A Zinc-dependent metalloproteinase in the intracellular adaptation of Brucella abortus in macrophages

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 of a Zinc-dependent metalloproteinase. Here, its contribution in the process of intracellular adaptation was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this Zinc-dependent metalloproteinase is a cytoplasmic protein that conforms an operon with a transcriptional regulator, which may constitute a type II toxin-antitoxin system. Functionally, this Zinc-dependent metalloproteinase participated neither in the adherence nor 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, reducing both its co-localization with calnexin, present in endoplasmic reticulum derived vesicles, and its intracellular replication within macrophages. Besides, B. abortus Δ270-infected macrophages produced significantly higher levels of TNF-α, IL-6, CD80 and CD86 than B. abortus 2308, even when several genes involved in virulence (vjbR, hutC, bvrR, virB1) were up-regulated in this mutant. Finally, its deletion significantly reduced the capacity of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, we discuss the role of this Zinc-dependent metalloproteinase in the regulation of the virulence of this pathogen, concluding that it contributes significantly to the intracellular adaptation of B. abortus 2308 during the infection of macrophages. Author summary Brucella abortus is the causative agent of the brucellosis, a highly contagious diseases. A Zinc-dependent metalloproteinase contributes significantly in the intracellular survival. Here, we demonstrate that this metalloproteinase has homology with ImmA/IrrE proteases, which are involved in the bacterial resistance to hostile environment. Furthermore, it conforms a gene pair with a transcriptional regulator, being required by B. abortus to escape from phagolysosomes, to achieve the endoplasmic reticulum and replicate within macrophages. Its deletion from B. abortus stimulated the macrophages, which produced higher levels of pro-inflammatory cytokines and co-stimulatory proteins. This pathogen showed a reduced ability to adapt and grow under acidic conditions, which would negatively affect its escape from phagolysosomes and consequently, stimulating macrophages. Therefore, this work describes how this Zinc-dependent metalloproteinase significantly contributes in the intracellular adaptation of B. abortus 2308 in macrophages.


Introduction
Macrophages destroys bacteria in the phagolysosome compartments and activates the innate 244 and adaptive immunity secreting pro-inflammatory cytokines (TNF-α and IL-6) and co-245 stimulatory proteins (CD80 and CD86), which were quantified at 6 and 24 h by ELISA and 246 flow cytometry, respectively. Macrophages infected with the B. abortus Δ270 strain at 6 h pi 247 produced levels of TNF-α significantly higher than the macrophages infected with wt (P < bvrR, virB1, virB2, virB5, vceA, vceC and btpA compared to wt strain (P < 0.05) (Figure 6C). 281 These results suggest that deletion of this Zn-dependent metalloproteinase reduces the capacity 282 of B. abortus to adapt and grow under acidic conditions and produces, directly or indirectly, 283 changes in the expression of diverse genes involved in its virulence and resistance to acidic 284 conditions.

290
B. abortus is a pathogen successfully adapted to survival and replicate in the intracellular 291 environment of macrophages [4,[8][9][10][11]. In these cells, it develops BCVs, which through of its in the pathogenicity of this bacteria is unknown, its characterization indicating is a cytoplasmic 299 protein that forms an operon with a transcriptional regulator containing a HTH domain of the 300 Xre family, which could constituted a type II Toxin-Antitoxin (TA II) system, which 301 participates in several aspects of bacterial physiology, including gene regulation, "growth 302 arrest" and survival under environmental stress, mechanisms contributing to bacterial 303 persistence [34,45]. Furthermore, this Zinc-dependent metalloproteinase has homology to 304 ImmA/IrrE proteases, which cleaves repressor proteins inhibiting the expression of several 305 genes involved in the horizontal transfer of mobile genetic elements or in the resistance to 306 adverse conditions [31][32][33]  However, in the infection process the gene vceA codifying of a protein secreted in the 325 cytoplasm of the macrophages and whose deletion in B. abortus promotes autophagy and 326 inhibit the apoptosis in human trophoblast cells [38]. These processes are keys in the 327 intracellular survival of this pathogen, because allows it to control the physiology of host cells.  could have contributed to make this strain less virulent, which reduced significantly its ability 335 to establish intracellularly and subsequently, to inhibit the expression of several proteins 336 involved in the host's innate and adaptive activation [39,40]. In this context, macrophages 337 infected with B. abortus Δ270 were significantly stimulated, which induced the production of high levels of TNF-α, IL-6, and the expression of CD80 and CD86 co-stimulatory proteins.

339
This process of activation could be associated to the co-localization of this mutant strain with  The intracellular environment is harsh for bacteria phagocytized, because they are exposed to 354 components such as the acidification, a factor that impair the bacterial growth and that activates 355 bactericidal proteases in the phagosome of the macrophages [44,45]  to acidic stress, a condition which this bacterium is exposed during its intracellular life cycle. 364 Therefore, is possible that, this Zinc-dependent metalloproteinase has a similar function to 365 ImmA/IrrE proteases, which cleaves repressor proteins impeding the expression of specific 366 genes, such as vceA, BAB1_0273 or BAB1_0627, which would modulate negatively the forms a type II TA system, then its deletion (toxin), turned the mutant strain less virulent and 376 less resistant to intracellular microbicide mechanisms, maybe this TA system provide tolerance 377 to stress, such as the bacterial SOS response or the production of guanosine pentaphosphate 378 ((p)ppGpp), which contribute to bacterial persistence [34,35]. Consequently, given the 379 importance of these TA systems, the function of this type II TA system present on the GI-3 in 380 the physiology of B. abortus during its interaction with the host must be clarified. Finally, is 381 possible that the regulatory functions associated to the type II TA system and the ImmA/IrrE-   Table 1. Primers for mutant strain are described in the S1 Table. All     Table 2). PCR products were visualized by agarose gel electrophoresis at 1%.

532
The production of these cytokines was quantified in the supernatants from macrophages     [26] pAKgfp1 Broad-host-range cloning vector pBBR1MCS4 (Amp r ) codifying of green fluorescence protein (GFP). [46] pColdII Vector derived from backbone plasmid pUC118 is used for cold shock-induced protein expression in E. coli followed by a 6xHis tag.
[58] 856 857 858     Results are expressed as the mean ±standard deviation. Values of P < 0.05 were considered as 914 statistically significant, where ns: non-significant differences and *, ** and *** denotes values 915 of P < 0.05, P < 0.01 and P < 0.001, respectively. All assays were made in triplicates.  infection of macrophages was calculated by the 2 -ΔΔCT method using qPCR assays at 24 h pi.

925
The housekeeping gyrA and 16s genes were used as reference genes. Results were expressed 926 as the mean ± standard deviation. Values of P < 0.05 were considered as statistically 927 significant, where ns denotes non-significant differences, *, *** and **** denotes values of P 928 < 0.05, P < 0.001 and P < 0.0001, respectively. All assays were made in triplicates.