The lipopolysaccharide of Brucella abortus BvrS/BvrR mutants contains lipid A modifications and has higher affinity for bactericidal cationic peptides

Abstract

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

FIG. 1.

The LPS of B. abortus bvrS and bvrR mutants shows higher affinity for bactericidal cationic peptides and contains an altered lipid A. (A) Binding of dansyl-polymyxin B by LPS from the wt and bvrS and bvrR mutant strains. LPS suspensions were incubated with increasing amounts of dansyl-polymyxin B, and the fluorescence (RFU) at 480 nm was determined. (B) HPTLC analysis of lipid A acylation in the wt strain, bvrS and bvrR mutant strains, and reconstituted bvrR⁺ strain. Lipid A was extracted from purified LPS preparations or from OM fragments (blebs). Lane Ec contained lipid A of E. coli MLK53 controls: hexa-acylated, penta-acylated, and tetra-acylated. (C) Maximum of the peak position of the symmetric stretching vibration ν_s(CH₂) versus temperature for the LPSs of the wt and bvrS and bvrR mutant strains in the absence or in the presence of polymyxin B. (D) Synchrotron radiation small-angle X-ray diffraction patterns of lipid A wt and bvrS mutant strain incubated with polymyxin B obtained at 40°C and high water content (90%). The logarithm of the scattering intensity (log I) is plotted versus the scattering vector s (= 1/d, where d = spacing).

FIG. 2.

Effect of polymyxin B on the viability of B. abortus chimeras. (A) wt and chimeric bacteria were exposed to increasing amounts of polymyxin B for 60 min at 37°C, and viability was assessed by plating on tryptic soy agar plates. Each experiment was run in triplicate, and the standard error at each point was less than 5% of the value. (B) Electron microscopy of polymyxin B-treated bacteria. Panels 1 and 2, untreated controls; 3 to 6, bacteria treated with 100 units of polymyxin B. The B. abortus wt strain (panels 1 and 3), bvrS mutant (panels 2 and 4), wt strain/bvrS mutant LPS chimera (panel 5), and bvrS mutant/wt strain LPS chimera (panel 6) are shown. The inserts in panels 4 and 5 show (×150,000) rolling of the inner membrane and blebbing. Arrowheads in panels 4, 5, and 6 show OM blebbing.

FIG. 3.

Cytokine induction by LPSs in murine bone marrow-derived macrophages. Macrophages were incubated with the indicated concentrations of LPS for 48 h, supernatants were collected, and the concentration of cytokines was determined by enzyme-linked immunosorbent assay. LPSs were obtained from Shigella flexneri serotype 5a, the B. abortus (Ba) wt, and the B. abortus bvrS mutant.

FIG. 4.

Mutants in the two-component system BvrS/BvrR are markedly sensitive to nonimmune serum. The survival of the B. abortus wt, bvrS mutant, bvrR mutant, reconstituted bvrR⁺ strain, and rough LPS mutant per and deep-rough LPS mutant manB_core strains in nonimmune bovine serum at 37°C is shown.