Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice

Abstract

In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

FIG. 1.

Replication kinetics of three bioluminescent B. melitensis strains, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091). A. Stationary-phase cultures (30 μl) were inoculated into 30 ml of brucella broth and grown at 37°C with shaking, and optical density at 600 nm (OD₆₀₀) was determined. B. RAW 264.7 macrophages were inoculated with a standardized bacterial suspension of different strains, and growth was monitored at specified times (hours postinfection [Hrs PI]). CFU values are presented as geometric means ± standard errors (error bars) for data from two independent experiments. Values that are statistically significantly different (P < 0.05) from the value for strain 16M are indicated by asterisks.

FIG. 2.

Schematic representation of EZ::Tnlux transposon insertion in the three attenuated bioluminescent B. melitensis strains, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091). Only relevant features are shown, and the picture is not drawn to scale. The EZ::Tnlux transposon is indicated as a closed hexagon relative to the site of insertion. The relevant ORFs upstream and downstream of the insertion are shown in open boxes with arrows indicating the direction of transcription and numbers corresponding to the B. melitensis 16M genome sequence. The orientation of the arrow below the transposon in each strain represents the direction of Lux expression on the basis of our sequence data. The sites for ClaI restriction enzyme used in Southern hybridization experiment are shown by the letter C.

FIG. 3.

Complementation of strain GR019 (virB4) with the virB operon fully restored growth in macrophages (A) and virulence in IRF-1^−/− mice (B and C). RAW 264.7 macrophages were inoculated with a standardized bacterial suspension of complemented strains GR019/pBBVirB4 and GR019/pBBVirB, and growth was monitored at specified times (hours postinfection [Hrs PI]). The CFU counts were log transformed, and values are averages ± standard errors (error bars) for data from two independent experiments. IRF-1^−/− mice were inoculated i.p. with 1 × 10⁷ CFU of complemented strains GR019/pBBVirB4 and GR019/pBBVirB, and mouse survival (B) and CFU from livers, spleens, and testes (C) were determined. The CFU counts from livers, spleens, and testes were presented as geometric means ± standard errors (error bars) for data for four mice.

FIG. 4.

GR024 (galE) and GR026 (BMEI1090-BMEI1091) protect IRF-1^−/− mice from virulent challenge. IRF-1^−/− mice (n = 9) immunized with the different attenuated B. melitensis strains (GRO19 (virB4), GR024 (galE), GR026 (BMEI1090-BMEI1091), and BM710 (1 × 10⁷ i.p./mouse) were challenged with virulent B. melitensis GR023 (1 × 10⁶) and monitored for survival.

FIG. 5.

A. Bioluminescent monitoring of virulent B. melitensis infection in vaccinated IRF-1^−/− mice. IRF-1^−/− mice vaccinated with GR019 (virB4), GR024 (galE), GR026 (BMEI1090-BMEI1091), and BM710 were imaged for 10 min following GR023 challenge. The numbers below the panels indicate the number of days postinfection, and images representing the same day postinfection from different groups are shown. The bioluminescent image of the same single mouse (n = 9) from each group at different times is shown. The rainbow scale represents approximate photon counts. B. Bioluminescence imaging of surviving IRF-1^−/− mice 44 days following challenge and the corresponding histological changes in livers and spleens. Livers were scored by the number of focal granulomas observed per field of view (fov) at a magnification of ×4. The average numbers of granulomas from 8 fov are indicated as follows: +, 1 to 8; ++, 9 to 16; +++, 17 to 24. Spleens were scored on the loss of white and red pulp architecture at a magnification of ×4 as follows: −, normal spleen or no noticeable changes; +, enlarged follicles, increased cellularity, and white pulp; ++, hyperplasia, with a significant increase in follicle size, and white pulp; +++, increased red pulp and loss of white pulp architecture.

FIG. 6.

A. Real-time analysis of attenuated bioluminescent B. melitensis strains in C57BL/6 mice. C57BL/6 mice were infected with 5 × 10⁷ CFU of B. melitensis strains GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) and imaged daily with a 10-min exposure. Numbers below the panels indicate the number of days postinfection, and images representing the same day postinfection from different groups are shown. The bioluminescent image of the same single mouse from each group is shown. B. Bioluminescent monitoring of the virulent B. melitensis infection in vaccinated C57BL/6 mice. C57BL/6 mice vaccinated with different attenuated strains were challenged with GR023 and imaged for 10 min. The rainbow scale represents approximate photon counts. The bioluminescent image of the same single mouse (n = 4 for each time point) at different time points from each group is shown.

FIG. 7.

CFU counts from livers (A) and spleens (B) of C57BL/6 mice vaccinated with GR019 (virB4), GR024 (galE), GR026 (BMEI1090-BMEI1091), BM710, or Rev-1 followed by virulent GR023 challenge. The number of CFU per gram of tissue was determined, and geometric means were derived from three or four mice at each time point (weeks postchallenge [Wks PC]). Error bars represent the range of CFU of the samples from each time point. Values that were statistically significantly different from the values for unvaccinated controls (P < 0.05 [*]) or from the values for BM710- and GR019 (virB4)-vaccinated groups (P < 0.05 [**]) are indicated.

FIG. 8.

Large grossly visible focal calcified granulomas in the livers of C56BL/6 mice vaccinated with Rev-1. (A) Liver from an unvaccinated mouse. The livers from unvaccinated mice or mice vaccinated with other attenuated mutants had no visible focal calcified granulomas at any time point, unlike the mice from Rev-1-vaccinated group. (B to D) Livers from Rev-1 vaccinated mice containing large focal granulomas with secondary changes, including a central area of necrosis, neutrophil infiltration, and fibrosis with calcification (C and D).