Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.
23Free-living amoebae might represent an evolutionary niche. In order to get more insights into 24 the potential amoebal training ground for Mycobacterium abscessus, we characterized its full 25 transcriptome in amoeba (Ac) and macrophages (Mf), as well as the Mycobacterium chelonae 26 intra-Ac transcriptome for comparison. Up-regulated genes in Ac allowed M. abscessus to 27 resist environmental stress and induce defense mechanisms, as well as showing switch from 28 carbohydrate carbon sources to fatty acid metabolism. Eleven genes implicated in the 29 adaptation to intracellular stress, were mutated, with all but one confirmed to be involved in 30 M. abscessus intra-Mf survival. Cloning two of these genes in M. chelonae increased its intra-31 Mf survival. One mutant was particularly attenuated in Mf that corresponded to the deletion 32 of an Eis N-acetyl transferase protein (MAB_4532c). Taken together, M. abscessus 33 transcriptomes revealed the intracellular lifestyle of the mycobacteria, with Ac largely 34 contributing to the enhancement of M. abscessus intra-Mf survival. such as Mycobacterium avium subsp paratuberculosis and M. avium subsp avium, 48Mycobacterium tuberculosis complex (MTBC) species and Mycobacterium leprae (Ahmed et 49 al., 2007; Stinear et al., 2008). 50Compared to other non-tuberculous mycobacteria (NTM), recovery of M. abscessus from the 51 environment is rare (Thomson et al., 2013). Information from its genome sequence indicates its 52 presence at the interface of soil, vegetation and water, an environment where free-living 53 amoebae (FLA) are commonly found (Ripoll et al., 2009). FLA have been isolated from habitats 54 in common with mycobacteria (Falkinham, 2009; Thomas and McDonnell, 2007) including 55 cold-drinking water systems (Eddyani et al., 2008; Thomas et al., 2006), hot water systems in 56 hospitals and cooling towers (Pagnier et al., 2008). FLA are ubiquitous organisms that feed on 57 bacteria, and these bacteria have likely developed adaptations to the intracellular lifestyle to 58 become amoeba-resistant bacteria (ARB) (Adékambi et al., 2006; Lamrabet et al., 2012). (White et al., 2010), allowing potential evolution toward pathogenicity by the acquisition 61 of virulence genes by horizontal gene transfer (Gutierrez et al., 2005; Ripoll et al., 2009). 62 2006)Finally, amoebae are often considered as an ancestral form of macrophages (Mf) sharing 63 similar cellular structures and biological features (Barker and Brown, 1994; Greub and Raoult, 64 2004; Siddiqui and Khan, 2012). 65 M. abscessus has been shown to be resistant to amoeba phagocytosis and encystment, a 66 property shared with all mycobacteria with the exception of the attenuated M. bovis BCG 67 vaccine strain (Adékambi et al., 2006; Salah et al., 2009; Bakala N'Goma et al., 2015). In 68 addition, co-culture of M. abscessus with Acanthamoeba castellanii (Ac) increases its virulence 69 when aerosolized in mice (Bakala N' Goma et al., 2015) suggesting the existence of an amoebal 70 'training ground' for opportun...
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