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Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

ABSTRACT: Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

SUBMITTER: Liu YC

PROVIDER: S-EPMC3335035 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Liu Yi-Chang YC Lin I-Hsuan IH Chung Wei-Jen WJ Hu Wensi S WS Ng Wailap Victor WV Lu Chi-Yu CY Huang Tsung-Yen TY Shu Hung-Wei HW Hsiao Kwang-Jen KJ Tsai Shih-Feng SF Chang Chuan-Hsiung CH Lin Chao-Hsiung CH

PloS one 20120420 4

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted prote ...[more]

PMID: 22536369

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Project description:The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two -10 boxes and a possible -35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both -10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.

Dataset Information

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Publications

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

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