A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication

Abstract

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

FIG. 1

Schematic representation of B. abortus 2308 virB operon and comparison with B. suis 1330 virB, A. tumefaciens virB, and B. pertussis ptl operons. The white bar denotes the 18-bp inversion of the B. abortus 2308 RS with respect to the B. suis 1330 virB operon. The homologues between ptl and virB are shown in parentheses.

FIG. 2

Schematic representation of the mutations introduced into the virB operon. The restriction sites where the cassettes were introduced are indicated. The hairpin indicates the transcriptional terminator of the kanamycin resistance cassette.

FIG. 3

virB operon transcriptional induction. (A) Expression of the virB10::lacZ fusions of B. abortus 2308 virB10::lacZ-Gm (●) and B. abortus 2308 virB1::Kan virB10::lacZ-Gm (○). β-Galactosidase activity was expressed as A₄₂₀/volume × A₆₀₀. (B) Growth curve of B. abortus 2308 virB10::lacZ-Gm and B. abortus 2308 virB1::Kan virB10::lacZ-Gm strains. Symbols are the same as in panel A. OD₆₀₀, optical density at 600 nm.

FIG. 4

Intracellular replication of parental B. abortus 2308 and virB1 polar mutant strain in HeLa cells. Monolayers of HeLa cells were inoculated with 5 × 10⁷ CFU of bacteria. After 1 h of incubation at 37°C, cells were washed as described in the text and streptomycin and gentamicin were added. CFU were determined at the indicated times. Each determination is the average of two independent experiments performed in duplicate. In all cases, the standard error of each mean was less than 5%. ●, B. abortus 2308; ○, B. abortus 2308 virB1::Kan; ⧫, B. abortus 2308 virB1::Km(pVK8.3); ◊, B. abortus 2308 virB1::Km(pBBR4-virB1).

FIG. 5

Intracellular replication of parental B. abortus 2308, virB10 mutants, and virB10 complemented mutant strains in HeLa cells. (A) B. abortus 2308 (○), B. abortus virB10::Kan (▴), and B. abortus virB10::Gm (●). (B) B. abortus 2308 (○), B. abortus 2308 virB10::Kan(pVK8.3) (▴), B. abortus 2308 virB10::Gm(pBBR-virB10) (●), and B. abortus 2308 virB10::Kan(pBBR-virB10) (□). Each value is the mean ± standard deviation of two independent experiments performed in duplicate. ∗, P < 0.05; ∗∗, P < 0.01 (compared with strain 2308). Statistical analysis was performed with a t test.

FIG. 6

Persistence of B. abortus 2308 and B. abortus virB10 mutants in spleens of infected mice. Mice were inoculated intraperitoneally with 10⁴ CFU of wild-type B. abortus 2308, the virB10 mutant strains, or the virB10 complemented mutant strains as indicated. Numbers of bacteria in spleens were determined at 15 days p.i. Values are expressed as means ± standard errors of the means (n = 5). ∗, P < 0.05 (compared with the control group). Statistical analysis was performed with a t test.