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Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

ABSTRACT: Brucella abortus, the aetiological agent of bovine brucellosis, is an intracellular pathogen whose virulence is completely dependent on a type IV secretion system. This secretion system translocates effector proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although much has been done in understanding how this secretion system participates in the virulence process, few effector proteins have been identified to date. We describe here the identification of a type IV secretion substrate (SepA) that is only present in Brucella spp. and has no detectable homology to known proteins. This protein is secreted in a virB-dependent manner in a two-step process involving a periplasmic intermediate and secretion is necessary for its function. The deletion mutant showed a defect in the early stages of intracellular replication in professional and non-professional phagocytes although it invades the cells more efficiently than the wild-type parental strain. Our results indicate that, even though the mutant was more invasive, it had a defect in excluding the lysosomal marker Lamp-1 and was inactivated more efficiently during the early phases of the intracellular life cycle.

SUBMITTER: Dohmer PH

PROVIDER: S-EPMC3945426 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

Döhmer Peter H PH Valguarnera Ezequiel E Czibener Cecilia C Ugalde Juan E JE

Cellular microbiology 20131103 3

Brucella abortus, the aetiological agent of bovine brucellosis, is an intracellular pathogen whose virulence is completely dependent on a type IV secretion system. This secretion system translocates effector proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although much has been done in understanding how this secretion system participates in the virulence process, few effector proteins have been iden ...[more]

PMID: 24119283

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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

Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

Publications

Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

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