Abstract
Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.
Highlights
Brucellae are facultative intracellular coccobacilli causing brucellosis, a widespread bacterial zoonosis, infecting wildlife animals, livestock and humans
To better understand the molecular mechanisms leading to acid resistance of B. microti at pH 4.5, we identified and characterized common and species-specific acid resistance determinants by comparative RNA-Seq-based transcriptome analysis of bacteria exposed to pH 4.5 and 7.0 in minimal medium
Whereas survival of B. suis and B. microti decreased in Gerhardt’s Minimal Medium (GMM) at pH 4.5 and 5.0, with a significantly more rapid decline for B. suis, growth was observed for B. microti at the same pH values in complex TS broth
Summary
Brucellae are facultative intracellular coccobacilli causing brucellosis, a widespread bacterial zoonosis, infecting wildlife animals, livestock and humans. Brucella melitensis, and Brucella suis are the most relevant species for human infections (Pappas et al, 2005) After their uptake by the host cell, brucellae establish a specific intracellular niche, the Brucella-containing vacuole (BCV), in which the bacteria survive and replicate (Celli, 2019). This vacuole undergoes endosomal maturation during the early phase of infection, interacting rapidly with early and late endosomes as well as lysosomes, resulting in transient acidification to a pH of 44.5 (Porte et al, 1999). This acidification is crucial for intracellular replication and induction of the major virulence factor of Brucella spp., the type IV secretion system VirB (O’Callaghan et al, 1999; Porte et al, 1999; Boschiroli et al, 2002; Köhler et al, 2002)
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