Osmoregulated Periplasmic Glucans Transmit External Signals Through Rcs Phosphorelay Pathway in Yersinia enterocolitica

Abstract

Fast response to environmental changes plays a key role in the transmission and pathogenesis of Yersinia enterocolitica. Osmoregulated periplasmic glucans (OPGs) are known to be involved in environmental perception of several Enterobacteriaceae pathogens; however, the biological function of OPGs in Y. enterocolitica is still unclear. In this study, we investigated the role of OPGs in Y. enterocolitica by deleting the opgGH operon encoding enzymes responsible for OPGs biosynthesis. Complete loss of OPGs in the ΔopgGH mutant resulted in decreased motility, c-di-GMP production, biofilm formation and smaller cell size, whereas the overproduction of OPGs through restoration of opgGH expression promoted c-di-GMP/biofilm production and increased antibiotic resistance of Y. enterocolitica. Gene expression analysis revealed that opgGH deletion reduced transcription of flhDC, ftsAZ, hmsT and hmsHFRS genes regulated by the Rcs phosphorelay system, whereas additional deletion of rcs family genes (rcsF, rcsC, or rcsB) reversed this effect and restored motility and c-di-GMP/biofilm production but further reduced cell size. Furthermore, disruption of the Rcs phosphorelay increased the motility and promoted the induction of biofilm and c-di-GMP production regulated by OPGs through upregulating the expression of flhDC, hmsHFRS, and hmsT. However, deletion of genes encoding the EnvZ/OmpR phosphorelay downregulated the flhDC, hmsHFRS and hmsT expression, leading to the decreased motility and prevented the induction of biofilm and c-di-GMP production regulated by OPGs. These results indicated that Rcs phosphorelay had the effect on OPGs-mediated functional responses in Y. enterocolitica. Our findings disclose part of the biological role of OPGs and the underlying molecular mechanisms associated with Rcs system in the regulation of the pathogenic phenotype in Y. enterocolitica.

Keywords: EnvZ/OmpR phosphorelay; Rcs phosphorelay; Yersinia enterocolitica; gene expression; osmoregulated periplasmic glucans; pathogenic phenotype.

FIGURE 1

Growth characteristics of the wild-type, ΔopgGH and ΔopgGH-P_BADopgGH strains of Yersinia enterocolitica. Bacteria were grown in LB (A), LBNS (B), and LNNS (C) media supplemented with 0.6 g/L L-arabinose. The data are presented as the mean ± SD of at least three independent experiments.

FIGURE 2

Purification and quantification of OPGs from Y. enterocolitica. OPGs were extracted from wild-type, ΔopgGH and ΔopgGH-P_BADopgGH strains grown to the exponential phase in LNNS medium supplemented with 0.6 g/L L-arabinose. (A) Gel filtration chromatography of OPGs. (B) Quantification of purified OPGs. The data are presented as the mean ± SD of at least three independent experiments. An asterisk indicates a significant difference with ****P < 0.0001.

FIGURE 3

Changes in swim motility of wild-type and mutant strains in LNNS medium. Y. enterocolitica was grown in LNNS semisolid plates supplemented with 0.6 g/L L-arabinose at 26°C. Swim diameters were measured after 48 h of incubation. The data are presented as the mean ± SD of at least three independent experiments. An asterisk indicates a significant difference with ***P < 0.001, **P < 0.01.

FIGURE 4

Biofilm formation in wild-type and mutant strains of Y. enterocolitica. Bacteria were cultured in LNNS medium supplemented with 0.6 g/L L-arabinose at 26°C. Biofilm formation was analyzed after 24, 48, and 72 h of incubation by staining with crystal violet and measuring absorbance at 595 nm. The data are presented as the mean ± SD of at least three independent experiments. An asterisk indicates a significant difference with ***P < 0.001, **P < 0.01, *P < 0.05.

FIGURE 5

The intracellular c-di-GMP concentration in wild-type and mutant strains of Y. enterocolitica. Bacteria were cultured in LNNS medium supplemented with 0.6 g/L L-arabinose at 26°C. Intracellular c-di-GMP levels were analyzed in bacteria grown to the exponential phase. The data are presented as the mean ± SD of at least three independent experiments. An asterisk indicates a significant difference with ***P < 0.001, **P < 0.01, *P < 0.05.

FIGURE 6

Changes in the cell size of wild-type and mutant strains. Bacteria were grown to the mid-log phase in LNNS medium supplemented with 0.6 g/L L-arabinose and cell size was measured using transmission electron microscopy. The data are presented as the mean ± SD of at least three independent experiments. An asterisk indicates a significant difference with ***P < 0.001, **P < 0.01.

FIGURE 7

Transcriptional changes in the mutant strains of Y. enterocolitica. Y. enterocolitica grown to the mid-log phase in LNNS medium and total RNA was extracted. Expression of opgGH, flhDC, ftsAZ, hmsHFRS, and hmsT were determined by RT-qPCR in vitro. The 16S rRNA gene was used as a normalization control. (A) Gene transcription in ΔopgGH and ΔopgGH-P_BADopgGH strains. (B) Gene transcription in ΔrcsF, ΔrcsC, ΔrcsB, ΔopgGH-ΔrcsF, ΔopgGH-ΔrcsC, and ΔopgGH-ΔrcsB mutant strains. (C) Gene transcription in ΔenvZ, ΔompR, ΔopgGH-ΔenvZ, and ΔopgGH-ΔompR mutant strains. (D) Gene transcription in ΔopgGH-ΔrcsF-P_BADopgGH, ΔopgGH-ΔrcsC-P_BADopgGH, ΔopgGH-ΔrcsB-P_BADopgGH, ΔopgGH-ΔenvZ-P_BADopgGH, and ΔopgGH-ΔompR-P_BADopgGH strains. The data are presented as the mean ± SD of at least three independent experiments.

FIGURE 8

Working model of the relationship between OPGs and the Rcs phosphorelay in Y. enterocolitica. (A) In the wild-type strain, the Rcs system represses cell motility, division and biofilm formation by inhibiting flhDC, ftsAZ, and hmsHFRS expression. (B) OPGs deficiency activates the Rcs phosphorelay and inhibits flhDC, ftsAZ, and hmsHFRS expression, resulting in further repression of cell motility, division, and biofilm formation. (C–E) Disruption of the Rcs phosphorelay pathway downregulates RcsB phosphorylation, restoring cell motility, division, and biofilm formation by enhancing the expression of flhDC, ftsAZ, and hmsHFRS.

FIGURE 9

Effects of Rcs and EnvZ/OmpR phosphorelays on OPGs-induced biofilm and c-di-GMP production in Y. enterocolitica. (A,B) OPGs overproduction induces the expression of hmsHFRS and hmsT, thus promoting biofilm and c-di-GMP production in the presence of both Rcs and EnvZ/OmpR phosphorelays. (C,D) Disruption of the Rcs phosphorelay upregulates the expression of flhDC, hmsHFRS and hmsT, resulting in increased motility and further promoted the induction of biofilm and c-di-GMP production regulated by OPGs. (E,F) Disruption of the EnvZ/OmpR phosphorelay decreases flhDC, hmsHFRS, and hmsT expression, inhibiting motility and preventing the induction of biofilm and c-di-GMP production regulated by OPGs.