Construction of Mycobacterium abscessus Defined Glycopeptidolipid Mutants: Comparison of Genetic Tools

Abstract

Mycobacterium abscessus is a rapidly growing mycobacterial species that can be involved in pulmonary and disseminated infections in immunosuppressed or young cystic fibrosis patients. It is an emerging pathogen and has attracted recent attention due to the numerous cases of infection; furthermore, genomic tools have been developed for this species. Nevertheless, the study of this species has until now been limited to spontaneous variants. We report here a comparison of three different mutagenesis systems-the ts-sacB, the phage, and the recombineering systems-and show that there are important differences in their efficiency for the construction of allelic-exchange mutants. We show, using the mmpL4b gene of the glycopeptidolipid pathway as a target, that allelic-exchange mutants can be constructed with a reasonable efficiency (ϳ7%) using the recombineering system. These observations will facilitate genetic and cellular microbiology experiments involving the construction and use of well-defined mutants to study the virulence determinant of this emerging pathogen.The mycobacterial genus contains plethora of species that are pathogenic for either humans or animals. The most wellknown are undoubtedly Mycobacterium leprae, M. tuberculosis, and M. ulcerans, the etiologic agents of human leprosy, tuberculosis, and Buruli ulcer, respectively (47-49). M. avium subsp. paratuberculosis, responsible for Johnes disease in ruminants, is also a serious health concern since it is suspected to be a threat to human via infected milk (9, 10). M. abscessus is an emerging pathogen involved in pulmonary and disseminated infection in young cystic fibrosis patients (26,36). M. abscessus can cause nosocomial infections of skin and soft tissues in immunosuppressed patients (28,35). It is also able to cross the blood-brain barrier and to cause meningoencephalitis (42). M. abscessus is phylogenetically related to M. chelonae and, indeed, these species have long been grouped together under the designation of the "M. abscessus-chelonae complex" (6). M. abscessus is a rapid grower that forms colonies in 5 days. Like other mycobacterial species, M. abscessus is equipped with a robust waxy cell wall that, as in other species, probably contributes to virulence (12). The emerging and growing interest in M. abscessus has led to its genome being sequenced (accession no. NC010397) (F. Ripoll et al., unpublished data) and to the development of DNA microarrays (Jean-Yves Coppée, unpublished data).The availability of genomic resources and animal models (32) makes M. abscessus a very attractive system. However, there is no defined mutagenesis system for this species and, to the best of our knowledge, no defined mutants have been constructed thus far. The consequence is that the study of this organism has been restricted to spontaneous variants. Utilization of spontaneous mutants has, nevertheless, allowed the characterization of morphotypically rough isolates that are hypervirulent both in vitro and in vivo (7,8,17). These rough isolates are low glycopep...

Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response

“…We have provided evidence that expression of GPL facilitates the M. abscessus colonization phenotype, but 'masks' underlying bioactive cell-wall lipids involved in virulence (Rhoades et al, 2009). In this study we have used genetic recombineering to delete the M. abscessus 390S mmpL4b gene, a gene coding for a membrane protein which has been found to play an essential role in GPL expression by NTM (Recht et al, 2000;Ripoll et al, 2007;Medjahed & Reyrat, 2009). Using this deletion mutant we provide direct evidence that GPL plays a role in both the colonization and invasion phenotypes of M. abscessus.…”

galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus

“…The key to this is a suitable counterselectable marker, such as sacB (conferring sucrose sensitivity), rpsL (conferring streptomycin susceptibility in a streptomycin-resistant background) or galK (conferring 2-deoxygalactose susceptibility) [14,[27][28][29]. Others have shown that sacB does not work in M. abscessus, and the rpsL system requires a pre-existing streptomycin resistance mutation in the choromosomal copy of rpsL, which complicates strain construction [16,23,29]. Additionally, in preliminary experiments (not shown), we found it difficult to isolate streptomycin-resistant mutants that could be used with our previously developed rpsL suicide vectors.…”

“…Unfortunately, M. abscessus is refractory to allelic exchange using the TM4 mycobacteriophage-based specialized transduction system [16]. Electroporation of dsDNA can work, either without [17] or with the expression of recombineering proteins [16,18], but the efficiency is low [16].…”

“…Unfortunately, M. abscessus is refractory to allelic exchange using the TM4 mycobacteriophage-based specialized transduction system [16]. Electroporation of dsDNA can work, either without [17] or with the expression of recombineering proteins [16,18], but the efficiency is low [16]. In addition, the specialized transduction or dsDNA electroporation methods cannot easily be used for allelic exchange of unmarked mutations such as point mutations, although they can be used with mutations marked with resolvable resistance markers [13,19].…”

“…Hence, the preferred method is a suicide vector system with a counterselectable marker, since it can be used for both marked and unmarked mutant alleles, either in-frame deletions or point mutations. A commonly used counterselectable marker in mycobacteria, sacB, which confers susceptibility to sucrose, does not work in M. abscessus for allelic exchange [16]. A recent report described the use of isoniazid counterselection for allelic exchange in M. abscessus [20].…”