A non‐RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis

The Snm secretion system is a crucial virulence determinant of Mycobacterium tuberculosis. Genes encoding all known components of this alternative secretion pathway are clustered at the same genetic locus, known as RD1. Here, we show that a mutant M. tuberculosis strain containing a transposon insertion in the Rv3615c gene, which is situated outside the RD1 locus, results in loss of Snm secretion. Complementation analysis revealed that both Rv3615c and the downstream gene Rv3614c are required for Snm secretion. Thus, we have renamed the two genes snm9 and snm10 respectively. The snm9::Tn mutant phenocopies bona fide snm mutants, exhibiting attenuation in mice, macrophage growth defects and failure to suppress cytokine induction. Furthermore, yeast two‐hybrid analysis revealed a physical interaction between Snm10 and Snm7 (Rv3882c), suggesting that Snm10 may function in complex with other Snm proteins during secretion. Thus, snm9 and snm10 are the first genes located outside the RD1 locus identified as critical components of Snm secretion. These data indicate that Snm secretion consists of an elaborate network of interactions that likely arose from multiple  duplication  events  during  the  evolution  of M.  tuberculosis.

[1]  S. Cole,et al.  Influence of ESAT-6 Secretion System 1 (RD1) of Mycobacterium tuberculosis on the Interaction between Mycobacteria and the Host Immune System1 , 2005, The Journal of Immunology.

[2]  J. Cox,et al.  A Protein Secretion Pathway Critical for Mycobacterium tuberculosis Virulence Is Conserved and Functional in Mycobacterium smegmatis , 2005, Journal of bacteriology.

[3]  D. Missiakas,et al.  EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Sherman,et al.  Tuberculous Granuloma Formation Is Enhanced by a Mycobacterium Virulence Determinant , 2004, PLoS biology.

[5]  S. H. Kaufmann,et al.  CFP10 discriminates between nonacetylated and acetylated ESAT‐6 of Mycobacterium tuberculosis by differential interaction , 2004, Proteomics.

[6]  J. Engel,et al.  A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT‐6 secretion , 2004, Molecular microbiology.

[7]  K. Derbyshire,et al.  The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Palmer,et al.  Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis , 2004, Clinical Diagnostic Laboratory Immunology.

[9]  P. Andersen,et al.  Protein-Protein Interactions of Proteins from the ESAT-6 Family of Mycobacterium tuberculosis , 2004, Journal of bacteriology.

[10]  D. Sherman,et al.  Individual RD1‐region genes are required for export of ESAT‐6/CFP‐10 and for virulence of Mycobacterium tuberculosis , 2004, Molecular microbiology.

[11]  Eugene V Koonin,et al.  Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging. , 2004, Nucleic acids research.

[12]  Christopher M. Sassetti,et al.  Genetic requirements for mycobacterial survival during infection , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  S. Raghavan,et al.  Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Eisenberg,et al.  The primary mechanism of attenuation of bacillus Calmette–Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Sherman,et al.  Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation. , 2003, The Journal of infectious diseases.

[16]  Priscille Brodin,et al.  Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti , 2002, Molecular microbiology.

[17]  Thomas M. Shinnick,et al.  Microarray Analysis of the Mycobacterium tuberculosis Transcriptional Response to the Acidic Conditions Found in Phagosomes , 2002, Journal of bacteriology.

[18]  Gary K. Schoolnik,et al.  ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response , 2002, Infection and Immunity.

[19]  S. Gordon,et al.  Conclusive Evidence That the Major T-cell Antigens of theMycobacterium tuberculosis Complex ESAT-6 and CFP-10 Form a Tight, 1:1 Complex and Characterization of the Structural Properties of ESAT-6, CFP-10, and the ESAT-6·CFP-10 Complex , 2002, The Journal of Biological Chemistry.

[20]  M. Pallen.  The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system? , 2002, Trends in microbiology.

[21]  S. Gordon,et al.  Conclusive Evidence That the Major T-cell Antigens of the Mycobacterium tuberculosis Complex ESAT-6 and CFP-10 Form a Tight, 1:1 Complex and Characterization of the Structural Properties of ESAT-6, CFP-10, and the ESAT-6 CFP-10 Complex IMPLICATIONS FOR PATHOGENESIS AND VIRULENCE* , 2002 .

[22]  R. Siezen,et al.  The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria , 2001, Genome Biology.

[23]  O. Schneewind,et al.  Protein secretion and the pathogenesis of bacterial infections. , 2001, Genes & development.

[24]  D. Collins,et al.  An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine. , 2000, Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[25]  William R. Jacobs,et al.  Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice , 1999, Nature.

[26]  C. Dye,et al.  Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. , 1999, JAMA.

[27]  G. Schoolnik,et al.  Comparative genomics of BCG vaccines by whole-genome DNA microarray. , 1999, Science.

[28]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[29]  G. Mahairas,et al.  Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis , 1996, Journal of bacteriology.

[30]  L. Guarente,et al.  Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. , 1981, Proceedings of the National Academy of Sciences of the United States of America.