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.
Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic balance under stress. In conclusion, our results provide a better understanding of the global metabolic adaptations of B. abortus associated with distinct environmental stresses. The identification of proteins necessary for stress resistance is crucial toward elucidating the infectious process in order to control brucellosis, and may facilitate the discovery of novel therapeutic targets and effective vaccines.
Abnormal development of central nervous system (CNS) caused by neural tube defects (NTDs) is not only remained the major contributor in the prevalence of stillbirths and neonatal deaths, but also represents a significant cause of lifelong physical disability in the surviving infants. Ethionine is a non-proteinogenic amino acid and antagonist of methionine. Methionine cycle is essential for the elimination of reactive oxygen species (ROS), while lysosomes are involved in the initiation of autophagy. However, its role in ethionine-induced cell death in neural tube defects, still need to be explored. In this study, we investigated the effect of ethionine on NTDs as well as the underlying mechanism involved in this process. Following the establishment of NTDs model using ethionine-induced C57BL/6 mice, ethionine was intraperitoneally injected at a dose of 500 mg/kg in E7.5. Our study revealed that ethionine has induced mitochondrial apoptosis in NTDs by reducing mitochondrial autophagy both in vivo and in vitro. These results provided a possible molecular mechanism for redox regulation of autophagic process.
Edema toxin (ET), which is composed of a potent adenylate cyclase (AC), edema factor (EF), and protective antigen (PA), is one of the major toxicity factors of Bacillus anthracis. In this study, we introduced mutations in full-length EF to generate alanine EF(H351A) and arginine EF(H351R) variants. In vitro activity analysis displayed that the adenylyl cyclase activity of both the mutants was significantly diminished compared with the wild-type EF. When the native and mutant toxins were administered subcutaneously in a mouse footpad edema model, severe acute swelling was evoked by wild-type ET, while the symptoms induced by mutant toxins were very minor. Systemic administration of these EF variants caused non-lethal hepatotoxicity. In addition, EF(H351R) exhibited slightly higher activity in causing more severe edema than EF(H351A). Our findings demonstrate that the toxicity of ET is not abolished by substitution of EF residue His351 by alanine or arginine. These results also indicate the potential of the mouse footpad edema model as a sensitive method for evaluating both ET toxicity and the efficacy of candidate therapeutic agents.
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