Role of lipid A acylation in Yersinia enterocolitica virulence

Abstract

Yersinia enterocolitica is an important human pathogen. Y. enterocolitica must adapt to the host environment, and temperature is an important cue regulating the expression of most Yersinia virulence factors. Here, we report that Y. enterocolitica 8081 serotype O:8 synthesized tetra-acylated lipid A at 37 degrees C but that hexa-acylated lipid A predominated at 21 degrees C. By mass spectrometry and genetic methods, we have shown that the Y. enterocolitica msbB, htrB, and lpxP homologues encode the acyltransferases responsible for the addition of C(12), C(14) and C(16:1), respectively, to lipid A. The expression levels of the acyltransferases were temperature regulated. Levels of expression of msbB and lpxP were higher at 21 degrees C than at 37 degrees C, whereas the level of expression of htrB was higher at 37 degrees C. At 21 degrees C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37 degrees C, an htrB mutant was the most susceptible. We present evidence that the lipid A acylation status affects the expression of Yersinia virulence factors. Thus, expression of flhDC, the flagellar master regulatory operon, was downregulated in msbB and lpxP mutants, with a concomitant decrease in motility. Expression of the phospholipase yplA was also downregulated in both mutants. inv expression was downregulated in msbB and htrB mutants, and consistent with this finding, invasion of HeLa cells was diminished. However, the expression of rovA, the positive regulator of inv, was not affected in the mutants. The levels of pYV-encoded virulence factors Yops and YadA in the acyltransferase mutants were not affected. Finally, we show that only the htrB mutant was attenuated in vivo.

FIG. 1.

Negative-ion MALDI-TOF mass spectrometry spectra for lipid A isolated from Y. enterocolitica serotype O:8 grown at 21°C (A) or at 37°C (B). Proposed structures corresponding to major peaks and acyl group positions follow previously reported structures for Yersinia and other Gram-negative bacteria. The results in both panels are representative of three independent lipid A extractions.

FIG. 2.

Negative-ion MALDI-TOF mass spectrometry spectra for lipid A isolated from Y. enterocolitica serotype O:8 acyltransferase mutants. msbB, YeO8-ΔmsbBGB2 grown at 21°C (A) or at 37°C (B); htrB, YeO8-ΔhtrBGB grown at 21°C (C) or at 37°C (D); lpxP, YeO8-ΔlpxPGB grown at 21°C (E) or at 37°C (F); lpxP-htrB, YeO8-ΔlpxPGB::pKNOCKYeinthtrB grown at 21°C (G) or at 37°C (H). The results in all panels are representative of three independent lipid A extractions.

FIG. 3.

The expression levels of Y. enterocolitica serotype O:8 msbB, htrB, and lpxP acyltransferases are temperature regulated. The levels of acyltransferase expression by Y. enterocolitica serotype O:8 strains carrying the fusion msbB::lucFF, lpxP::lucFF, or htrB::lucFF and grown at 21 or 37°C were analyzed. Data are presented as means ± SD (n = 3). *, results are significantly different (P < 0.05; one-tailed t test) from the results for 21°C.

FIG. 4.

Motility and flhDC and yplA expression patterns are altered in Y. enterocolitica serotype O:8 acyltransferase mutants. (A) Results from a motility assay on a semisolid agar plate. Shown are the motilities after 24 h of incubation at RT. Data are presented as means ± SD (n = 3). Strains are listed according to the relevant genotype. (B) Analysis of flhDC expression by strains carrying the transcriptional fusion flhDC::lucFF. Results are given in RLU per OD₅₄₀. (C) β-Galactosidase activity production by yplA′::lacZYA present in different strains (β-galactosidase values are given in Miller units and are means ± SD [n = 3]). *, results are significantly different (P < 0.05; one-tailed t test) from the results for YeO8 (wild-type strain).

FIG. 5.

inv expression is altered in Y. enterocolitica serotype O:8 acyltransferase mutants. (A) AP activities exhibited by wild-type (YeO8), YeO8-ΔmsbBGB2 (ΔmsbB), YeO8-ΔlpxPGB (ΔlpxP), YeO8-ΔhtrBGB (ΔhtrB), YeO8-ΔmsbBGB2/pTMYemsbB (ΔmsbB/pTMYemsbB), and YeO8-ΔhtrBGB/pTMYehtrB (ΔhtrB/pTMYehtrB) strains carrying an inv::phoA translational fusion (AP activity is expressed in enzyme units per OD₆₀₀ unit, and data are means ± SD [n = 3]). (B) Analysis of rovA expression by strains carrying the transcriptional fusion rovA::lucFF. Results are given in RLU per OD₅₄₀. *, results are significantly different (P < 0.05; one-tailed t test) from the results for YeO8 (wild-type strain).

FIG. 6.

The levels of expression of Yops and YadA are not affected in Y. enterocolitica serotype O:8 acyltransferase mutants. (A) SDS-PAGE analysis (the acrylamide concentration was 4% in the stacking gel and 12% in the separation one) and Coomassie brilliant blue staining of proteins from the supernatants of Ca²⁺-deprived cultures. (B) SDS-PAGE analysis (the acrylamide concentration was 4% in the stacking gel and 12% in the separation one) and Coomassie brilliant blue staining of OM proteins purified from strains grown in LB medium either at 21°C or at 37°C. The white arrow marks YadA protein. MW, molecular weight marker; YeO8, wild type; ΔmsbB, YeO8-ΔmsbBGB2; ΔlpxP, YeO8-ΔlpxPGB; ΔhtrB, YeO8-ΔhtrBGB. The results in both panels are representative of three independent experiments.

FIG. 7.

Bacterial counts for mouse organs at 3 days postinfection (A) or 7 days postinfection (B). Mice were infected orally with 100 μl of a bacterial mixture containing 10⁹ bacteria of the wild type (YeO8; □) or the acyltransferase mutant YeO8-ΔmsbBGB2 (ΔmsbB; •), YeO8-ΔhtrBGB (ΔhtrB; ▿), or YeO8-ΔlpxPGB (ΔlpxP; ▴). Results were reported as log numbers of CFU per gram of tissue. Symbols indicate results for individual mice, and horizontal lines indicate means for groups.