Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection

Abstract

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.Brucella spp. are found in association with a large number of wild and domesticated animal species, where they can cause persistent infection and abortion. Zoonotic transmission of the organism to humans can lead to a chronic febrile disease known as brucellosis or Malta fever. A characteristic of both natural and zoonotic forms of the disease is extended survival of the organism in tissues of the reticuloendothelial system, such as the spleen, lymph nodes, and bone marrow.In a mutant screen for Brucella abortus virulence factors required for survival in the murine reticuloendothelial system, the virB operon, encoding homologs of Agrobacterium tumefaciens and Bordetella pertussis type IV secretion systems (24), was found to be essential for persistence in mice (18). The importance of these genes for intracellular survival was demonstrated in tissue culture models of infection as well (12,15,20,24,30). These genes were subsequently found to be expressed intracellularly by Brucella suis in macrophages (8), where they are required for localization of B. abortus to an intracellular niche that is associated with the endoplasmic reticulum (9), the same location where B. abortus was earlier shown to replicate in nonprofessional phagocytes (13,14,27) and in the ruminant placenta (2, 23). For B. abortus, the virB genes have been implicated in the initial interactions between the bacterium and the host cell during entry into macrophages (33).Based on studies performed with A. tumefaciens, both VirB1 and VirB2 are predicted to be accessible on the surface of B. abortus. VirB2 is predicted to form a pilus-like structure, and VirB1 is predicted to have two domains-a lytic transglycosylase and a second domain that is released to the extracellular medium and remains a...

VirB1* Promotes T-Pilus Formation in the vir -Type IV Secretion System of Agrobacterium tumefaciens

“…Both nopaline and octopine strains process and secrete a nopaline VirB1 variant [VirB(Q/S)], where the VirB1* processing site was altered to conform to the amino acid sequence of octopine VirB1 in this region. Recently, a VirB1*candidate has been identified in the cell lysate of Brucella abortus (20). Finally, homology searches identified a set of VirB1 homologs that exhibit sequence similarity in their C termini in addition to the region of lytic transglycosylase similarity.…”

Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

“…The mutants of purine biosynthesis pathway genes purL, purD, and purE (1) displayed significant attenuation in BALB/c mice, but live brucellae remained viable after 12 weeks, suggesting that their virulence was not sufficiently attenuated for adoption as a livestock vaccine. The aromatic amino acid pathway aroC mutant of B. suis (14), the B. abortus lipid A fatty acid-transporting gene bacA mutant (12,26), the type IV secretion virB mutant (11), and the ferrochelatase hemH mutant (2) showed highly attenuated virulence, but their protective efficacy has yet to be reported. B. abortus nicotinamidase pncA (22) and exopolysaccharide transporter exsA (33) mutants showed substantial protection, but these vaccine strains still colonized the host even after 6 weeks.…”

Desenvolvimento e avaliação de uma cepa knockout de Brucella abortus obtida pela deleção do gene virB10

“…Em experimento realizado utilizando mutantes para os genes virB1 e virB2 de B. abortus observou-se que VirB1 e VirB2 são essenciais para a replicação intracelular em macrófagos do tipo J774 (Hartigh et al 2004). Em estudos com mutantes para o gene virB4 de B. abortus foi verificado que este possui importante função no crescimento intracelular e na virulência em camundongos (Watarai et al 2002).…”

“…Sieira et al (2000) em seu trabalho com ΔvirB10 2308 de B. abortus observou que o número de bactérias recuperadas do baço de camundongos inoculados com mutantes apolares de virB10 foi significativamente mais baixo (p<0,05) do que o número recuperado de camundongos inoculados com a cepa selvagem, o que está de acordo com o encontrado em nossos resultados sendo o mutante ΔvirB10 S2308 também apolar. Hartigh et al (2004) visando determinar o modo como os genes virB1 e virB2 são necessários para a persistên-cia de B. abortus in vivo testaram a habilidade de estabelecimento da infecção e persistência em camundongos BALB/c de mutantes polares e apolares para estes genes. A recuperação de UFC bacteriana foi observada uma, três e oito semanas após a inoculação.…”

“…Esses resultados de clearance estão de acordo com resultados obtidos em experimentos com outros genes relacionados à virulência de Brucella spp. (Hartigh et al 2004). Yang et al (2006) relataram que a cepa mutante ΔznuA foi atenuada em camundongos BALB/c.…”