The identification of a second quorum-sensing regulatory protein suggests a greater level of complexity to the Brucella
quorum-sensing network than has been previously proposed. While BlxR and VjbR are both involved in regulation of the VirB type IV secretion system and flagella, microarray analysis identified other systems that may be involved in the quorum-sensing network. They include nine transcriptional-regulatory proteins and seven proteins predicted to be involved in cell envelope biogenesis. Although the targets of the transcriptional-regulatory proteins are currently unidentified, the lysR
family transcriptional regulator encoded at locus BMEI1573 is known to be involved in virulence in mice (21
). BMEI1573 is located near genes for purine catabolism and may be involved in their regulation. Another gene known to be involved in pathogenicity was identified among the cell envelope biogenesis proteins. BacA (BMEI1553) is involved in very long-chain fatty acid modification of lipid A (17
) and has been shown to play a role in the persistence of infection (34
). These microarray results, combined with the very different effects of vjbR
deletions on in vivo dissemination and pathogenicity, suggest that the quorum-sensing network of Brucella
regulates numerous systems associated with host-pathogen interaction. However, much of the network remains to be elucidated.
Our data raise questions regarding the regulation of the type IV secretion system in broth culture versus macrophage infection. It was previously reported that the VirB8 protein was undetectable in a VjbR mutant during growth in broth, and it was suggested that the phenotype of the VjbR mutant was due to loss of the type IV secretion complex (13
). Although virB
transcription was dependent on both the BlxR and VjbR proteins, deletion of either regulatory-protein gene did not produce a phenotype consistent with complete loss of type IV secretion during macrophage infection. Intracellular bacterial counts of 16MΔblxR
increased steadily between 6 and 48 h at a slightly lower rate than that of wild-type 16M. The numbers of intracellular bacteria were not significantly different at 24 h postinfection. However, by 48 h, the intracellular bacterial counts of 16MΔblxR
were 0.96 log units lower than those of 16M, and the intracellular bacterial counts of 16MΔvjbR
were 1.34 log units lower than those of 16M. These results are consistent with previously published reports for a vjbR
mutant B. melitensis
strain at 48 h postinfection (13
). However, previously published results for the growth of virB
mutant B. melitensis
are considerably different (46
). The virB
mutant exhibited a continuing drop in intracellular numbers after the initial decrease, reaching 4 log units lower than the wild-type 16M by 48 h postinfection.
A lower level of virB
transcription in the mutants may still allow assembly of sufficient functional type IV secretion complexes for correct trafficking of the Brucella
-containing vacuole in early infection. However, it is possible that the level of attenuation of virB
expression observed in the regulatory-gene mutants grown in broth is not representative of quorum-sensing regulation during macrophage infection. The VirB proteins are expressed constitutively in B. melitensis
during growth in rich media but are upregulated by incubation at pH 4.5 (50
). The environment within the phagocytic vacuole is nutritionally poor (38
), and survival within the macrophage involves exposure to many stresses, including transient low pH (2
). Therefore, gene regulation may be much different within macrophages than during growth in rich medium.
Although the effects of the vjbR and blxR deletions on intracellular growth of B. melitensis were similar, the in vivo effects of these deletions were strikingly different. The vjbR mutant failed to disseminate early in infection and was fully attenuated in IRF-1−/− mice. In contrast, the blxR deletion only delayed the death of infected mice compared to infection with wild-type B. melitensis 16M and did not affect dissemination. Overall, these observations suggest that the two regulatory proteins are not redundant in function. Although the regulatory networks are convergent, their different contributions to pathogenesis demonstrate that certain elements of the two networks are independent.
An autoinducer binding domain mutation in VjbR rendered B. melitensis
unresponsive to the C12-HSL autoinducer (56
), demonstrating that VjbR responds to C12-HSL, but also suggesting that BlxR does not. The interaction of heterologous LuxR homologs has been reported in Erwinia carotovora
, where ExpR1 and ExpR2 act cooperatively to repress the negative regulator RsmA in the presence of autoinducer signal (53
). ExpR1 and ExpR2 bind preferentially to different autoinducer molecules, allowing the quorum-sensing system to respond to multiple signals (53
). In the quorum-sensing pathway of Brucella
, loss of either protein is sufficient for suppression of the type IV secretion system and flagellar genes, suggesting that the two transcriptional regulators may provide a way for genes at the intersection of the regulatory pathways to be suppressed in response to different environmental stimuli.