A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages

Abstract

Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying for a zinc-dependent metalloproteinase (ZnMP). Here, its contribution in the intracellular adaptation of B. abortus was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this ZnMP did not participated in either the adherence or the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus Δ270 with phagolysosomal cathepsin D and reduced its co-localization with calnexin present in endoplasmic reticulum (RE)-derived vesicles. Although B. abortus Δ270 showed an upregulated expression of genes involved in virulence (vjbR, hutC, bvrR, virB1), it was insufficient to reach a successful intracellular replication within macrophages. Furthermore, its attenuation favored in macrophages infected the production of high levels of cytokines (TNF-α and IL-6) and co-stimulatory proteins (CD80 and CD86), signals required in T cell activation. Finally, its deletion significantly reduced the ability of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, and considering that this ZnMP has homology with ImmA/IrrE proteases, we discuss its role in the virulence of this pathogen, concluding that ZnMP is required in the intracellular adaptation of B. abortus 2308 during the infection of macrophages.

Keywords: attenuation; gene regulation; host-pathogen interaction; toxin-anti-toxin systems; virulence.

FIGURE 1

Modeling and purification of Zn-dependent metalloproteinase. (A) ProSA-web Z-score plot for 3D structure of the hypothetical protein after refinement. The Z-score of the final model is −4.52. The black dot represents the hypothetical protein. (B) 3D structure according to the I-TASSER server after being refined and visualized by Chimera PyMOL. (C) Purification of the ZnMP codifying for ORF BAB1_0270 of B. abortus 2308. ZnMP sequences were cloned in the expression vector pColdII (pColdII-ZnMP), and the recombinant vector was used to transform the Escherichia coli BL21 (DE3) strain. Western blot was performed to visualized protein using anti-6x-HisTag antibodies. MW: molecular weight marker in kDa; 1: recombinant protein purified from E. coli.

FIGURE 2

A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II toxin-antitoxin. Identification of the transcriptional unit (operon) constituted by ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in (A) the genomic DNA and (B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). (C) Hypothetical Type II toxin-antitoxin (TA) model for operon constitute by ZnMP and transcriptional regulator. Toxin (ZnMP) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress conditions, cleaving the anti-toxin, which increases the levels of toxin-free, inducing various biological functions in bacteria. Predicted promoter at site -35 and -10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides shared between final part of transcriptional factor and metalloproteinase codified for ORF BAB1_0270.

FIGURE 3

Adhesion and intracellular trafficking of B. abortus strains in RAW267.4 macrophages. (A) Macrophages were treated with cytochalasin D (0.5 mg/ml) and infected with the B. abortus 2308 and B. abortus Δ270 strains for 30 min. Image shows the proximity of B. abortus strains with the RAW264.7 macrophages, where cyan represents bacteria with a proximity less than 5 mm from macrophages, purple represents bacteria with a proximity greater than 5 mm distance from macrophages, and yellow represents bacteria adhered to macrophages. (B) Adherence is expressed by the percentages of bacteria (yellow) adhered to RAW264.7 macrophages. (C,E,G) are representative of confocal images showing the co-localization of B. abortus-GFP strains with EEA1 at 5, 10, and 15 min pi, cathepsin D at 1 and 12 h pi and calnexin at 1 and 12 h pi. (D,F,H) show the percentages of the co-localization of B. abortus-GFP strains with EEA1 at 5, 10, and 15 min pi; cathepsin D at 1 and 12 h pi, and calnexin at 1 and 12 h pi. Results are expressed as the mean ± standard deviation. Values of P < 0.05 were considered statistically significant, where ns: non-significant differences and *, **, and *** denote values of P < 0.05, P < 0.01, and P < 0.001, respectively. All assays were performed in triplicate.

FIGURE 4

Intracellular survival and gene expression of B. abortus strains in macrophages. (A) Intracellular bacteria were obtained from macrophages infected at 1:10 MOI with B. abortus 2308, B. abortus Δ270, or B. abortus Δ270C at 6 and 24 h pi. Intracellular survival was recorded as mean ± standard deviation of bacterial counts as CFU/mL. (B) The relative expression of vjbR, hutC, bvrR, virB1, virb2, virB5, vceA, vceC, btpA, and the ORFs BAB1_0273 (GI-3) and BAB1_0627 induced in B. abortus 2308 (wt) and B. abortus Δ270 during macrophage infection was calculated by the 2^−ΔΔCT method using qPCR assays at 24 h pi. The housekeeping genes gyrA and 16s were used as reference genes. Results were expressed as the mean ± standard deviation. Values of P < 0.05 were considered statistically significant, where ns denotes non-significant differences, *, **, ***, and **** denote values of P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. All assays were performed in triplicate.

FIGURE 5

Attenuation of B. abortus by deletion of ZnMP in macrophages. Expression of secondary signals for T cells from B. abortus strain-infected macrophages at MOI 1:10. (A) Production of TNF-α (pg/ml) and (B) production of IL-6 (pg/ml) from macrophages infected with Brucella strains were measured by Sandwich ELISA at 6 and 24 h pi with standard curves using TNF-α and IL-6 recombinant proteins. (C,E) Expression of co-stimulatory proteins CD80 and CD86 from infected macrophages measured by flow cytometry at 6 h pi (blue) and 24 h pi (red). Fluorescence minus one (FMO) was used as a negative control. (D,F) show the mean ± standard deviation of the mean fluorescence intensity (MFI) of CD80 and CD86. Data represent unstimulated (PBS-treated) macrophages, B. abortus 2308-infected macrophages, B. abortus Δ270-infected macrophages and B. abortus 2308i (inactivated strain) stimulated macrophages. Results were expressed as mean ± standard deviation. Values of P < 0.05 were considered as statistically significant, where **, ***, and **** denote values of P < 0.01, P < 0.001, and P < 0.0001, respectively. All assays were performed in triplicate.

FIGURE 6

Brucella strains growth and gene expression under acidic stress. (A) Growth curves of B. abortus 2308, B. abortus Δ270, and B. abortus Δ270C strains cultured in acidic medium (pH 5.5). (B) Linear regression analysis of pH changes in the medium during the growth of Brucella strains. (C) The relative expression of vjbR, hutC, bvrR, virB1, virb2, virB5, btpA, vceA, and vceC induced in B. abortus 2308 (wt) and B. abortus Δ270 cultured in medium with pH 5.5. The gene expression was calculated by 2^−ΔΔCT method using qPCR assays at 24 h. The housekeeping genes gyrA and 16s were used as reference genes. Results were expressed as the mean ± standard deviation. P value < 0.05 were considered statistically significant, where * denotes values of P < 0.05 and **** denotes values of P < 0.0001. All assays were performed in triplicate.