The Three Flagellar Loci of Brucella ovis PA Are Dispensable for Virulence in Cellular Models and Mice

Abstract

Brucella ovis is a facultative intracellular bacterium that causes a non-zoonotic ovine brucellosis mainly characterized by male genital lesions and is responsible for important economic losses in sheep farming areas. Studies about the virulence mechanisms of Brucella have been mostly performed with smooth (bearing O-polysaccharide in lipopolysaccharide) zoonotic species, and those performed with B. ovis have revealed similarities but also relevant differences. Except for few strains recently isolated from unconventional hosts, Brucella species are non-motile but contain the genes required to assemble a flagellum, which are organized in three main loci of about 18.5, 6.4, and 7.8 kb. Although these loci contain different pseudogenes depending on the non-motile Brucella species, smooth B. melitensis 16M builds a sheathed flagellum under particular culture conditions and requires flagellar genes for virulence. However, nothing is known in this respect regarding other Brucella strains. In this work, we have constructed a panel of B. ovis PA mutants defective in one, two or the three flagellar loci in order to assess their role in virulence of this rough (lacking O-polysaccharide) Brucella species. No relevant differences in growth, outer membrane-related properties or intracellular behavior in cellular models were observed between flagellar mutants and the parental strain, which is in accordance with previous results with B. melitensis 16M single-gene mutants. However, contrary to these B. melitensis mutants, unable to establish a chronic infection in mice, removal of the three flagellar loci in B. ovis did not affect virulence in the mouse model. These results evidence new relevant differences between B. ovis and B. melitensis, two species highly homologous at the DNA level and that cause ovine brucellosis, but that exhibit differences in the zoonotic potential, pathogenicity and tissue tropism.

The Transcriptional Regulator MucR, but Not Its Controlled Acid-Activated Chaperone HdeA, Is Essential for Virulence and Modulates Surface Architecture and Properties in Brucella ovis PA

“…Nevertheless, this observation is not in disagreement with the proposed role of MucR as repressor of ftcR since the predicted binding site of FtcR upstream of fliF ( 42 ) is missing in B. ovis due to two independent deletion events accounting for 283 bp ( 11 ). Even if there was a MucR-mediated regulation of flagellar gene expression in B. ovis PA, deletion of mucR would not have a relevant impact in flagellum-dependent bacterial properties since it has been demonstrated that the entire three main flagellar loci are dispensable for B. ovis PA virulence, and their deletion does not modify any of the evaluated bacterial characteristics ( 15 ). Upregulation of flagellar genes detected in B. canis RM6/66 Δ mucR ( 32 ) was also in accordance with the results described for the B. melitensis 16M mutant ( Table 4 ), but the expression of flagellar genes in B. abortus 2308 does not seem to be dependent on MucR ( 20 ) ( Table 4 ).…”

“…The microbiological procedures were reviewed and approved by the Biosecurity Committee of the University of Salamanca, Spain. For non-polar deletion of mucR in B. ovis PA, the wildtype chromosomal gene was replaced by the inactivated gene, following a procedure previously described (11,15). Briefly, two PCR reactions were performed to amplify the 5 ′ and 3 ′ ends of mucR, together with about 700 bp located upstream or downstream the gene, respectively.…”

“…Thus, some virulence factors described in smooth brucellae, such as the type IV secretion system encoded by the virB operon, the quorum-sensing transcriptional regulator VjbR, ß-1,2 glucans, and core LPS glycosyltransferases or pyruvate phosphate dikinase, are also required for virulence in B. ovis ( 11 – 13 ). However, some other proteins that are essential in smooth B. melitensis or B. abortus for full virulence, such as Omp10, Omp19, BepC, SP41, BacA, or the flagellar apparatus, are not required in B. ovis ( 11 , 14 , 15 ), while an ATP-binding cassette transporter, absent in the main zoonotic smooth brucellae, is a virulence factor in B. ovis ( 16 ).…”

Brucella abortus BspJ Is a Nucleomodulin That Inhibits Macrophage Apoptosis and Promotes Intracellular Survival of Brucella

“…Brucella has a full set of genes coding for proteins that are assembled into functional flagella, but not all coding genes are expressed ( Delvecchio et al, 2002 ). In some virulent strains of Brucella , fully formed flagella can be observed ( Sidhu-Muñoz et al, 2020 ). Therefore, T4SS and flagella may play a regulatory role together in Brucella ( Delrue et al, 2005 ).…”

Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays

“…pilH is embedded in an operon along with pilG , pilI , and pilJ 84 . The same applies for the flagellar proteins fliE, fliG, fliI, fliJ, fliM, fliN and more 85 .…”