Investigation of Rifampicin Resistance Mechanisms in Brucella abortus Using MS-Driven Comparative Proteomics

Abstract

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

“…In this study, we utilized a proteomics approach to identify Brucella-specific proteins and pathways affected by changes in global c-di-GMP levels. This approach detected over half the orfeome (ϳ1,900 proteins) of Brucella, significantly more than previously reported (typically Ͻ500 proteins) (38)(39)(40). In the attenuated, high-c-di-GMP-expressing ⌬bpdA mutant, proteins involved in cell wall biogenesis were upregulated compared to both 16M and the ⌬cgsB mutant.…”

Antimicrobial drug resistance affects broad changes in metabolomic phenotype in addition to secondary metabolism

“…Notably in these adaptive strategies, a small number of mutations ultimately result in complex and significant global changes in gene expression and protein synthesis. For instance, rpoB mutations foster the up-regulation of proteins involved in central metabolism (including nucleoside and nucleotide, amino acid, carbohydrate, lipid, and phospholipid biosynthesis), detoxification, signal transduction, protein synthesis, and cell envelope processes, to name a few, whereas cell division proteins are generally down-regulated (6,7). Streptomycin resistance, via rpsL mutation (encoding for ribosomal protein S12, a subunit of the 30S ribosome), leads to increased translational accuracy, slower overall translation, and has demonstrated late growth phase protein synthesis increases in comparison with the wild type (8).…”

Emergence of antibiotic-resistant extremophiles (AREs)

“…The proteomic profile of rifampicin-resistant Brucella abortus was examined and compared to that of a non-resistant strain using MS-drive comparative proteomics; the resistant strain contained a mutation in the rpoB gene. In the same study, 12 836 MS/MS spectra identified 6,753 peptides corresponding to 456 proteins, of which 39 were differentially regulated in the resistant strain and were involved in various metabolic pathways (Sandalakis et al 2012). From this study, it can be hypothesized that various proteins, such as pyruvate decarboxylase, urocanatehydratase, bifunctional protein FolD, and an aminotransferase class-III: maltose-binding protein could potentially regulate antibiotic resistance (Sandalakis et al 2012).…”

“…In the same study, 12 836 MS/MS spectra identified 6,753 peptides corresponding to 456 proteins, of which 39 were differentially regulated in the resistant strain and were involved in various metabolic pathways (Sandalakis et al 2012). From this study, it can be hypothesized that various proteins, such as pyruvate decarboxylase, urocanatehydratase, bifunctional protein FolD, and an aminotransferase class-III: maltose-binding protein could potentially regulate antibiotic resistance (Sandalakis et al 2012). Using an MS/MS technique, Piras et al (2012) were able to identify the proteins that were differentially regulated in a multidrug-resistant E. coli as those involved in the metabolic pathways, including malate dehydrogenase, fructose-bisphosphatealdolase, triosephosphateisomerase, phosphoglucomutase, and others.…”