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.
Highlights
Brucella abortus is the causative agent of brucellosis, a highly contagious zoonosis transmitted from bovines to humans
The open reading frame (ORF) BAB1_0273, codifying for a hypothetical transcriptional regulator, and the ORF BAB1_0627, codifying for a hypothetical protein of unknown functions, were downregulated in B. abortus 270. These results demonstrate that deletion of zinc-dependent metalloproteinase (ZnMP) negatively affects the intracellular survival of B. abortus 270, which expresses a dysregulation in various genes involved in the infection of macrophages
At 24 h pi the expression of CD80 and CD86 in macrophages infected with B. abortus 270 was significantly higher compared to wt, inactivated or unstimulated macrophages (Figures 5C–F). These results demonstrate that deletion of this ZnMP attenuated the virulence of B. abortus, favoring the production of cytokines (IL-6 and TNF-α) and co-stimulatory (CD80 and CD86) proteins, which are secondary signals required in the activation of T cells and bacterial clearance
Summary
Brucella abortus is the causative agent of brucellosis, a highly contagious zoonosis transmitted from bovines to humans. It shows a tropism toward the erythritol present in the Brucella Metalloproteinase and Intracellular Adaptation placenta and genital organs, producing abortions in females and infertility in males (de Figueiredo et al, 2015; Letesson et al, 2017) To reach these organs, B. abortus survives within neutrophils, macrophages and dendritic cells, using them as “Trojan horses” to disseminate themselves systemically (Martirosyan et al, 2011; Miraglia et al, 2018; GutiérrezJiménez et al, 2019). B. abortus survives within neutrophils, macrophages and dendritic cells, using them as “Trojan horses” to disseminate themselves systemically (Martirosyan et al, 2011; Miraglia et al, 2018; GutiérrezJiménez et al, 2019) Within these cells, endosomal Brucella containing vesicles (eBCVs) develop, which interact with early/late endosomes and lysosomes, exposing B. abortus to the activity of proteases, reactive oxygen and nitrogen species (ROS and RNS, respectively) and to low pH (von Bargen et al, 2012). The rBCVs can be engulfed by autophagosome-like structures originating autophagic BCVs (aBCVs), facilitating the exit of B. abortus from infected cells and completion of its intracellular cycle (von Bargen et al, 2012; Roop et al, 2013; Celli, 2019)
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