Release of Periplasmic Proteins of Brucella suis upon Acidic Shock Involves the Outer Membrane Protein Omp25

Abstract

The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.Bacteria of the genus Brucella are gram-negative facultative intracellular pathogens of various wild and domestic mammals and are able to cause severe zoonotic infections in humans. Traditionally, three major species are distinguished by their predilections for certain animal hosts: Brucella abortus for cattle, Brucella melitensis for caprines, and Brucella suis for hogs. Whereas B. abortus is the livestock pathogen with the greatest economic impact, B. melitensis and B. suis account for most clinical cases in humans (1, 2, 11).To evade host defenses, Brucella can inhibit neutrophil degranulation and block tumor necrosis factor (TNF) production by macrophages. It has been shown that membrane integrity, in terms of both smooth lipopolysaccharide and outer membrane proteins, is required for such virulent behavior. Furthermore, studies using transposon or signature-tagged mutagenesis have unraveled, with respect to Brucella virulence, the crucial role of an operon homologous to the virB operon of Agrobacterium tumefaciens encoding a type IV secretion system (16, 21, 28). The virulence regulon of A. tumefaciens is triggered in response to chemical signals released at the plant wound site, such as acetosyringone and low pH. Type IV secretion system production is potentiated by monosaccharides (galactose and arabinose) through binding to the periplasmic multiple sugar binding protein ChvE, as well as by low pH (6). However, it was found that under neutral conditions, this secretory system is already produced in B. melitensis or B. abortus, while ...

Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis, Horizontal Gene Transfer, and Virulence Gene Expression

“…The most highly upregulated annotated gene in the ⌬exoR mutant with empirically demonstrated functions is aopB (Agrobacterium outer-membrane protein), with an FC of 4.99. This gene is highly similar to a family of outer membrane proteins in the Rhizobiaceae that have been shown to influence outer membrane stability, permeability, and topology (40)(41)(42). Homologues of aopB are regulated by a variety of environmental conditions, including low pH (23,40,41), peptides (42), and calcium (43).…”

“…This gene is highly similar to a family of outer membrane proteins in the Rhizobiaceae that have been shown to influence outer membrane stability, permeability, and topology (40)(41)(42). Homologues of aopB are regulated by a variety of environmental conditions, including low pH (23,40,41), peptides (42), and calcium (43). The most dramatic decrease in gene expression in the ⌬exoR mutant is for fliN, which encodes the flagellar motor switch protein (log 2 FC ϭ Ϫ4.58) (see Table S3 in the supplemental material).…”

BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential forBrucella abortusVirulence

“…Virulent WT B. abortus naturally lacks functional genes for group 3 Omp31 and Omp25b (23), showing that the absence of one or two members of this family is not decisive for virulence. All this indirect evidence has to be considered with caution because B. melitensis and B. abortus do not have the same profile of group 3 Omps and there are contradictory reports on the level of group 3 Omps in B. suis mutants with mutations in bvrR and/or bvrS (3,37). Nevertheless, the compensatory hypothesis would explain why mutations in a single Omp do not cause marked phenotypic changes, while dysfunction in BvrR/BvrS has a profound influence, at least in B. abortus.…”

Genomic Island 2 of Brucella melitensis Is a Major Virulence Determinant: Functional Analyses of Genomic Islands

“…GI-2 encodes 20 ORFs, with a majority of these ORFs having no known function. The island includes two hypothetical sugar transferases, BMEI0997 and BMEI0998, that are likely involved in LPS biogenesis (27,41,43), a hypothetical secretion activator protein (BMEI0995), a 25-kDa outer membrane precursor protein implicated in Brucella virulence (7,14), and a tRNA-ribosyltransferase (BMEI1003) (Fig. 3).…”