RESUMO
The ESX-1 locus is a region critical for full virulence in Mycobacterium tuberculosis, which encodes two secreted proteins as well as other genes involved in their secretion. The mechanism of secretion of the two proteins, ESAT-6 and CFP-10, and their function remain unknown. Using proteomic methods to search for additional proteins secreted by the ESX-1 locus, we discovered that a protein encoded by a chromosomally unlinked gene, espA, is also secreted by strains that contain the ESX-1 locus but not by strains with ESX-1 deletions. Mutations in individual ESX-1 genes, including those that encode ESAT-6 and CFP-10, were found to block EspA secretion. Surprisingly, mutants that lack espA reciprocally failed to secrete ESAT-6 and CFP-10 and were as attenuated as ESX-1 mutants in virulence assays. The results indicate that secretion of these proteins, which are each critical for virulence of pathogenic mycobacteria, is mutually dependent. The results further suggest that discerning the nature of the interaction and the structure of macromolecular complexes will provide insights into both an alternative mechanism of protein secretion and mycobacterial virulence.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Western Blotting , Vetores Genéticos/genética , Espectrometria de Massas , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Proteômica , VirulênciaRESUMO
An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.