Intracellular rescuing of a B. melitensis 16M virB mutant by co-infection with a wild type strain.

Brucella is a broad-range, facultative intracellular pathogen that can survive and replicate in an endoplasmic reticulum (ER)-derived replication niche by preventing fusion of its membrane-bound compartment with late endosomes and lysosomes. This vacuolar hijacking was demonstrated to be dependent on the type IV secretion system VirB but no secreted effectors have been identified yet. A virB mutant is unable to reach its ER-derived replicative niche and does not multiply intracellularly. In this paper, we showed that, by co-infecting bovine macrophages or HeLa cells with the wild type (WT) strain of Brucella melitensis 16M and a deletion mutant of the complete virB operon, the replication of DeltavirB is rescued in almost 20% of the co-infected cells. Furthermore, we demonstrated that co-infections with the WT strains of Brucella abortus or Brucella suis were equally able to rescue the replication of the B. melitensis DeltavirB mutant. By contrast, no rescue was observed when the WT strain was given 1h before or after the infection with the DeltavirB mutant. Finally, vacuoles containing the rescued DeltavirB mutant were shown to exclude the LAMP-1 marker in a way similar to the WT containing vacuoles.

The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection.

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.