Search

Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs observed in bacteria, which is crucial for functional regulations of many prokaryotic and eukaryotic organisms. Mycobacterium bovis bacillus Calmette–Guérin (BCG) vaccine has been recognized as an indispensable tool in the global fight against tuberculosis (TB) worldwide over several decades. Nevertheless, analysis of glycoprotein profiles of BCG has not been clearly investigated. In this study, we performed O‐mannosylated protein analysis in BCG bacteria using gel‐based and gel‐free approaches. In total, 1,670 hexosylated peptides derived from 754 mannosylated proteins were identified. Furthermore, 20 novel protein products supported by 78 unique peptides not annotated in the BCG database were detected. Additionally, the translational start sites of 384 proteins were confirmed, and 78 proteins were validated through the extension of translational start sites based on N‐terminus‐derived peptides. The bioinformatic analysis of the O‐mannosylated proteins was performed and the expression profiles of four randomly selected proteins were validated through Western blotting. A number of proteins involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis, oxidative phosphorylation, and two‐component system, are discussed. Taken together, these results offer the first O‐mannosylated protein analysis of a member of mycobacteria reported to date by using complementary gel‐based and gel‐free approaches. Some of the proteins identified in this study have important roles involved in metabolic pathways, which could provide insight into the immune molecular mechanisms of this recognized vaccine strain. [ABSTRACT FROM AUTHOR]

Abstract Background New strategies for high-throughput sequencing are constantly appearing, leading to a great increase in the number of completely sequenced genomes. Unfortunately, computational genome annotation is out of step with this progress. Thus, the accurate annotation of these genomes has become a bottleneck of knowledge acquisition. Results We exploited a proteogenomic approach to improve conventional genome annotation by integrating proteomic data with genomic information. Using Shigella flexneri 2a as a model, we identified total 823 proteins, including 187 hypothetical proteins. Among them, three annotated ORFs were extended upstream through comprehensive analysis against an in-house N-terminal extension database. Two genes, which could not be translated to their full length because of stop codon 'mutations' induced by genome sequencing errors, were revised and annotated as fully functional genes. Above all, seven new ORFs were discovered, which were not predicted in S. flexneri 2a str.301 by any other annotation approaches. The transcripts of four novel ORFs were confirmed by RT-PCR assay. Additionally, most of these novel ORFs were overlapping genes, some even nested within the coding region of other known genes. Conclusions Our findings demonstrate that current Shigella genome annotation methods are not perfect and need to be improved. Apart from the validation of predicted genes at the protein level, the additional features of proteogenomic tools include revision of annotation errors and discovery of novel ORFs. The complementary dataset could provide more targets for those interested in Shigella to perform functional studies.

Abstract Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. Conclusions In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction.

Abstract Background Trichophyton rubrum is the most common dermatophyte causing fungal skin infections in humans. Asexual sporulation is an important means of propagation for T. rubrum, and conidia produced by this way are thought to be the primary cause of human infections. Despite their importance in pathogenesis, the conidia of T. rubrum remain understudied. We intend to intensively investigate the proteome of dormant T. rubrum conidia to characterize its molecular and cellular features and to enhance the development of novel therapeutic strategies. Results The proteome of T. rubrum conidia was analyzed by combining shotgun proteomics with sample prefractionation and multiple enzyme digestion. In total, 1026 proteins were identified. All identified proteins were compared to those in the NCBI non-redundant protein database, the eukaryotic orthologous groups database, and the gene ontology database to obtain functional annotation information. Functional classification revealed that the identified proteins covered nearly all major biological processes. Some proteins were spore specific and related to the survival and dispersal of T. rubrum conidia, and many proteins were important to conidial germination and response to environmental conditions. Conclusion Our results suggest that the proteome of T. rubrum conidia is considerably complex, and that the maintenance of conidial dormancy is an intricate and elaborate process. This data set provides the first global framework for the dormant T. rubrum conidia proteome and is a stepping stone on the way to further study of the molecular mechanisms of T. rubrum conidial germination and the maintenance of conidial dormancy.

Abstract Background Compelling evidence indicates that Shigella species, the etiologic agents of bacillary dysentery, as well as enteroinvasive Escherichia coli, are derived from multiple origins of Escherichia coli and form a single pathovar. To further understand the genome diversity and virulence evolution of Shigella, comparative genomic hybridization microarray analysis was employed to compare the gene content of E. coli K-12 with those of 43 Shigella strains from all lineages. Results For the 43 strains subjected to CGH microarray analyses, the common backbone of the Shigella genome was estimated to contain more than 1,900 open reading frames (ORFs), with a mean number of 726 undetectable ORFs. The mosaic distribution of absent regions indicated that insertions and/or deletions have led to the highly diversified genomes of pathogenic strains. Conclusion These results support the hypothesis that by gain and loss of functions, Shigella species became successful human pathogens through convergent evolution from diverse genomic backgrounds. Moreover, we also found many specific differences between different lineages, providing a window into understanding bacterial speciation and taxonomic relationships.

Objective: To detect the mutation sites of exons 2, 20, 11A and 11B in Chinese patients with breast cancer., Methods: A total of 86 patients with breast cancer without blood relationship were randomly selected. Polymerase chain reaction (PCR) and double-strand DNA direct sequencing were applied., Results: No mutations, especially deletions were found in exons 2, 20 and 11 with carefully checking the sequencing results, although they were reported frequently in Europe populations with breast cancer. We found one polymorphism in exon 11, with high frequency, and in the test of chi-square, the frequencies of two alleles had no significant difference between the patients and controls., Conclusion: The above results suggest this SNP may not be associated with the breast cancer in Chinese population, and indicates that the gene sequence of what we have studied doesn't account much for occurrence of the breast cancer in the population of China.