Abstract
The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.
Highlights
Brucellosis is the collective name of a group of zoonotic diseases afflicting a wide range of domestic and wild mammals (Whatmore, 2009; Zheludkov and Tsirelson, 2010)
The results show that, whereas Brucella LptA modifies the lipid A, this is not the case for lpxE and lpxO, the former encoding a putative phosphatase acting on an unidentified outer membrane (OM) molecule and the latter for an enzyme with OlsC activity
Our data strongly suggest that B. melitensis LptA is involved in the addition of pEtN to lipid A, homologous proteins carrying out this function are not uncommon in Gram-negative pathogens and modulate the properties of lipid A
Summary
Brucellosis is the collective name of a group of zoonotic diseases afflicting a wide range of domestic and wild mammals (Whatmore, 2009; Zheludkov and Tsirelson, 2010). Concerning the LPS, most bacteria carry C1 and C4 glucosamine disaccharides with C12 and C14 acyl and acyl-oxyacyl chains This highly amphipathic structure, named lipid A, is adjacent to additional negatively charged groups of the core oligosaccharide, namely the heptose phosphates and 2-keto-3-deoxyoctulosonate carboxyl groups (Kastowsky et al, 1992; Moriyón, 2003). This lipid A-core PAMP is so efficiently detected by the innate immunity system that some pathogens partially conceal it by removing phosphate groups or substituting them with arabinosamine and/or ethanolamine, or by hydroxylating the acyl chains (Takahashi et al, 2008; Lewis et al, 2013; Moreira et al, 2013; Needham and Trent, 2013; Llobet et al, 2015; Trombley et al, 2015). We investigated in Brucella the role of gene homologs to phosphatases, phospho-ethanolamine (pEtN) transferases, and acyl hydroxylases (Figure 1) that have been shown in other Gram-negative pathogens to act on LPS and to contribute to overcoming innate immunity defenses
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