Metabolic Control of Virulence Genes inBrucella abortus: HutC CoordinatesvirBExpression and the Histidine Utilization Pathway by Direct Binding to Both Promoters

Abstract

Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (P virB ). Using different procedures, we isolated a 27-kDa protein that binds specifically to P virB . This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.Type IV secretion systems (T4SS) are multicomponent machineries central to the pathogenesis of many bacterial genera (e.g., Brucella, Agrobacterium, Helicobacter, Legionella, and Bordetella) (4). T4SS function comprises recognition and translocation of specific substrates across the bacterial cell envelope. The nature of the translocated substrates varies from proteins to DNA-protein complexes. In addition to the wellstudied Agrobacterium transferred DNA (T-DNA) and Bordetella pertussis toxin, several translocated effectors have been identified for Helicobacter, Legionella, and Brucella (7). In every case, the translocated molecules alter cellular processes in such a way that allows the pathogen to overcome host defenses.Brucella is a gram-negative bacterium that causes brucellosis, a worldwide zoonosis that affects domestic mammals. Different Brucella species vary in their host preferences. Brucella abortus, Brucella suis, and Brucella melitensis infect cattle, pigs, and goats, respectively, but also infect humans. In animals, the symptoms of the disease are sterility in males and abortion in pregnant females (6). In humans, brucellosis causes undulant fever during the acute phase and, if it reaches chronicity, can lead to endocarditis, osteoarthritis, and neurological damage.Brucella is an intracellular parasite that persists and replicates within host macrophages. After internalization, the bacterium actively controls the matur...

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…As growth proceeds, HutC is eventually diluted by cell growth to a level that no longer activates virB. This would result in a delayed and transient expression of virB, which is in fact the pattern of virB expression during infection (146). This appears to argue for a direct effect of the Hut system on pathogenicity.…”

“…Sieira et al have shown that in Brucella abortus, the HutC (Hut repressor) protein binds to the promoter of the virB operon, which encodes the type 4 secretion system (146). Binding of HutC to the virB promoter results in an increase in virB transcription that is not seen in vitro or in vivo if hutC is deleted.…”

“…It is not clear whether this increase results from a direct activation or from interference with a negative regulator. However, several features of the system have been established (146). The affinity of the virB promoter for HutC is 30 times lower than that of the hut promoter.…”

The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB inBrucella abortus

“…Several molecular systems have been reported to modulate or impact VirB expression. Among them, a direct interaction with the promoter region of VirB has been demonstrated only for integration host factor (IHF), VjbR, and HutC (2,9,27,28).…”

“…Several molecular systems have been reported to modulate or impact VirB expression. Among them, a direct interaction with the promoter region of VirB has been demonstrated only for integration host factor (IHF), VjbR, and HutC (2,9,27,28).Both the Brucella T4SS VirB and the TCS BvrRS have homologs present in alphaproteobacterial endosymbionts and pathogens of plants and animals such as Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bartonella (4,19,30). Indeed, ChvG/ChvI and ExoS/ChvI are also two-component systems devoted to the control of critical functions during Agrobacterium tumefaciens parasitism and Sinorhizobium meliloti endosymbiosis, respectively.…”