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°C but that hexa-acylated lipid A predominated at 21°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°C than at 37°C, whereas the level of expression of htrB was higher at 37°C. At 21°C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37°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.

Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

“…Significant differences are indicated by lines, and P values shown are based on one-way ANOVA with the Bonferroni correction. The attenuation of virulence noted with our msbB mutants in both rodent models was thought to be due mainly to the production of the less toxic penta-acylated lipid A moiety of LPS, which is considered a common mechanism of reduced virulence for several other pathogenic bacteria (11,14,16,39,54,56,57). However, indistinguishable levels of cytokines/chemokines produced in the host during infection with both the WT bacteria and our msbB mutants at early stages of infection (i.e., 24 and 48 h p.i.)…”

“…Based on the published sequences for the Y. pestis genomes (20)(21)(22)(23), only two late acyltransferases, LpxP and LpxM (MsbB), have been identified. The absence of LpxL and the inactivity of LpxP and MsbB at high temperatures are believed to be responsible for the lack of hexa-acylated lipid A in yersiniae at 37°C (9,16,18,20). The acyltransferase MsbB in yersiniae is responsible for the addition of C 12 to lipid A, while in other enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, MsbB is responsible for the addition of C 14 to lipid A (16).…”

“…Interestingly, the change from tetra-acylated lipid A at 37°C to hexa-acylated lipid A at 21 to 27°C is the characteristic trait for all yersiniae (9,(16)(17)(18)(19)(20), and it is the absence of hexa-acylated lipid A in yersiniae at 37°C that prevents activation of Toll-like receptor 4 (TLR-4) (9,16,18,20). Based on the published sequences for the Y. pestis genomes (20)(21)(22)(23), only two late acyltransferases, LpxP and LpxM (MsbB), have been identified.…”

Analysis of the Networks Controlling the Antimicrobial-Peptide-Dependent Induction of Klebsiella pneumoniae Virulence Factors

“…Isolation and analysis of lipid A. Lipid A's were extracted using an ammonium hydroxide/isobutyric acid method and subjected to negative-ion matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis (16,53). Briefly, lyophilized bacteria (10 mg) were resuspended in 400 l of isobutyric acid-1 M ammonium hydroxide (5:3 [vol/vol]) and incubated in a screw-cap test tube at 100°C for 2 h, with occasional vortexing.…”

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

“…Y. pestis synthesizes a rough LPS devoid of the O antigen and exists in different acylated forms depending upon bacterial growth temperatures (20,(24)(25)(26)(27)(28)(29)(30). For example, lipid A of Y. pestis LPS shifts from a hexa-acylated form at 21°C to 27°C (flea temperature) to a tetra-acylated form at 37°C (human temperature), due in part to the inactivity of MsbB at 37°C, which prevents the activation of TLR-4 (20)(21)(22)(23).…”