Production of the Type IV Secretion System Differs among BrucellaSpecies as Revealed with VirB5- and VirB8-Specific Antisera

ABSTRACTExpression of the virBoperon, encoding the type IV secretion system required for Brucella suisvirulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suisVirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suisfrom which chvEwas deleted was fully virulent in a mouse model. Comparative analyses of various Brucellaspecies revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canisand B. suis, as well as the Brucella abortus-and Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortusand B. melitensisstrains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virBexpression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virBoperon are not uniformly regulated in different Brucellaspecies. Since VirB proteins were shown to modulate Brucellaphagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.