A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M

Abstract

Background Brucella spp. are Gram-negative, facultative intracellular pathogens that cause brucellosis in both humans and animals. The B. abortus vaccine strain 104 M is the only vaccine available in China for the prevention of brucellosis in humans. Although the B. abortus 104 M genome has been fully sequenced, the current genome annotations are not yet complete. In addition, the main mechanisms underpinning its residual toxicity and vaccine-induced immune protection have yet to be elucidated. Mapping the proteome of B. abortus 104 M will help to improve genome annotation quality, thereby facilitating a greater understanding of its biology.ResultsIn this study, we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of B. abortus 104 M using high-resolution mass spectrometry. In total, 1,729 proteins (56.3% of 3,072) including 218 hypothetical proteins were identified using the culture conditions that were employed this study. The annotations of the B. abortus 104 M genome were also refined following identification and validation by reverse transcription-PCR. In addition, 14 pivotal virulence factors and 17 known protective antigens known to be involved in residual toxicity and immune protection were confirmed at the protein level following induction by the 104 M vaccine. Moreover, a further insight into the cell biology of multichromosomal bacteria was obtained following the elucidation of differences in protein expression levels between the small and large chromosomes.ConclusionsThe work presented in this report used a proteogenomic approach to perform whole-proteome analysis and genome reannotation in B. abortus 104 M; this work helped to improve genome annotation quality. Our analysis of virulence factors, protective antigens and other protein effectors provided the basis for further research to elucidate the mechanisms of residual toxicity and immune protection induced by the 104 M vaccine. Finally, the potential link between replication dynamics, gene function, and protein expression levels in this multichromosomal bacterium was detailed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3800-9) contains supplementary material, which is available to authorized users.

Proteomics of Brucella

“…The multiplexing of samples and the MS/MS-based quantification of iTRAQ reporter ions [35,36], or alternatively with Isotope Coded Protein Labeling (ICPL) [32] has been pursued. In-solution digestion [37][38][39][40][41], or in-gel digestion [42,43], and LC-MS/MS were sometimes employed.…”

“…NCBI ORF finder [88] detected possible alternative ORFs 200 bp up-or downstream of genes and predicted pseudogenes. In another study, fractionation and shotgun proteomics yielded a comprehensive set of 1729 proteins for the Brucella vaccine strain 104M [42]. Six novel ORFs were identified and three existing ORF models revised.…”

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

“…The acquired raw MS data files were then analyzed and the spectral files matched to 27,076 unique peptides with an FDR of 1.0%. We mapped the unique peptides to the B. abortus 104-M UniProt database (3,072 protein sequences), and only proteins that were identified by at least two unique peptides were confirmed (Zai et al, 2017 ). A total of 2,289 proteins were identified by two repeats, which represents approximately 74.5% coverage of the predicted proteome (Figure 2A ).…”

“…The protein samples were prepared as described previously (Zai et al, 2017 ). Briefly, cells cultured in each condition were harvested by centrifugation (7,000 × g for 15 min), and then washed three times with phosphate-buffered saline (PBS).…”

“…The initial allowed mass deviation of the precursor ion was set to 15 ppm, and the allowed value for the fragment mass was set to 0.02 Da. Protein identifications that contained at least two identified peptides were accepted with a false discovery rate (FDR) less than 1.0% (Zai et al, 2017 ).…”