The transcriptional repressor FarR is not involved in meningococcal fatty acid resistance mediated by the FarAB efflux pump and dependent on lipopolysaccharide structure

Abstract

Free fatty acids are important antimicrobial substances regulating the homeostasis of colonizing bacteria on epithelial surfaces. Here, we show that meningococci express a functional farAB efflux pump, which is indispensable for fatty acid resistance. However, other than in Neisseria gonorrhoeae, the transcriptional regulator FarR is not involved in regulation of this operon in Neisseria meningitidis. We tested the susceptibility of 23 meningococcal isolates against saturated and unsaturated long-chain fatty acids, proving that meningococci are generally highly resistant, with the exception of serogroup Y strains belonging to sequence type 23. Using genetically determined lipopolysaccharide (LPS)-truncated mutant strains, we show that addition of the LPS core oligosaccharide and hexa-acylation of its membrane anchor lipid A are imperative for fatty acid resistance of meningococci. The sensitivity of the serogroup Y strains is due to naturally occurring mutations within the lpxL1 gene, which is responsible for addition of the sixth acyl chain on the LPS membrane anchor lipid A. Therefore, fatty acid resistance in meningococci is provided by both the active efflux pump FarAB and by the natural permeability barrier of the Gram-negative outer membrane. The transcriptional regulator FarR is not implicated in fatty acid resistance in meningococci, possibly giving rise to a constitutively active FarAB efflux pump system and thus revealing diverse mechanisms of niche adaptation in the two closely related Neisseria species.

FIG. 1.

Fatty acid resistance of N. meningitidis. Clinical isolates and carrier strains (Table 1) covering seven serogroups and unencapsulated strains as well as several sequence types were tested for their susceptibility to 150 μg/ml palmitic (A) or 10 μM linoleic (B) acid. Resistance is shown as the ratio of growth on agar plates with supplementation to growth on control plates (as a percentage) of at least three independent experiments. The error bars indicate the standard deviations.

FIG. 2.

Structural model of meningococcal LPS. Depicted is the chemical structure of the LPS of MC58 (serogroup B, ST-32, immunotype L3). Genes responsible for the additional molecules beyond the particular linkage are indicated and labeled with an arrow. The numbers in brackets denote the number of carbon atoms of the acyl chain, and dashed connections indicate phase variability. The PEA at the 3′ position of Hep II can be replaced phase variably by an α(3,1)-linked glucose residue. GlcN, glucosamine; KDO, 2-keto-3-deoxyoctulosonic acid; Glc, glucose; GlcNAc, N-acetylglucosamine; Gal, galactose; NeuNAc, N-acetylneuraminic acid.

FIG. 3.

Susceptibility of meningococcal LPS mutants to fatty acids. N. meningitidis serogroup B, ST-32 wild-type (wt) strains (H44/76 or MC58), the unencapsulated MC58 ΔsiaD strain or unencapsulated mutant strains with various truncations within their LPS molecules were tested for their susceptibility to 150 μg/ml (585 μM) palmitic (A) or 10 μM linoleic (B) acid. Resistance is shown as the ratio of growth on agar plates with supplementation to growth on control plates (as a percentage) of at least three independent experiments. The error bars indicate the standard deviations.

FIG. 4.

Impact of naturally occurring mutations in the lpxL1 gene on fatty acid resistance. (A) Depiction of the inactivating mutations found in the three serogroup Y, ST-23 strains DE 6853, α 24, and DE 7671. Alignment with the reference strain MC58 displayed a deletion of an adenosine in a stretch of seven adenosines (type V mutation) in strain DE 6853 and a deletion of 10 nucleotides (type VI mutation) in strains α 24 and DE 7671 (12). The position within the gene is indicated above, sequence accordance is shown in gray, nucleotide deletions are indicated by hyphens, and the point mutation is boxed. (B) The unencapsulated wild-type MC58 ΔsiaD strain and a corresponding lpxL1 deletion mutant, as well as strain α 24 and a complemented α 24 derivative in which the type VI mutation was replaced by wild-type lpxL1, were tested for their susceptibility to 150 μg/ml (585 μM) palmitic or 10 μM linoleic acid. Resistance is shown as the ratio of growth on agar plates with supplementation to growth on control plates (as a percentage) of at least three independent experiments. The error bars indicate the standard deviations. (C) FACS analysis of the incorporation of BODIPY-labeled palmitic acid to MC58 ΔsiaD (black line) and MC58 ΔsiaD ΔlpxL1 (gray line). Unlabeled bacteria were used as controls (wild type, gray shading; ΔlpxL1 mutant, black line [left side of the graph]).

FIG. 5.

Role of the FarAB system in meningococcal fatty acid resistance. (A) Under normal conditions, FarR and IHF act in concert to repress transcription of the farAB operon in N. gonorrhoeae. (B) In the absence of the repressor FarR, the farAB operon is transcribed, and the efflux pump, consisting of the FarB membrane transporter protein and the FarA membrane fusion protein, is expressed at the cell surface. MtrE acts as an outer membrane protein channel. OM, outer membrane; IM, inner membrane. (C) The impact of the FarAB system on meningococcal fatty acid resistance was analyzed by testing the susceptibility of the wild-type (wt) strain MC58, an isogenic ΔfarR strain, a ΔkdtA strain without the polysaccharide moiety of meningococcal LPS or a ΔkdtA ΔfarR double mutant strain, a ΔfarAB strain, and a ΔsiaD ΔkdtA ΔfarAB mutant strain to palmitic or linoleic acid. Resistance is shown as the ratio of growth on agar plates with supplementation to growth on control plates (as a percentage) of at least three independent experiments. The error bars indicate the standard deviations.