An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine

183

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.

mentioning

confidence: 77%

General secretion signal for the mycobacterial type VII secretion pathway

Daleke

Ummels

Bawono³

et al. 2012

183

224

“…Therefore, it is reasonable to hypothesize that RD1 was the primary attenuating mutation, which resulted in the generation of BCG from M. bovis. An attempt to restore virulence to BCG with an RD1-complementing clone was only partially successful (10) although the deletion of the esat-6 gene from M. bovis resulted in an attenuated phenotype in guinea pigs (11). A recent study has also determined that deletion of RD1 from M. tuberculosis results in attenuation of virulence (12), but there has been no definitive study looking at the mechanism of attenuation or at the function of each of the genes that comprise RD1.…”

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

658

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...

“…The resulting phage ( WhiB3) was used for the transduction of H37Rv, as described (20). Disruption of whiB3 in M. bovis was accomplished by electroporating alkali-denatured pYUBwhiB into M. bovis ATCC35723 and identifying an allelic exchange mutant by Southern blotting in a way similar to that described for disruption of another gene (21).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

Steyn

Collins

Hondalus

et al. 2002

223

210

Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-5153 His) in the 4.2 domain of RpoV. We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M. tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-5153 His) allele. Infection of mice and guinea pigs with a M. tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model. In contrast, an M. bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs. However, we found that immunocompetent mice infected with the M. tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M. tuberculosis. Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection. Our results imply that M. tuberculosis replication per se is not a sufficient condition for virulence in vivo. They also indicate a different role for M. bovis and M. tuberculosis whiB3 genes in pathogenesis generated in different animal models. We propose that M. tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host.T he increased susceptibility of HIV-infected individuals and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) results in the death of 2-3 million people each year (1) and underscores the urgency of deciphering the molecular mechanisms of virulence of this pathogen. The highly variable protective efficacy of Mycobacterium bovis bacillus Calmette-Guérin in adults (0-80%; ref. 2) emphasizes the urgency for developing second-generation antituberculosis antimicrobial agents and vaccines. With these aims in mind, research stimulated by the advances in mycobacterial genetics (3, 4) has led to the identification of several genes that have been implicated in virulence (5-12).MTB requires sophisticated genetic mechanisms to recognize appropriate environmental signals and to convey this information to the transcriptional apparatus of the organism. The activation of bacterial sigma factors to regulate gene expression is an effective response mechanism that enables pathogens to respond instantly to a multitude of environmental signals. Bacterial 70 -type sigma factors are composed of four major regions, called regions 1, 2, 3 and 4 (13). Region 4 is subdivided further into sub regions 4.1 and 4.2; the latter is known to interact with the Ϫ35 region of promoters (13) and other transcription factors. Mutations in or close to the helix-turn-helix (HTH) motif in region 4.2 can result in either positive or negative effects on activation by transcri...