls the activity of HmsD that in turn regulates the intracellular c-di-GMP levels and ultimately biofilm formation.