A specific secretion system mediates PPE41 transport in pathogenic mycobacteria

Mycobacterial genomes contain two unique gene families, the so‐called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT‐6‐like secretion system. This cluster, designated ESX‐5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX‐5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX‐5 mutant of M. marinum was affected in spreading to uninfected macrophages.

[1]  T. Parish,et al.  Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. , 2003, FEMS microbiology letters.

[2]  M. Chase,et al.  Mutually dependent secretion of proteins required for mycobacterial virulence. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. Hasnain,et al.  PPE Antigen Rv2430c of Mycobacterium tuberculosis Induces a Strong B-Cell Response , 2003, Infection and Immunity.

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

[5]  R M Warren,et al.  Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c. , 2001, Tuberculosis.

[6]  B. Gicquel,et al.  A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences. , 1998, FEMS microbiology letters.

[7]  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.

[8]  Martin Phillips,et al.  Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Ottenhoff,et al.  Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance , 2001, Cellular microbiology.

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

[11]  S. Falkow,et al.  Mycobacterium marinum persists in cultured mammalian cells in a temperature-restricted fashion , 1994, Infection and immunity.

[12]  W. Jacobs,et al.  Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis , 1990, Molecular microbiology.

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

[14]  K. Papavinasasundaram,et al.  A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. , 2000, Microbiology.

[15]  Y. Akhter,et al.  Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: Evidence that PE Rv2431c is co‐transcribed with PPE Rv2430c and their gene products interact with each other , 2006, FEBS letters.

[16]  A. Kolk,et al.  Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. , 1984, Clinical and experimental immunology.

[17]  T. Hirst,et al.  Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin. , 1999, Vaccine.

[18]  Eugene Rosenberg,et al.  Adherence of bacteria to hydrocarbons: A simple method for measuring cell‐surface hydrophobicity , 1980 .

[19]  Martin Wu,et al.  A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice , 2004, Cellular microbiology.

[20]  Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6 , 2004, Infection and Immunity.

[21]  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.

[22]  Wilbert Bitter,et al.  Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections , 2003, Cellular microbiology.

[23]  E. Rubin,et al.  Genes required for mycobacterial growth defined by high density mutagenesis , 2003, Molecular microbiology.

[24]  W. Jacobs,et al.  Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Brennan,et al.  The PE multigene family: a 'molecular mantra' for mycobacteria. , 2002, Trends in microbiology.

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

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

[28]  Liem Nguyen,et al.  Protein Kinase G from Pathogenic Mycobacteria Promotes Survival Within Macrophages , 2004, Science.

[29]  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.

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

[31]  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.

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

[33]  M. Hattori,et al.  The complete genomic sequence of Nocardia farcinica IFM 10152. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[34]  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.

[35]  Karl W. Broman,et al.  A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: Application to Mycobacterium tuberculosis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  C. Vandenbroucke-Grauls,et al.  Mycobacterium marinum Strains Can Be Divided into Two Distinct Types Based on Genetic Diversity and Virulence , 2004, Infection and Immunity.

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