Temporal analysis of pathogenic events in virulent and avirulent Brucella melitensis infections

Abstract

Despite progress in mouse models of brucellosis, much remains unknown regarding Brucella dissemination and tissue localization. Here, we report the dynamics of Brucella infection in individual mice using bioluminescent Brucella melitensis. Bioluminescent imaging of infected interferon regulatory factor-1 knockout (IRF-1(-/-)) mice identified acute infection in many tissues. Brucella was found to replicate in the salivary glands of IRF-1(-/-) and wild-type C57BL/6 mice suggesting a previously unknown tissue preference. Establishing a niche in this region may have relevance in humans where infection can result from ingestion of few bacteria. Sublethal infection of IRF-1(-/-) mice resulted in chronic Brucella localization in tail joints, an infection parallel to osteoarticular brucellosis in humans. Importantly, bioluminescent imaging rapidly identified attenuated EZ::TN/lux mutants in infected mice and revealed differences in dissemination, thereby defining the contribution of Brucella genes to virulence and tissue localization. Surprisingly, a virB mutant, though defective in persistence, disseminated similarly to virulent Brucella, suggesting bacterial spread is independent of VirB proteins that are important for intracellular survival. Together, our results reveal kinetics of acute and chronic Brucella infection in individual mice that parallels human infection as well as readily identified attenuated bacteria. Our approach facilitates identifying virulence determinants that may control tissue specific replication and may help develop therapeutics to overcome Brucella-induced chronic debilitating conditions.

Putative Quorum-Sensing Regulator BlxR ofBrucella melitensisRegulates Virulence Factors Including the Type IV Secretion System and Flagella

“…The mice were imaged every 2 days using an in vivo imaging system (Xenogen). Dissemination of the wild-type bioluminescent strain was consistent with previously published reports for this strain in IRF-1 Ϫ/Ϫ mice (47,48), and dissemination of the blxR mutant (ARL02) was similar to that of the wild type (GR023) (Fig. 6A and B).…”

“…Replacement of target genes was verified by PCR using sets of primers internal to the Km r cassette and flanking the deleted genes (see Table S2 in the supplemental material). Southern hybridization was performed to confirm the replacement of vjbR and blxR using a DNA fragment internal to vjbR (250 bp) and blxR (273 bp) amplified from B. melitensis 16M DNA, as well as a 600-bp DNA fragment internal to the Km r cassette, by North2South Detection (Pierce) as described previously (47).…”

Nondividing but Metabolically Active Gamma-IrradiatedBrucella melitensisIs Protective against VirulentB. melitensisChallenge in Mice

“…We used a bacterial lux operon (32) as a gene reporter to evaluate the effects of irradiation on brucella transcriptional and translational activity. Bioluminescent B. melitensis (GR023) expresses lux genes under a constitutive promoter (BMEI0101) (32), and change in the luminescence intensity relates directly to the transcription or translation ability under similar medium environments. Consistent with the previous finding, irradiated but not heat-killed bacteria preserved light emission evaluated by biophotonic imaging (Fig.…”

The mglA gene and its flanking regions in Brucella: The role of mglA in tolerance to hostile environments, Fe-metabolism and in vivo persistence

“…3, Table 1) can be interpreted as a result of ongoing genetic rearrangements in this region of chromosome II. Such mechanisms have been described as a cause for the generation of strain and species-specific differences within the genus Brucella ( Chain et al, 2005, Rajashekara et al, 2005and Tsoktouridis et al, 2003.…”