Candong Wei

The use of comparative genomic hybridization to characterize genome dynamics and diversity among the serotypes of Shigella

BackgroundCompelling 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.ResultsFor 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.ConclusionThese 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.

Proteomic profile of dormant Trichophyton Rubrum conidia

BackgroundTrichophyton 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.ResultsThe 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.ConclusionOur 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.

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%–80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on the mycobacterial secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

BackgroundTuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the worlds 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.ResultsUsing 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.ConclusionsIn 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.

A comprehensive proteomic analysis of Mycobacterium bovis bacillus Calmette–Guérin using high resolution Fourier transform mass spectrometry

Since 1921, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important vaccine to prevent tuberculosis worldwide. Nonetheless, a global analysis of BCG proteome has not been clearly investigated. In this study, we performed an in-depth proteomic analysis of BCG under an in vitro cultivation condition using SDS-PAGE and high resolution Fourier transform mass spectrometry. In total, 3434 proteins (35,259 unique peptides) including 512 transmembrane proteins were identified, covering ~87% of the predicted BCG proteome. Seven pseudogene protein products were also obtained and validated by RT-PCR at gene transcript level. Additionally, translational start sites of 832 proteins were confirmed and 186 were extended using N-terminus-derived peptides. The physicochemical characteristics of all identified proteins were determined. Some predominant proteins, including PE and PPE family proteins, lipoproteins, heat shock proteins, transport proteins and low molecular weight protein antigens, are discussed, which represent potential prominent antigens in the humoral and cellular immune response. This study represents the most comprehensive BCG proteome to date, which will likely facilitate the design of vaccination and immunodiagnostic strategies against TB.

DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity

ABSTRACT The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed of the PB1, PB2, and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nuclei of infected cells. In this study, we newly identified host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with the bPB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, while the J domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to enter the nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner. IMPORTANCE The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaptation. In this study, we newly identified a host protein, DnaJA1/Hsp40, that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nuclei of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into the nucleus along with the nuclear import of the PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, which is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of the Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs.

Subproteomic tools to increase genome annotation complexity

Comprehensive and precise annotations of short protein‐coding genes are always a challenging task. Here we propose a new design to facilitate the characterization of previously overlooked short protein‐coding genes by integrating a shotgun proteomics method and oligonucleotide array analysis. Using Shigella flexneri as a model, we validate 163 annotated ORFs and 51 hypothetical or putative transcripts at the protein level, and discover four novel short ORFs. This strategy will contribute significantly to comprehensive and accurate genome‐wide annotation, and to our understanding of prokaryotic genome structure.

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein-protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

Proteogenomic Analysis of Trichophyton rubrum Aided by RNA Sequencing

Infections caused by dermatophytes, Trichophyton rubrum in particular, are among the most common diseases in humans. In this study, we present a proteogenomic analysis of T. rubrum based on whole-genome proteomics and RNA-Seq studies. We confirmed 4291 expressed proteins in T. rubrum and validated their annotated gene structures based on 35 874 supporting peptides. In addition, we identified 323 novel peptides (not present in the current annotated protein database of T. rubrum) that can be used to enhance current T. rubrum annotations. A total of 104 predicted genes supported by novel peptides were identified, and 127 gene models suggested by the novel peptides that conflicted with existing annotations were manually assigned based on transcriptomic evidence. RNA-Seq confirmed the validity of 95% of the total peptides. Our study provides evidence that confirms and improves the genome annotation of T. rubrum and represents the first survey of T. rubrum genome annotations based on experimental evidence. Additionally, our integrated proteomics and multisourced transcriptomics approach provides stronger evidence for annotation refinement than proteomic data alone, which helps to address the dilemma of one-hit wonders (uncertainties supported by only one peptide).

The first succinylome profile of Trichophyton rubrum reveals lysine succinylation on proteins involved in various key cellular processes

BackgroundDermatophytes, the most common cause of fungal infections, affect millions of individuals worldwide. They pose a major threat to public health because of the severity and longevity of infections caused by dermatophytes and their refractivity to therapy. Trichophyton rubrum (T. rubrum), the most common dermatophyte species, is a promising model organism for dermatophyte research. Post-translational modifications (PTMs) have been shown to be essential for many biological processes, particularly in the regulation of key cellular processes that contribute to pathogenicity. Although PTMs have important roles, little is known about their roles in T. rubrum and other dermatophytes. Succinylation is a new PTM that has recently been identified. In this study, we assessed the proteome-wide succinylation profile of T. rubrum. This study sought to systematically identify the succinylated sites and proteins in T. rubrum and to reveal the roles of succinylated proteins in various cellular processes as well as the differences in the succinylation profiles in different growth stages of the T. rubrum life cycle.ResultsA total of 569 succinylated lysine sites were identified in 284 proteins. These succinylated proteins are involved in various cellular processes, such as metabolism, translation and epigenetic regulation. Additionally, 24 proteins related to pathogenicity were found to be succinylated. Comparison of the succinylome at the conidia and mycelia stages revealed that most of the succinylated proteins and sites were growth-stage specific. In addition, the succinylation modifications on histone and ribosomal proteins were significantly different between these two growth stages. Moreover, the sequence features surrounding the succinylated sites were different in the two stages, thus indicating the specific recognition of succinyltransferases in each growth phase.ConclusionsIn this study, we explored the first T. rubrum succinylome, which is also the first PTM analysis of dermatophytes reported to date. These results revealed the major roles of the succinylated proteins involved in T. rubrum and the differences in the succinylomes between the two major growth stages. These findings should improve understanding of the physiological and pathogenic properties of dermatophytes and facilitate future development of novel drugs and therapeutics for treating superficial fungal infections.