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. 2015 Sep;83(9):3638-47.
doi: 10.1128/IAI.00482-15. Epub 2015 Jul 6.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

Kévin Quintard  1 , Amélie Dewitte  2 , Angéline Reboul  2 , Edwige Madec  3 , Sébastien Bontemps-Gallo  3 , Jacqueline Dondeyne  3 , Michaël Marceau  2 , Michel Simonet  2 , Jean-Marie Lacroix  4 , Florent Sebbane  5
Affiliations

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

Kévin Quintard et al. Infect Immun. 2015 Sep.

Abstract

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.

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Figures

FIG 1
FIG 1
Y. pseudotuberculosis does not produce detectable OPGs, despite the fact that its opgGH operon encodes functional active glucosyltransferases in E. coli and D. dadantii. (A) Eluate fractions of bacterial samples (prepared for OPG extraction, as described in Materials and Methods) stained with anthrone-sulfuric acid reagent. Blue color indicates the presence of OPGs. In these experiments, WT Y. pseudotuberculosis, E. coli, and D. dadantii strains and their derivative opgGH- or opgG-negative strains harboring (or not) the Y. pseudotuberculosis opgGH operon and its own promoter on a plasmid (pNF400; +opgGHYpst) were grown in NaCl-free LB. (B) Swimming motility of WT E. coli and D. dadantii strains (black bars) and their OPG-negative derivative strains harboring pNF400 (+opgGHYpst) (gray bars) or not (white bars), after a 24-hour incubation on a LB soft agar plate at 30°C. The photos are representative of bacteria after 24 h of swimming, and the swim diameter (mean ± SEM) was determined from three independent experiments. The swim diameters of the ΔopgGH E. coli strain and the ΔopgG D. dadantii strain were significantly lower than those of the corresponding WT strain and the respective OPG-deficient mutant strain expressing opgGH from Y. pseudotuberculosis (P < 0.05 in a one-way analysis of variance). However, partial complementation was observed in D. dadantii, presumably due to plasmid instability in this species. (C) Virulence of D. dadantii on chicory leaves after 48 h of contact. Leaves were infected with 107 CFU of WT D. dadantii, ΔopgG mutant, or ΔopgG mutant harboring pNF400 (+opgGHYpst). *, P < 0.05.
FIG 2
FIG 2
Y. pseudotuberculosis constitutively expresses the opgGH operon under various growth conditions. (A) Fold change expression of opgGH in Y. pseudotuberculosis (strain 2777) grown under various conditions and in Y. pseudotuberculosis harboring a recombinant pBR322 plasmid containing its galUF operon or a recombinant pUC18 plasmid containing its opgGH operon, compared with the opgGH expression level in Y. pseudotuberculosis grown under low-osmolarity and aerobic conditions and at 28°C. galUF and opgGH were under the control of their own respective promoters. Under each condition, the level of opgGH transcripts was normalized to that of crr transcripts. The data are means and SEM from two independent experiments. (B) Immunoblot of a whole-cell lysate of Y. pseudotuberculosis (strain 2777), its isogenic ΔopgGH mutant and WT E. coli grown in LB at 30°C. E. coli was used as a positive control. The blot was immunostained with a polyclonal antibody against E. coli OpgG. Arrowheads indicate the positions of the Y. pseudotuberculosis OpgG protein.
FIG 3
FIG 3
In contrast to other proteobacteria, Y. pseudotuberculosis lacking a functional opgGH operon does not show the pleiotropic phenotype resulting from the activity of the OPGs through the Rcs signaling pathway. Data on swimming motility on an LB soft agar plate after a 24-hour incubation at 21°C (A), biofilm formation (relative to the WT strain) after a 24-hour incubation at 21°C with shaking in LB supplemented with Ca2+ and Mg2+ (B), rates of survival within RAW 264.7 macrophages up to 10 h after internalization (C), the time course of colonization of the Peyer's patches and mesenteric lymph nodes after intragastric inoculation (109 bacteria) (D), and the time course of colonization of the spleen and liver after intravenous inoculation (103 bacteria) by the WT strain 2777 and its derivative ΔopgGH strain (E) are shown. The photos are representative of bacteria after 24 h of swimming. The means and SEM from three independent experiments (A and B) and two independent experiments (C) are shown. The time course of organ colonization was determined from one experiment using groups of six animals (D and E); horizontal lines indicate the medians of the individual data points. Bacterial loads in organs did not vary significantly according to the presence or absence of opgGH (P > 0.05 in a one-way analysis of variance).
FIG 4
FIG 4
Y. pestis is smaller than Y. pseudotuberculosis when grown at 37°C (but not at 21°C), presumably because the bacterium lost opgGH during its emergence. The lengths of Y. pseudotuberculosis WT strain 2777, the ΔopgGH strain, the complemented mutant, and Y. pestis KIM6+ expressing opgGH from Y. pseudotuberculosis (+opgGH) or not (WT) were measured during exponential-phase growth in LB supplemented with glucose at 21°C and at 37°C. The data are means and SEM from five independent experiments. Regardless of the growth temperature, the ΔopgGH Y. pseudotuberculosis strain (but not the complemented mutant strain) was significantly smaller than the WT strain (P < 0.05 in one way analysis of variance). When grown at 37°C, Y. pestis was significantly smaller than Y. pseudotuberculosis except when it expressed opgGH from Y. pseudotuberculosis (P < 0.05 in one way analysis of variance). *, P < 0.05.
FIG 5
FIG 5
Y. pestis expressing opgGH from Y. pseudotuberculosis is fully competent in fleas. (A) Proportion of fleas became blocked in the 4 weeks following an infected blood meal; (B) proportion of infected fleas; (C) bacterial loads immediately after and 27 days after the infectious meal. Experiments were performed with Y. pestis WT KIM6+ (WT) and Y. pestis KIM6+ harboring a chromosomally integrated mini-Tn7 encompassing the opgGH operon from Y. pseudotuberculosis (+opgGH). Data in panels A and B are means and standard deviations (SD) from two independent experiments. In panel C, the data correspond to samples from two independent experiments. Each circle indicates the bacterial load of an individual flea, and the horizontal lines indicate the median CFU per flea.
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