## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Protozoa-Mycobacteria Symbiosis  
Similarly to pathogenic mycobacteria and their ability to evade phagolysosomes and escape the lytic activity exerted by mammalian cells, namely the macrophages, NTM have the extraordinary ability to survive phagocytosis by protozoa such as amoebae and additionally grow as endosymbionts (Figure 4) [154; 155; 156; 157; 158]. NTM are preferentially predated by protozoa since these microorganisms graze biofilms, the usual niche of NTM in both natural and engineered environments [154; 155; 156; 157; 158]. Several works have highlighted the association of free-living amoebae with mycobacteria, with _M. leprae_, _M. avium_,M. marinum, M. ulcerans, M. simiae, M. hubane, M. gordonae, M kansasi, and M. xenopi_ revealing the capacity of intra-amoebal survival inside vacuoles [159]. Work performed with _M. avium_ suggests that genes organized in a pathogenicity island (PI) are connected to the bacterial capacity of invading amoeba, once mutants lacking the PI exhibited defects in _Acanthamoeba castellanii_ invasion [160]. Moreover, _M. avium_ persistence inside amoeba is associated with the up-regulation of several genes involved in transcription regulation, metabolic pathways, degradation of macromolecules, and membrane proteins, with a few being also related to the survival process inside macrophages [161]. The cell surface hydrophobicity and composition are known to play a crucial role in phagocytosis by amoeba and protozoa, including in pathogens other than mycobacteria [162]. Indeed, co-culture works developed with _M. marinum_ and amoebae suggested the presence of anti-phagocytosis factors in the cell wall composition [163]. Furthermore, a recent work linked the action of three phosphatases (PtpA, PttpB, and SapM) to the mechanisms of intracellular replication of _M. marinum_ in amoeba, revealing their combination to reduce vacuole acidification and to enable escaping the vacuole towards the host cytosol [164]. For _M. abscessus_, the ESX-4 type VII secretion system has been implicated in resistance to amoeba phagocytosis, with mutants in _esx-4_ genes exhibiting a reduction in intra-amoeba replication [165]. Furthermore, a study analyzing the transcriptomic profile of _M. abscessus_ in amoeba revealed a group of 45 up-regulated genes that allow mycobacteria to resist environmental stress and induce defense mechanisms [166]. The possibility to grow in endosymbiosis with protozoa protect NTM from extreme environmental conditions, namely the pressure exerted by natural antimicrobial compounds and starvation, especially due to NTM survival in encystment structures [154; 155; 156; 157; 158]. The persistence within amoeba has been related to the increase in the infectivity and virulence of _M. avium_ and _M. abscessus_[167]. Additionally,  
Figure 4: Mycobacteria–protozoa symbiosis. Pathogenicity islands (PI) promote mycobacteria invasion into amoeba, leading to phagocytosis. Three outcomes can result from this phagocytosis process: mycobacteria evade the lysosome and multiply, causing amoeba lysis; mycobacteria cannot evade the lysosome, causing mycobacteria death; or a symbiotic relationship occurs, with phosphatases encoded by the _ptpA_, _ptpB_, _sapM_ genes being produced and excreted by the ESX-4 type VII secretion system. The symbiosis can lead to mycobacteria encystment or escapement from the amoeba. The information presented in this figure was combined from several studies regarding various non-tuberculous mycobacteria species. this symbiotic relationship also benefits the phagocytic protozoa, for instance in the _Tetrahymena_ spp. that acquire an evolutive advantage by ease access to lipids provided by intracellular NTM [168]. Although nowadays amoebae are recognized as an important reservoir [169], the underlying implications in the ecological, epidemiological, and public health patterns of NTM are unclear.