Dataset Information

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

ABSTRACT: Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.

SUBMITTER: Soler-Llorens P

PROVIDER: S-EPMC4107470 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

Soler-Lloréns Pedro P Gil-Ramírez Yolanda Y Zabalza-Baranguá Ana A Iriarte Maite M Conde-Álvarez Raquel R Zúñiga-Ripa Amaia A San Román Beatriz B Zygmunt Michel S MS Vizcaíno Nieves N Cloeckaert Axel A Grilló María-Jesús MJ Moriyón Ignacio I López-Goñi Ignacio I

Veterinary research 20140717

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the ortholog ...[more]

PMID: 25029920

Similar Datasets

Project description:Brucella ovis is a non-zoonotic Brucella species lacking specific vaccine. It presents a narrow host range, a unique biology relative to other Brucella species, and important distinct surface properties. To increase our knowledge on its peculiar surface and virulence features, and seeking to develop a specific vaccine, multiple mutants for nine relevant cell-envelope-related genes were investigated. Mutants lacking Omp10 plus Omp19 could not be obtained, suggesting that at least one of these lipoproteins is required for viability. A similar result was obtained for the double deletion of omp31 and omp25 that encode two major surface proteins. Conversely, the absence of major Omp25c (proved essential for internalization in HeLa cells) together with Omp25 or Omp31 was tolerated by the bacterium. Although showing important in vitro and in vivo defects, the Δomp10Δomp31Δomp25c mutant was obtained, demonstrating that B. ovis PA survives to the simultaneous absence of Omp10 and four out seven proteins of the Omp25/Omp31 family (i.e., Omp31, Omp25c, Omp25b, and Omp31b, the two latter naturally absent in B. ovis). Three multiple mutants were selected for a detailed analysis of virulence in the mouse model. The Δomp31Δcgs and Δomp10Δomp31Δomp25c mutants were highly attenuated when inoculated at 106 colony forming units/mouse but they established a persistent infection when the infection dose was increased 100-fold. The Δomp10ΔugpBΔomp31 mutant showed a similar behavior until week 3 post-infection but was then totally cleared from spleen. Accordingly, it was retained as vaccine candidate for mice protection assays. When compared to classical B. melitensis Rev1 heterologous vaccine, the triple mutant induced limited splenomegaly, a significantly higher antibody response against whole B. ovis PA cells, an equivalent memory cellular response and, according to spleen colonization measurements, better protection against a challenge with virulent B. ovis PA. Therefore, it would be a good candidate to be evaluated in the natural host as a specific vaccine against B. ovis that would avoid the drawbacks of B. melitensis Rev1. In addition, the lack in this attenuated strain of Omp31, recognized as a highly immunogenic protein during B. ovis infection, would favor the differentiation between infected and vaccinated animals using Omp31 as diagnostic target.

Project description:The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential for extra- and intracellular survival in mice.

Dataset Information

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

Publications

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets