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.

Unraveling the Role of the Zinc-Dependent Metalloproteinase/HTH-Xre Toxin/Antitoxin (TA) System of Brucella abortus in the Oxidative Stress Response: Insights into the Stress Response and Virulence

“…A search in the Toxin-Antitoxin Database TADB2.0 [ 33 ] allowed us to identify at least four potential type II TA systems present in the chromosome I of B. abortus biovar 1 strain 9-941 ( , accessed on 29 August 2023) and B. abortus strain 2308 ( , accessed on 29 August 2023) ( Table 1 ). One of these TA systems is composed of the ZnMP (toxin) coded in the ORF BAB1_0270 (WP_002965518.1) present in a genomic island called “genomic island 3”, which is shared by some Brucella species, such as B. abortus 2308 and 9-941 strains [ 31 , 32 , 34 ]. This protein is homologous to the ImmA/IrrE metalloproteinases family, which is characterized by conserved domains COG2856 and motif HEXXH (pfam06114).…”

“…This evidence demonstrates that several regulatory factors participate in the adaptation of B. abortus during its intracellular lifecycle in professional and non-professional phagocytic cells. Interestingly, our group described the roles of a zinc-dependent metalloproteinase (ZnMP), which forms an operon with an HTH-Xre regulator, constituting a putative type II TA system in B. abortus [ 31 ]. Results showed that deletion of ORF BAB1_0270 (ZnMP) significantly affected the capacity of this pathogen to survive under acidic conditions, to escape from phagolysosomes to reach its replicative niche in ER-derived vesicles, to survive within phagocytic cells and to colonize mice tissues [ 31 , 32 ].…”

“…Interestingly, our group described the roles of a zinc-dependent metalloproteinase (ZnMP), which forms an operon with an HTH-Xre regulator, constituting a putative type II TA system in B. abortus [ 31 ]. Results showed that deletion of ORF BAB1_0270 (ZnMP) significantly affected the capacity of this pathogen to survive under acidic conditions, to escape from phagolysosomes to reach its replicative niche in ER-derived vesicles, to survive within phagocytic cells and to colonize mice tissues [ 31 , 32 ]. Notably, the deletion of BAB1_0270 (ZnMP) significantly reduced the expression of various genes coding for transcriptional regulators (e.g., vjbR , hutC , bvrR ), T4SS VirB proteins (e.g., virB1 , virb2 , virB5 ) and their effectors (e.g., btpA , vceA and vceC ) [ 31 ].…”

“…Results showed that deletion of ORF BAB1_0270 (ZnMP) significantly affected the capacity of this pathogen to survive under acidic conditions, to escape from phagolysosomes to reach its replicative niche in ER-derived vesicles, to survive within phagocytic cells and to colonize mice tissues [ 31 , 32 ]. Notably, the deletion of BAB1_0270 (ZnMP) significantly reduced the expression of various genes coding for transcriptional regulators (e.g., vjbR , hutC , bvrR ), T4SS VirB proteins (e.g., virB1 , virb2 , virB5 ) and their effectors (e.g., btpA , vceA and vceC ) [ 31 ]. Therefore, considering that these TA systems have been scarcely studied in B. abortus, this work aimed to evaluate the roles of the type II ZnMP/HTH-Xre TA system in the survival of B. abortus 2308 subjected to oxidative conditions.…”

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

“…Several virulence factors determine Brucella survival in macrophages, including zinc-dependent metalloproteinase (ZnMP). Its deletion causes an increase in the co-localization of bacteria with phagosomal Cts D, reducing intracellular replication of the pathogen within RAW 264.7 cells (Goḿez et al, 2020). Importantly, B. abortus persistence in macrophages is associated with the presence of anti-inflammatory cytokine, IL-10 (Xavier et al, 2013).…”