Poly-N-acetylglucosamine expression by wild-type Yersinia pestis is maximal at mammalian, not flea, temperatures

Abstract

Numerous bacteria, including Yersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis by Y. pestis have been reported to be maximal at 21 to 28°C or "flea temperatures," facilitating the regurgitation of Y. pestis into a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation by Y. pestis have used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WT Y. pestis. Extracts of WT sylvatic Y. pestis strains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WT Y. pestis is maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection.

Importance: Yersinia pestis transitions from low-temperature residence and replication in insect vectors to higher-temperature replication in mammalian hosts. Prior findings based primarily on an avirulent derivative of WT (wild-type) KIM, named KIM6+, showed that biofilm formation associated with synthesis of poly-N-acetylglucosamine (PNAG) is maximal at 21 to 28°C and decreased at 37°C. Biofilm formation was purported to facilitate the transmission of Y. pestis from fleas to mammals while having little importance in mammalian infection. Here we found that for WT strains KIM and CO92, maximal PNAG production occurs at 37°C, indicating that temperature regulation of PNAG production in WT Y. pestis is not mimicked by strain KIM6+. Additionally, we found that Congo red binding does not always correlate with PNAG production, despite its widespread use as an indicator of biofilm production. Taken together, the findings show that a role for PNAG in WT Y. pestis infection should not be disregarded and warrants further study.

FIG 1

(A) Confocal microscopic analysis of PNAG expression by avirulent Y. pestis strain KIM6+ or KIM6 (Δpgm) grown at either 28 or 37°C overnight in BHIB. (B) Reactivity of Y. pestis strain KIM6+ grown at 28°C with MAb F598-AF488 to PNAG after treatment of bacterial cells with either chitinase (control) or the PNAG-degrading enzyme dispersin B. Bars, 10 µm.

FIG 2

(A) Confocal microscopic analysis of PNAG expression by WT Y. pestis strain KIM or CO92 grown at either 28 or 37°C overnight in BHIB. (B) Reactivity of Y. pestis strain KIM or CO92 grown at 37°C with MAb F598-AF488 to PNAG after treatment of bacterial cells with either chitinase (control) or the PNAG-degrading enzyme dispersin B. Bars, 10 µm.

FIG 3

(A) Expression of PNAG by the indicated strains of Y. pestis grown in BHIB at the temperatures indicated in individual panels as determined by FACS. Dark gray trace, reactivity with control MAb F429 to P. aeruginosa alginate. Light gray trace, reactivity with PNAG-specific MAb F598. (B) Quantitative analysis of fluorescence intensity of Y. pestis strains reacted with either control MAb F429 (black bars) or MAb F598 to PNAG (gray bars). Bars indicate means, and error bars indicate the standard errors of the means.

FIG 4

Colony morphology on Congo red agar of four Y. pestis strains grown overnight at the temperatures indicated. Only pgm/hms-positive strains produced dark-staining colonies at 28°C and not at 37°C.

FIG 5

Confocal microscopic analysis of PNAG expression by avirulent Y. pestis strain KIM6+ or KIM6 (Δpgm) grown at either 28 or 37°C overnight on Congo red agar. Reactivity with MAb F598-AF488 to PNAG after treatment of bacterial cells with either chitinase or the PNAG-degrading enzyme dispersin B. Bars, 10 µm. No binding of control MAb F429-AF488 was observed (not shown).

FIG 6

Confocal microscopic analysis of PNAG expression by WT Y. pestis strain KIM or CO92 grown at either 28 or 37°C overnight on Congo red agar. Reactivity with MAb F598-AF488 to PNAG after treatment of bacterial cells with either chitinase or the PNAG-degrading enzyme dispersin B. Bars, 10 µm. No binding of control MAb F429-AF488 was observed (not shown).

FIG 7

(A) Expression of PNAG by the indicated strains of Y. pestis grown on Congo red agar at the temperatures indicated in individual panels as determined by FACS analysis. Dark gray trace, reactivity with control MAb F429 to P. aeruginosa alginate. Light gray trace, reactivity with PNAG-specific MAb F598. (B) Quantitative analysis of fluorescence intensity of Y. pestis strains reacted with either control MAb F429 (black bars) or MAb F598 to PNAG (gray bars). Bars indicate means, and error bars indicate the standard errors of the means.