Abstract
Brucella ovis is a non-zoonotic bacterium causing contagious epididymitis and other genital lesions in rams and responsible for significant economic losses in sheep-breeding areas. It is a naturally rough (without O-chains in the lipopolysaccharide) Brucella species whose virulence mechanisms have been less explored than those of zoonotic smooth brucellae (bearing O-chains that mask other outer membrane molecules). Considering the rough nature of Brucella ovis, the influence of surface components other than O-chains on its biological properties may be greater than in smooth Brucella species. Here we describe the construction and characterization of the mucR deletion mutant of virulent B. ovis PA, which is defective in a transcriptional regulator, affecting surface properties and virulence in smooth brucellae. This mutant showed increased amounts of three proteins identified as HdeA (acid-activated chaperone), Omp25d (outer membrane protein undetectable in the parental strain), and BOV_A0299 (hypothetical protein of unknown function). This observation correlated with the enhanced transcription of the corresponding genes and constitutes the first report on this type of proteome alteration in Brucella ΔmucR mutants. The upstream regions of the three genes contained AT rich domains with T-A steps described as binding sites for MucR in the Brucella abortus 2308 babR promoter (gene also upregulated in B. ovis ΔmucR), which suggests that hdeA, omp25d, and BOV_A0299 expression could be repressed by MucR through a direct binding to their promoter regions. Relative quantification of transcripts of several other genes selected according to the transcriptome of smooth brucellae ΔmucR mutants revealed not only similarities but also relevant differences among strains, such as those detected in flagellar and virB genes. Periplasmic HdeA has been related to the resistance of B. abortus to acidic pH, conditions encountered by Brucella inside phagocytes, but the deletion of hdeA in B. ovis PA and the ΔmucR mutant did not modify any of the evaluated properties of these strains. The B. ovis PA ΔmucR and ΔmucRΔhdeA mutants had defective in vitro growth and altered surface properties and architecture, exemplified by detectable amounts of Omp25d. Moreover, they showed virulence attenuation but established persistent splenic infection in mice, which encourages their evaluation as specifical attenuated vaccines against B. ovis.
Highlights
The genus Brucella includes facultative intracellular Gram-negative bacterial pathogens that differ, among other phenotypic and genotypic characteristics, in pathogenicity and host preference (1, 2)
A protein of low molecular mass showed a remarkable intensity in the mucR mutant (Figure 1) and was identified as the product of the gene BOV_A0312, which codes for a protein annotated as acid-activated periplasmic chaperone HdeA in the genome sequence of B. ovis 63/290 (GenBank accession number NC_009504) and that contributes to acid resistance in B. abortus 2308 (23)
Several upregulated or downregulated genes at the transcription level were found in the corresponding mucR mutants, but apart from some flagellar proteins in B. melitensis 16M (21), no reports exist regarding whether the modification of the transcription rate of affected genes effectively leads to increased or reduced levels of the encoded proteins
Summary
The genus Brucella (https://lpsn.dsmz.de/genus/brucella) includes facultative intracellular Gram-negative bacterial pathogens that differ, among other phenotypic and genotypic characteristics, in pathogenicity and host preference (1, 2). Ovine brucellosis is mainly caused by Brucella melitensis and Brucella ovis, species that share high levels of homology at the DNA level (3) but exhibit relevant differences regarding pathogenicity. B. melitensis induces abortion in the natural host, is the most relevant zoonotic Brucella species (4), and is defined as smooth because it bears O-polysaccharide chains in the lipopolysaccharide (LPS), which are required for full virulence (5, 6). B. ovis is rough (lacks O-chains in the LPS), has never been reported as a human pathogen, and rarely induces abortion in sheep. B. ovis causes contagious epididymitis and other genital lesions in rams that originate important economic losses worldwide (7, 8) but lack a specific vaccine
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