Identification and functional analysis of the cyclopropane fatty acid synthase of Brucella abortus

The brucellae are facultative intracellular pathogens of mammals that are transmitted by contact with infected animals or contaminated materials. Several major lipidic components of the brucella cell envelope are imperfectly recognized by innate immunity, thus contributing to virulence. These components carry large proportions of acyl chains of lactobacillic acid, a long chain cyclopropane fatty acid (CFA). CFAs result from addition of a methylene group to unsaturated acyl chains and contribute to resistance to acidity, dryness and high osmolarity in many bacteria and to virulence in mycobacteria. We examined the role of lactobacillic acid in Brucella abortus virulence by creating a mutant in ORF BAB1_0476, the putative CFA synthase gene. The mutant did not incorporate [(14)C]methyl groups into lipids, lacked CFAs and synthesized the unsaturated precursors, proving that BAB1_0476 actually encodes a CFA synthase. BAB1_0476 promoter-luxAB fusion studies showed that CFA synthase expression was promoted by acid pH and high osmolarity. The mutant was not attenuated in macrophages or mice, strongly suggesting that CFAs are not essential for B. abortus intracellular life. However, when the mutant was tested under high osmolarity on agar and acid pH, two conditions likely to occur on contaminated materials and fomites, they showed reduced ability to grow or survive. Since CFA synthesis entails high ATP expenses and brucellae produce large proportions of lactobacillic acyl chains, we speculate that the CFA synthase has been conserved because it is useful for survival extracellularly, thus facilitating persistence in contaminated materials and transmission to new hosts

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of Brucella abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure

Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope.

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

Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models

The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ppdK and mae mutants was severely impaired and growth of the double fbp- glpX mutant was reduced. In macrophages, only the ppdK and mae mutants showed reduced multiplication, and studies with the ppdK mutant confirmed that it reached the replicative niche. Similarly, only the ppdK and mae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines

Relation between the lipid composition of Brucella membrane and the resistance to cationic peptides

Póster presentado en el Annual Meeting of the German Biophysical Society, celebrado en Göttingen (Alemania) del 23 al 26 de septiembre de 2012.The members of the genus Brucella are α-2 Proteobacteria that cause brucellosis, an important zoonosis. These bacteria trigger only low proinflammatory responses during early infection that allows them to reach sheltered intracellular niches before effective immunity activation. The outer membranes (OM) of brucellae are of critical importance in this strategy. The OM of B. abortus is unusually resistant to antimicrobial peptides (AMPs). Brucella lipopolysaccharide (LPS) is implicated in this property and there is evidence that other lipids (such as phosphatidylcholine) also contribute. Furthermore, these bacteria have acyl chains of average number of carbon units which leads an increase in hydrophobicity, proposed as a biophysical factor underlining AMPs resistance.Peer reviewe

A modified brucella vaccine strain for the treatment of brucellosis

The present application provides a modified Brucella strain, its use as a medicament, and its use as a medicament for the treatment and!or prevention ofbmcellosis. The Brucella strain has been modífied through an inactivation ofthe wzm gene. Further, the present application provides a pharmaceutical composition which comprises the modífied Brucella strain, its use as a medicament, and its use as a medicament for the treatment and!or prevention ofbmcellosis. The present application also provides a kit which comprises the modífied Brucella strain and a pharmaceutically acceptable carrier or diluent and its use for the treatment and!or prevention of bumcellosisPeer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Pública de NavarraA1 Solicitud de patente con informe sobre el estado de la técnic

A modified brucella vaccine strain for the treatment of brucellosis

The present application provides a modified Brucella strain, its use as a medicament, and its use as a medicament for the treatment and!or prevention ofbmcellosis. The Brucella strain has been modífied through an inactivation ofthe wzm gene. Further, the present application provides a pharmaceutical composition which comprises the modífied Brucella strain, its use as a medicament, and its use as a medicament for the treatment and!or prevention ofbmcellosis. The present application also provides a kit which comprises the modífied Brucella strain and a pharmaceutically acceptable carrier or diluent and its use for the treatment and!or prevention of bmcellosisPeer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Pública de NavarraA1 Solicitud de patente con informe sobre el estado de la técnic

A modified brucella vaccine strain for the treatment of brucellosis

The present application provides a modified Brucella strain, its use as a medicament, and its use as a medicament for the treatment andlor prevention of brucellosis. The Brucella strain has been modified through an inactivation of the wzm gene. Further, the present application provides a pharmaceutical composition which comprises the modified Brucella strain,its use as a medicament, and its use as a medicament for the treatment and/or prevention of bmcellosis. The present application also provides a kit which comprises the modified Brucella strain and a pharmaceutically acceptable carrier or diluent and its use for the treatment and/or prevention of brucellosisPeer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Pública de NavarraA1 Solicitud de patente con informe sobre el estado de la técnic