Membrane subproteomic analysis of <b><i>Mycobacterium bovis</i></b> bacillus Calmette‐Guérin

Zheng, Jianhua; Wei, Candong; Leng, Wenchuan; Jie, Dong; Li, Rui; Li, Weijun; Wang, Jin; Zhang, Zongde; Jin, Qi

doi:10.1002/pmic.200700342

Cited by 14 publications

(11 citation statements)

References 72 publications

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“…For example, GroES is necessary for the correct folding of a variety of proteins. Certain ribosomal proteins can serve as potent immunogens and have been applied in the skin's delayed-type hypersensitivity response (73).…”

Section: Mammalian Cell Entry Familymentioning

confidence: 99%

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

Zheng

Ren

Wei

et al. 2013

Molecular & Cellular Proteomics

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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-Gué rin (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 superna-

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Section: Mammalian Cell Entry Familymentioning

confidence: 99%

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

Zheng

Ren

Wei

et al. 2013

Molecular & Cellular Proteomics

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“…They have been proposed to be the main targets of ethambutol (EMB). Disruption of these proteins would inhibit the formation of the mycolyl-arabinogalactan-peptidoglycan complex and may lead to increased permeability of the cell wall and drug uptake [33]. Therefore, they may serve as targets for the development of therapeutic approaches against TB.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

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BackgroundTuberculosis 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.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.

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“…The distribution of these TMHs is shown in Figure 1. The grand average of hydropathy(GRAVY) value, which is used to evaluate the hydrophilicity and hydrophobicity of a protein along with its amino acid sequence[18], was minus 0.96. There are 21 proteins with GRAVY scores ≥ 0.4, which are so hydrophobic that they are susceptible to precipitation during isoelectric focusing and impossible to be detected by 2-DE.…”

Section: Resultsmentioning

confidence: 99%

Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155

Buck

2010

BMC Microbiology

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BackgroundThe usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis.ResultsA proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. smegmatis. An enzymatic cell surface shaving method was used to determine the surface-exposed proteins. As a result, a total of 390 cell wall proteins and 63 surface-exposed proteins were identified. Further analysis of the 390 cell wall proteins provided the theoretical molecular mass and pI distributions and determined that 26 proteins are shared with the surface-exposed proteome. Detailed information about functional classification, signal peptides and number of transmembrane domains are given next to discussing the identified transcriptional regulators, transport proteins and the proteins involved in lipid metabolism and cell division.ConclusionIn short, a comprehensive profile of the M. smegmatis cell wall subproteome is reported. The current research may help the identification of some valuable vaccine and drug target candidates and provide foundation for the future design of preventive, diagnostic, and therapeutic strategies against mycobacterial diseases.

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Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette‐Guérin

Cited by 14 publications

References 72 publications

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

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

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

Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155

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