Abstract
Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.
Highlights
Mycobacterium abscessus subsp. abscessus (M. abscessus) is an emerging pathogen increasingly recognized as a serious threat to cystic fibrosis (CF) patients who show a marked vulnerability to infections with this bacterium, which is exacerbated by M. abscessus’s extraordinary intrinsic tolerance toward many antibiotics
These results suggest (i) that homologous recombination occurs at a higher frequency in M. abscessus than that reported for other commonly investigated mycobacterial species, such as M. smegmatis (Pavelka and Jacobs, 1999), M. bovis BCG (Sander et al, 2001) and M. tuberculosis (Parish et al, 1999) and (ii) that illegitimate recombination events between pUX1 and the M. abscessus chromosome occurs at a very low level or not at all
In comparison to the large number of studies dedicated to delineating the pathophysiology of M. tuberculosis, nontuberculous mycobacteria (NTM) have been largely neglected as pathological organisms
Summary
Mycobacterium abscessus subsp. abscessus (M. abscessus) is an emerging pathogen increasingly recognized as a serious threat to cystic fibrosis (CF) patients who show a marked vulnerability to infections with this bacterium, which is exacerbated by M. abscessus’s extraordinary intrinsic tolerance toward many antibiotics (van Dorn, 2017). New approaches to combat M. abscessus infections may rely on the discovery of completely new chemical entities that inhibit essential pathways in this bacterium (Viljoen et al, 2017) This may involve the improvement of available compounds to better inhibit putative molecular targets in M. abscessus, which may have slightly different structures or functions compared to orthologs efficiently inhibited in other bacteria. M. abscessus shares many virulence features of M. tuberculosis (Ripoll et al, 2009; Choo et al, 2014), recent studies have highlighted common genetic vulnerabilities and drug resistance mechanisms between the two bacteria involving unrelated compounds (Dupont et al, 2016; Halloum et al, 2017; Kozikowski et al, 2017) These data further warrant studies aimed at deciphering the physiology of M. abscessus from the genetic level upwards. This has led to the identification of a yet small number of genes that are downright essential for virulence or intracellular survival (Bernut et al, 2014, 2016; Halloum et al, 2016) or playing more important roles during certain stages of infection, such as the establishment of infection (Bakala N’Goma et al, 2015) or during the chronic stage of infection (Viljoen et al, 2016)
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