Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette‐Guérin Attenuation

649

750

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Gué rin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting ⌬RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis ⌬RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the ⌬RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Gué rin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. B acillus Calmette-Guérin (BCG) was first isolated fromMycobacterium bovis after serial subculturing in ox bile medium (1, 2), when Drs. Calmette and Guérin set out to test the hypothesis that a bovine tubercle bacillus could transmit pulmonary tuberculosis after oral administration (1, 3, 4). However, unexpectedly after the 39th passage, the strain was unable to kill experimental animals (1, 2), and showed no reversion to virulence even after the authors had performed over 200 passages (3), which is consistent with the attenuating mutation being a deletion mutation. In proceeding studies, BCG was determined to be able to protect animals receiving a lethal challenge of virulent tubercle bacilli (5), and in 1921 was first used as an anti-tuberculous vaccine (6). Presently, an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, yet the mechanism that causes the attenuation of BCG remains unknown.Mahairas et al. (6) first compared the genomic sequences of BCG and M. bovis, by using subtractive hybridization, and found that there were three regions of difference (designated RD1, RD2, and RD3) present in the genome of M. bovis, but missing in BCG. Behr et al. (7), and others (8), later identified 16 large deletions, including RD1-RD3, which were present in the Mycobacterium tuberculosis genome but absent in BCG. Eleven of these 16 deletions were unique to M. bovis whereas the remaining 5 del...

Section: B Acillus Calmette-guérin (Bcg) Was First Isolated Frommentioning

confidence: 99%

The primary mechanism of attenuation of bacillus Calmette–Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue

Hsu

Hingley‐Wilson

Chen

et al. 2003

649

750

“…The secreted proteins encoded within the locus are potent T cell antigens (7,10,11). More significantly, Mycobacterium tuberculosis strains with RD1 deletions are far less virulent in mice than strains that are otherwise isogenic (12)(13)(14). They replicate poorly and cause less pathology in both immunocompetent and immunocompromised mice, although the latter ultimately succumb to infection.…”

Section: Esat-6 Protein ͉ Mycobacterium Tuberculosis ͉ Virulence Factormentioning

confidence: 99%

Mutually dependent secretion of proteins required for mycobacterial virulence

Fortune

Jaeger²,

Sarracino³

et al. 2005

349

410

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.ESAT-6 protein ͉ Mycobacterium tuberculosis ͉ virulence factor I nteractions between bacterial pathogens and their hosts occur at the surface of cells. Many pathogenic bacteria secrete proteins involved in virulence (1). These proteins often interfere with normal host defenses and allow bacteria to survive and multiply. Strikingly, many of these virulence factors are secreted by nonclassical secretory systems that, in some cases, facilitate translocation of proteins across host cell membranes (2, 3).During the evolution of Mycobacterium bovis Bacille Calmette-Guérin (BCG), the attenuated bacillus widely used as vaccine against tuberculosis, a major deletion removed a set of nine ORFs (4). Similar deletions are found in Mycobacterium microti (5) and the Dassie bacillus (6), other relatively avirulent mycobacteria. The BCG deletion, known as RD1, includes two genes that encode the abundantly secreted proteins, ESAT-6 and CFP-10. The RD1 deletion encompasses most but not all of a larger, 15-gene region known as ESX-1 (7), which is hypothesized to encode an alternative secretory system (8, 9).The proteins encoded by the ESX-1 locus clearly play an important role in virulence. The secreted proteins encoded within the locus are potent T cell antigens (7, 10, 11). More significantly, Mycobacterium tuberculosis strains with RD1 deletions are far less virulent in mice than strains that are otherwise isogenic (12-14). They replicate poorly and cause less pathology in both immunocompetent and immunocompromised mice, although the latter ultimately succumb to infection. RD1-deleted strains of Mycobacterium marinum elicit only very modest macrophage aggregation and granuloma formation in zebrafish (15). Additional work suggests the RD1-encoded proteins may disrupt host cell membranes. In M. tuberculosis, these proteins may be required for lysis of infected cells (13) and c...

“…For instance, the primary cause of attenuation of the Mycobacterium bovis bacille Calmette-Guérin vaccine strain is attributed to a deletion of nine genes from the ESX-1 locus (7,16,17). Consistently, deletion of ESX-1 genes from the genomes of M. tuberculosis or M. bovis results in diminished virulence (18,19). Of the other ESX loci, both ESX-3, which is involved in iron acquisition, and ESX-5 have been demonstrated to encode functional secretion systems (20)(21)(22).…”

mentioning

confidence: 99%

General secretion signal for the mycobacterial type VII secretion pathway

Daleke

Ummels

Bawono³

et al. 2012

178

224

Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes.