Identification of outer membrane proteins of Mycobacterium tuberculosis.
The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis.
Song
Houhui
H
Department of Microbiology, University of Alabama at Birmingham, 609 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294, USA.
Sandie
Reatha
R
Wang
Ying
Y
Andrade-Navarro
Miguel A
MA
Niederweis
Michael
M
D000465:Algorithms; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D015153:Blotting, Western; D002473:Cell Wall / Q000737:chemistry*; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D017433:Protein Structure, Secondary
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins