Corresponding author and reprint requests: M. Drancourt, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Faculté de Médecine-27, Boulevard Jean Moulin, Marseille, Cedex 5, France E-mail: Michel.Drancourt@univmed.fr
Mycobacterium species evolved from an environmental recent common ancestor by reductive evolution and lateral gene transfer. Strategies selected through evolution and developed by mycobacteria resulted in resistance to predation by environmental unicellular protists, including free-living amoebae. Indeed, mycobacteria are isolated from the same soil and water environments as are amoebae, and experimental models using Acanthamoeba spp. and Dictyostelium discoideum were exploited to analyse the mechanisms for intracellular survival. Most of these mechanisms have been further reproduced in macrophages for mycobacteria regarded as opportunistic and obligate pathogens. Amoebal cysts may protect intracellular mycobacteria against adverse conditions and may act as a vector for mycobacteria. The latter hypothesis warrants further environmental and clinical studies to better assess the role of free-living amoebae in the epidemiology of infections caused by mycobacteria.
Mycobacteria Evolved from an Environmental Ancestor
Mycobacteria are mycolic acid-containing, high guanine-cytosine-content bacterial organisms belonging to the Actinobacteria phylum . Comparative genomics indicated that the most recent common ancestor of Mycobacteria was an environmental species [2,3] which evolved towards soil-borne mycobacteria, waterborne mycobacteria such as Mycobacterium avium, Mycobacterium intracellulare, and host-associated species including M. avium subsp. paratuberculosis and Mycobacterium avium subsp. avium , Mycobacterium tuberculosis complex (MTC) species, and Mycobacterium leprae. Some species retained genes for dual lifestyle as in the case of Mycobacterium marinum . In the latter species, genome synteny and size are more related to the saprophyte Mycobacterium smegmatis than to M. tuberculosis, despite the fact that M. marinum and M. tuberculosis share 80% of coding sequences . This evolution mainly relied upon irreversible deletions of genomic fragments, illustrated by downsizing of the genome from environmental, generalist mycobacteria (Mycobacterium vanbaalenii genome, 6.49 Mb) towards host-associated, specialized species (M. leprae genome, 3.26 Mb) (Table 1). Current genomic data suggest that reductive evolution occurred independently in several Mycobacterium phyla . Lateral gene transfer has also been demonstrated in some Mycobacterium species such as the M. tuberculosis complex  and Mycobacterium abscessus . Transfer of the IS6110 insertion sequence between Mycobacterium smegmatis and the M. tuberculosis complex species, and its discovery in another environmental Mycobacterium sp. strain JLS, suggested that these species once co-occupied a single environmental niche . Reductive evolution of specialized mycobacteria is illustrated by the agent of Buruli ulcer, Mycobacterium ulcerans, which derived from an ancestor in common with M. marinum after genome size reduction from 6.63 to 5.63 Mb  and the acquisition of a 174-kb plasmid encoding a polyketide toxin mycolactone . Of 140 species currently described in this genus, 133 (95%) are environmental organisms found in soil , air , and water [12–14], whereas seven are host-associated organisms (http://www.bacterio.cict.fr/m/mycobacterium.html).
Table 1. Comparative genome evolution of Mycobacterium sp.
In water and in soil, the most recent common ancestor of Mycobacteria may have encountered free-living predators including free-living amoebae (FLA). The classification of FLA is mainly based on 18S rRNA gene sequencing (Fig. 1). These organisms are cell-wall free, unicellular eukaryotes that switch from a motile trophozoite phase towards an immobile cyst phase (Fig. 2) during starvation, drying, hypoxia and fluctuation in temperature. Trophozoites are professional phagocytes that engulf any particle with a diameter ≥0.5 μm into phagocytic vacuole with further lysosome fusion and destruction. Mycobacteria have evolved mechanisms to withdraw and resist such unicellular protist predators, including secreted toxins, and the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. Mycobacteria gained the advantage of being protected from adverse conditions by the amoebal cyst. Encystement consists in the formation of a cell wall comprising one layer as in the case of Entamoeba, two layers as in the case of Acanthamoeba and Hartmannella vermiformis , and an even more complex structure as in the case of Giardia . In Acanthamoeba, the formation of the first layer (i.e. ectocyst) results in an immature cyst, and further apposition of a second, internal layer (i.e. endocyst) forms a mature cyst. The cell wall is composed of polysaccharides with β-configuration, which are cellulose in the case of Acantamoeba cysts  (Fig. 3). Cysts are resistant to alterations in osmolarity and pH, desiccation, freezing, high concentration of hydrochloric acid, moist heat, chemical antimicrobial agents and biocides [18,19]. Moreover, cysts have been shown to survive in the laboratory for at least 20 years . Excystment refers to the process by which trophozoites emerge through ostioles of the amoebal cyst, after the mechanical expulsion of the opercalum  (Fig. 2) and requires the action of cellulase of the Glycosyl Hydrolase family 5 as in Dictyostelium discodium (Glycosyl Hydrolase family 9). Both encystement and excystement were found to be dependant on serine protease function .
During their evolution from the MRCA, mycobacteria have evolved strategies to avoid predation by FLA and these strategies have been further used to resist macrophage destruction, which is a key step for pathogenicity.
Mycobacteria- Free Living Amoebae Interactions (Table 2)
Table 2. Environmental Mycobacterium species isolated by amoebal coculture
FLA have been isolated from habitats in common with mycobacteria [23,24] including cold drinking water systems [25,26], hot water systems in hospitals , cooling towers , and contact lens storage cases . Direct observation of mycobacteria within amoebae collected in such environments has not been reported; however, mycobacteria have been isolated from FLA collected in such environments, including the isolation of 13 different Mycobacterium species in parallel to amoebae related to Hartmannella, Echinamoeba, Acanthamoeba and Naegleria . In a hospital water network, the presence of FLA including H. vermiformis, Acanthamoeba polyphaga, and an unidentified protist was significantly correlated with the presence of mycobacteria in the same water specimen, including Mycobacterium gordonae, M. xenopi and M. kansasii subtype 1 . In Benin, ten different Mycobacterium species, but not Mycobacterium ulcerans have been isolated from protected and unprotected water sources in which six different species of Acanthamoeba, Naegleria and Tetramitus amoeba have been detected  (Table 2). In most studies, waterborne mycobacteria were isolated by co-culturing a specimen with A. polyphaga [23,28,30] as in the case of soil mycobacteria . The same approach has been used to isolate Mycobacterium massiliense from sputum  and M. avium subsp. paratuberculosis from colon biopsies collected from patients with Crohn’s disease .
Experimental studies confirmed the reality of FLA-mycobacteria interactions. These studies used waterborne amoebae, including Acanthamoeba polyphaga [33–36], Acanthamoeba castellanii [37–41], Hartamnella vermiformis [42,43], and the social soil amoeba Dictyostelium discodium [44–48]. The ciliated protozoan Tetrahymena pyriformis has also been used to study the intracellular growth of the M. avium complex species . Amoeba models allowed the study of surface receptors , phagocytosis , and bactericidal mechanisms , using organisms easy to cultivate with short generation times, for which specific genetic, biochemical and cell biological tools were available. Acanthamoeba organisms are less well characterized genetically than D. discodium, whose genome is available , but they remain viable and grow at temperature above 25°C (32–37°C), in the temperature range for natural mycobacteria-macrophage interactions . Therefore, Acanthamoeba is an important member of the FLA and has served as a model organism to study the in vitro interactions with bacteria; for eukaryotic transcription in vitro, and more recently in vivo, this model facilitates the study of cytoskeletal and mitochondrial functions . Variable multiplicity of infection (MOI) from 1:100  to 1:10 [45,54] is most often used with removal of extra-amoebal mycobacteria using buffer , buffer in addition to antibiotic such as amikacin , streptomycin  or HCl 3%  (Table 3). After excystment the extraction of an intra-amoebal fraction with SDS 0.5% and culture on standard medium made it possible to obtain evidence of intracystic survival. Intra-amoebal multiplication could be measured by microscopic counting of intra-amoebal mycobacteria after Ziehl  (Fig. 4) or fluorescent acid-fast staining , electron microscopy counting  (Fig. 5), counting of mycobacteria colonies, or real-time PCR.
Table 3. Experimental model of interaction between mycobacteria and protists
Evidences of Intra-amoebal
Extra-cellular mycobacteria treatment
Am, Amikacine (100 μg/2 h); NR, Not Reported; CFU, Colony Forming Unity; M. a.a, Mycobacterium avium subsp. avium; M. a .p, Mycobacterium avium subsp. paratuberculosis; EM, Electronic Microscopy; LM, Light Microscopy; ZN, Zhiel Nelseen staining; FAM, Fluorescent acid-fast microscopy.
All the Mycobacterium species under study have been shown to be phagocytosed and to penetrate amoebal trophozoites into vacuoles, including M. tuberculosis , M. bovis and BCG strains , M. leprae [41,56], M. marinum [37,47], M. avium [34,37,45], M. avium subsp. paratuberculosis [33,54], Mycobacterium kansasii , M. xenopi , Mycobacterium fortuitum , M. smegmatis [37,38], and 26 additional non-tuberculous species  (Table 3). All these Mycobacterium species have been shown to persist in trophozoites, with the exceptions of M. smegmatis [37,38] and the two BCG strains (Pasteur and Japan) of M. bovis, which were killed within A. castellanii trophozoites, contrary to the M. bovis parent strain . After phagocytosis, FLA-resisting mycobacteria grow inside trophozoites, as demonstrated for M. avium [34,37], M. avium subsp. paratuberculosis , M. fortuitum , M. marinum , M. xenopi . Intra-amoebal growth may depend on the temperature because M. avium and M. fortuitum grew and replicated equally at 32°C and 37°C whereas M. marinum replicated only at 32°C and was killed at higher temperatures . Only a portion of the inoculum may adapt and survive within trophozoites, as shown for M. avium subsp. paratuberculosis . Likewise, the replication fate of M. avium within A. castellanii was found to vary with strains . Moreover, it has been shown that inta-amoebal growth of M. avium increased its entry and replication within both the epithelial cell line HT-29 and human monocyte-derived macrophages, as well as its virulence in a mice model . Likewise, the level of inta-amoebal replication of M. kansasii within A. castellanii correlated with the virulence of strains, regardless of the genetic subtype . Measurement of intra-amoebal growth could be used as a test predictive of virulence for this opportunistic pathogen. Finally, a parachamber experiment indicated that M. avium may replicate saprozoically on products secreted by amoeba .
In further experiments, a few Mycobacterium species were shown to survive within the amoebal cyst, as in the case of M. xenopi , M. avium subsp. paratuberculosis , and 26 additional non-tuberculous species . Likewise, it has been suggested that M. bovis was able to survive encystement . Interestingly, these mycobacteria were observed in the cyst wall and, more precisely, the exocyst layer of the cyst. An increased oxygen uptake during the first 10 h of Hartamnella encystment has been shown; subsequently, oxygen consumption gradually decreased to an immeasurable value when the cyst formation was complete . In fact, a strict hypoxic condition can be tolerated by some free-living protists but many of them tolerate a very low level of oxygen. For example, it has been shown that the strict, non-sporulated bacterium Mobiluncus curtisii was able to grow within Acanthamoeba culbertsoni and other Acanthamoeba strains which were maintained under an aerobic atmosphere . This anaerobic condition during a long period of time (more than 20 years) may be favourable to intracellular mycobacteria, which could survive in dormancy, and the amoebal cyst could be used as a model to study this particular state of mycobacteria. Indeed, despite the fact that M. tuberculosis is an aerobe bacterium, it can survive extended periods of anaerobiosis by induction of over 60 genes known as the dormancy regulon, also activated during human latent infection .
Free-living Amoebae: Models for Macrophage-Mycobacterium Interactions
The observation of FLA-mycobacteria interactions contributed to the understanding of the mechanisms that sustain the resistance of mycobacteria to the human macrophage used to study host-mycobacteria interactions [60,61]. Regarding the entry of mycobacteria using the complement receptors CR1, CR3 and CR4, and the manose receptor, it has been observed that M. avium mutants lacking a pathogenicity island exhibited highly similar defects in invading A. castellanii and human macrophages . In this model, 20 genes initially found to be up-regulated in trophozoites, were further found to be also up-regulated in human macrophages; these genes encoded metabolic pathways, protein transcription and translation and macromolecule degradation . Also, M. xenopi was found to survive and to multiply within A. polyphaga trophozoites  and was found to survive and to replicate, ten-fold in 48 h, within human peripheral macrophages . Likewise, it was observed that A. castellanii phagosomes containing M. smegmatis fused with lysosomes as soon as 5 min post-inoculation, whereas the majority of mycobacteria are partially degraded 30 min post-inoculation in the absence of persistence . This has been confirmed in a further experiment indicating that a double mutant lacking the mspA and mspC porin genes exhibited enhanced persistence, demonstrating the role of mycobacteria outer membrane permeability in intracellular persistence .
Survival within amoebal trophozoites may train environmental mycobacterial species to adapt and survive within the hostile human macrophages by avoiding degradation by the host cell lysosome. After pathogen uptake by phagocytosis, the host cell enters into lag phase that reflects the early and active transformation of phagosomes containing pathogens into real replication niches, implicating the protein MAG24  (Fig. 6) (Table 4). M. tuberculosis, M. bovis, M. marinum and M. avium have been shown to prevent the maturation of the phagosome into a phagolysosome, thus residing in a replicative early phagosome  .
Table 4. Molecular determinants implicated in Mycobacterium-amoeba interactions. D. discodium: Dictyostelium discodium
II. Mycobacterial determinants
Rise of cell wall Permeability → Reduce intracellular persistence
Fine electron microscopy observations indicated that mycobacteria prevented phagosome maturation only when they established and maintained a close apposition with the phagosome membrane, which is probably required for the inhibition of the phagosome conversion , a situation seen only when mycobacteria enter as isolated, non-clumped organisms . In D. discoideum, phagosome maturation takes place 90 min after uptake of mycobacteria, and includes a sequence of events beginning with the accumulation of vacuolar H+ATPase (VatA), the endosomal protein p80, and the vacuolin (VacB)  (Table 4). Further gradual accumulation of VacB surrounding cathepsin D is followed by morphological changes in the replication niches and enhanced proliferation of the pathogen . In these experiments, M. marinum has been used as a model organism for M. tuberculosis , being closely related . In the course of M. marinum infection, granulomas are indistinguishable from those caused by M. tuberculosis  and the intracellular proliferation niches of both organisms are very similar . Several M. marinum virulence mutants have phenotypes similar to M. tuberculosis mutants and can be completed by M. tuberculosis genes [71–73]. The most prominent characteristics of the M. tuberculosis phagosome are its incomplete luminal acidification  by exclusion of the vacuolar proton H+-ATPase  and the absence of mature lysosomal hydrolases. The accessibility to transferrin-bound iron  and trafficking of transferrin receptors through mycobacterial phagosome  indicate that the mycobacterial phagosome is not a static organelle, despite the blockade in the acquisition of lysosomal compartments. Iron supply by the transferrin is required for Mycobacteria replication . M. tuberculosis manipulates the small rab GTPase function to colonize the host . Maturation of mycobacterium-containing phagosomes is blocked at the Rab5-positive stage (early phagosome). Contrary to the retention of the small GTPase Rab5, mycobacterial phagosomes do not recruit Rab7, a molecule required for phagosome conversion. Rab5 and Rab7 are central players in the biogenesis of the endosomal pathway . Because phagosome maturation is blocked at the Rab5-stage, Rab5 effectors, which are involved in the phagosome maturation process, are recruited into mycobacterial phagosomes, with the notable exception of a Rab5 effector early endosomal autoantigen 1 (EEA1) [79,80].
Moreover, it has been shown that phosphatidylinositol 3-phosphate (PI3P), a critical regulator of Rab5 effector recruitment, such as EEA1 , is reduced or absent in phagosomes containing M. tuberculosis . PI3P is generated by the cellular enzyme VPS34, a type III phosphatidylinositol 3-kinase (PI3K) . PI3P generation is inhibited by two M. tuberculosis products, including the lipid liparabinomannan (LAM)  and a PI3P phosphatase SapM . The inhibition of PI3P generation by LAM and SapM represents a double mechanism required to ‘hijack’ the maturation of the M. tuberculosis-containing phagosomes, thus minimizing the possibility of PI3P-dependent maturation into phagolysosome.
It has also been reported that M. tuberculosis, as well as M. marinum and M. leprae, could escape from the phagosomes and grow within the cytosol of host cells; however, it is not required for replication [85,86]. The efficient translocation from vacuole to cytosol depends on an intact region of difference (RD), one locus [86,87] which encodes a type 7 secretion system and essential secreted effectors . The effector ESAT-6 was demonstrated to be secreted by the ESX-1 secretion system and to be responsible for breakage of the replication niche and pore forming which facilitate the exit of M. marinum and M. tuberculosis into the cytosol . Intracellular growth of mycobacterial species involves a unique trafficking pathway, which blocks maturation of the phagolysosome and establishes a replicative compartment that maintains vesicular contact with the plasma membrane surface . This mechanism of persistence to phagocytosis of some Mycobacterium species, such as M. avium or M. marinum, in both amoebal and mammalian cells, has put into question the role of some host cell proteins. One crucial protein was coronin/TACO (Tryptophan aspartate-containing coat protein), a phagosomal protein implicated in intracellular mycobacterial trafficking through the inhibition of phagolysosme fusion and the activation of mycobacterial growth . Mutation in the coronin gene permitted the isolation of strains defective in particle phagocytosis, fluid phase uptake, migration, and cytokinesis . However, the M. marinum/D. discoideum model showed that the absence of coronin did not enhance intra-amoebal replication, suggesting that other amoebal factors were required for intra-amoebal replication of M. marinum . Further host factors important in restricting mycobacterial growth include the NRAMP-1 transporter on the phagosome, presumably due to limitation of divalent cations within this compartment . The flotillin homologue vacuolin B and p80, a predicted copper transporter, accumulates at the vacuole during pathogen replication until it finally ruptures and the bacteria are released into the host cytosol . Flotilin-1 accumulation at the replication niche and its rupture were also observed in human blood monocytes. Infection of various Dictyostelium mutants demonstrates that the absence of one of the two Dictyostelium vacuolin isoforms renders the host more immune to M. marinum. Conversely, the absence of the small GTPase RacH renders the host more susceptible to M. marinum proliferation but inhibits cell-to-cell spreading  (Table 4).This protein factor was implicated also in regulation of the actin cytoskeleton and in endosomal membrane trafficking and acidification. Like M. tuberculosis, M. marinum evades the endocytic pathway after uptake by host cells , and factors associated with the intracellular growth of this organism also appear to play a role in M. tuberculosis pathogenesis . Recently, it has been discovered that M. tuberculosis and M. Marinum, but not M. avium, were ejected from the Dictyostelium trophozoite, owing to an actin-based structure, the ejectosome. This conserved nonlytic spreading mechanism requires a host cytoskeleton regulator and an intact mycobacterial ESX-1 secretion system . The mycobacterium-induced blockage of phagosome-lysosome fusion is an essential prerequisite for such escape into the cytosol. Escape of mycobacteria outside the phagosome occurs after a significant delay  and requires the secreted effector ESAT-6, recently linked to niche breackage and pore forming activity . This phenomenon has been well described for M. marinum, which escapes outside the phagosome and is propelled by actin-based motility into the cytoplasm . The coronin mutants of D. discodium were used to determine the genetic basis of mycobacterial trafficking in mammalian macrophages . Vacuolar escape has been linked to the ESX-1 secretion system locus. Moreover, M. marinum undergoes actin-based motility in the cytosol, allowing the spread of the organizm to neighbour cells . Wiskott-Aldrich syndrome family proteins are necessary for this actin-based motility and intercellular spreading [85,94]. However, escape into the cytosol does not seem to be required for mycobacterium replication, contrary to what has been observed for Listeria monocytogenes .
Conclusions and Perspectives
Among several species of free-living amoebae, only members of the genus Acanthamoeba, Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea are the opportunistic pathogens  mainly responsible for keratitis  and encephalitis . Moreover, FLA, including ‘Limax amoeba’, A. polyphaga, A. castellanii, Hartmanella vermiformis and Naegleria gruberi, with a composition comparable to that of inanimate environment of the individuals have been isolated from the human nasal mucosa . Hartmanella species have been also isolated from human throats . Among these FLA demonstrated to infect humans, Acanthamoeba spp. are known to host the facultative and obligate bacterial pathogens  Parachlamydiae , Salmonella typhimurium , Escherichia coli O157 , Francisella tularensis , Burkholderia pseudomallae , Coxiella burnetii , Helicobacter pylori , Legionella pneumophila , Chlamydia , Vibrio parahaemolyticus  as well as the fungus Cryptococcus neoformans . This review indicates that all mycobacteria known to be obligate or facultative human pathogens should be added to the growing list of amoeba-resistant bacteria . Such amoeba-resistant bacteria have been recovered from human nasal swabs using amoebal co-culture . One could therefore hypothesize that FLA may be vectors for mycobacteria and may facilitate their penetration into the host and facilitate their pathogenic role, as was suggested more the 30 years ago for M. leprae , with recent experimental confirmation . Indeed, intra-amoebal location has been demonstrated to exacerbate the virulence of M. avium  and to protect M. avium and M. xenopi against antibiotics and biocides [35,54]. Environmental and clinical studies are warranted to further test this hypothesis.
All co-authors report that they have no conflicts of interest relevant to this article.
Methods for Literature Review and Selection
We performed the bibliographic research in the PubMed database by using the following keywords: interaction between amoeba and Mycobacterium, amoeba and Mycobacterium coculture, association of Mycobacterium and amoeba, survival of mycobacteria in amoeba. We then selected English publications published in the period 1967–2009.