Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

Abstract

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.Tuberculosis exacts enormous human suffering and death on a global basis and is also a widespread cause of animal morbidity and mortality. The primary human pathogen is Mycobacterium tuberculosis, although in many regions a significant amount of human disease results from infection with the very closely related organisms Mycobacterium africanum and the major animal pathogen Mycobacterium bovis. Together with a few less important species, these organisms are collectively referred to as the M. tuberculosis complex. The high degree of similarity in the pathogenesis of disease caused by these pathogens and the recent findings from genomic studies that the vast majority of genes within these species are identical or nearly so (16,22) clearly indicate that the majority of their virulence genes are shared in common. There is particular interest in discovering the identity of these genes, so that new strategies including improved vaccines and antibacterials can be developed for combating tuberculosis.Over the last 15 years, there has been continual development of molecular genetic techniques that can be applied to the M. tuberculosis complex (2). This enabled the discovery of individual tuberculosis virulence genes from 1995 onwards (7, 38) and led to the application over the last 5 years of a number of global techniques for identifying large numbers of genes that are essential for virulence or whose expression is altered during different stages of infection and disease. Among these global techniques, signature tag mutagenesis (18) is widely recognized as a powerful tool for investigating pathogenic organisms because of its ability to directly identify mutants that have reduced virulence (24). This identific...

Transposon Mutagenesis of Mb0100 at theppe1-nrpLocus inMycobacterium bovisDisrupts Phthiocerol Dimycocerosate (PDIM) and Glycosylphenol-PDIM Biosynthesis, Producing an Avirulent Strain with Vaccine Properties At Least Equal to Those ofM. bovisBCG

“…Whether or not a modified protocol can be devised that has sufficient resolving power awaits further experimentation. In addition, it is possible that a double- knockout mutant comprising a Mb0100 deletion and a deletion within another gene (11,12) may produce a vaccine strain that can be shown to be significantly better than BCG even using our present protocol.…”

“…Plasmids were propagated at 37°C in Escherichia coli XL1 Blue MR in Luria-Bertani broth or Luria-Bertani agar (Difco) supplemented with kanamycin (50 g/ml). M. bovis strains examined were the following: WAg200, a virulent wild-type isolate from New Zealand cattle, and its transposon mutant, WAg533; WAg201, also a virulent wild-type isolate from New Zealand cattle, and its transposon mutant, WAg537; and a signature tag mutant derived from WAg201, WAg579, that carries a disruption in pks15/1 (11). M. bovis wild-type and transposon mutant strains were cultured at 37°C in Middlebrook 7H9 (Difco) liquid medium and on Middlebrook 7H11 (Difco) solid medium as previously described (12).…”

“…5D for both wild-type strains but was not present in either of the transposon mutant strains, we utilized a pks15/1-disrupted M. bovis mutant (WAg579) derived from WAg201 by selection for reduced virulence in a guinea pig model of bovine tuberculosis (11). We expected this mutant to produce PDIMs but not produce glycosylphenol-PDIM, based upon results from a similar M. bovis BCG pks15/ 1-disrupted mutant (13).…”

“…Extensive wildlife culling operations over many years have failed to eliminate infected possums from many parts of the country, and vaccination of wildlife against tuberculosis is being investigated. Any vaccine developed for tuberculosis control in the New Zealand environment must be compatible with largescale vaccination of animals, and this requirement has directed research towards the development of rationally attenuated strains of M. bovis with vaccine efficacy (9,11,12). Moredetailed investigation of the attenuation of some of these strains (10, 38) is also contributing to our understanding of the molecular determinants required for tuberculosis pathogenesis.…”

The mycobacterial acyltransferase PapA5 is required for biosynthesis of cell wall-associated phenolic glycolipids

“…1). An overwhelming body of evidence has accumulated that demonstrates that PGLs and PDIMs play important roles in host-pathogen interaction and/or virulence in various mycobacterial pathogens (Alibaud et al, 2011; AstarieDequeker et al, 2009;Brodin et al, 2010;Camacho et al, 1999;Collins et al, 2005;Cox et al, 1999;Dhungel et al, 2008;Murry et al, 2009;Ng et al, 2000;Rambukkana et al, 2002; Reed et al, 2004;Robinson et al, 2008;Ruley et al, 2004;Sinsimer et al, 2008;Tsenova et al, 2005). Moreover, production of these compounds has been shown to correlate with decreased antimicrobial drug susceptibility (Alibaud et al, 2011;Chavadi et al, 2011b).…”