Acanthamoeba polyphaga-Enhanced Growth of Mycobacterium smegmatis

Background Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival. Methodology/Principal Findings Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc2 155, M. smegmatis ATCC 19420T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth. Conclusions/Significance Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.


Introduction
Mycobacteria are mycolic-acid containing, high GC% bacterial organisms belonging to the phylum Actinobacteria. They are recovered from soil and fresh water environments where freeliving amoeba (FLA) are also living [1,2,3]. Co-isolation of mycobacteria and FLA collected from such environmental sources has been reported [4,5]. Several experiments further demonstrated the ability of most environmental mycobacteria to survive in the amoebal trophozoites and to further reside into the amoebal cysts [6,7,8]. We recently showed that this holds true also for some of the Mycobacterium tuberculosis complex mycobacteria [9]. FLA have been therefore regarded as ''Trojan horses'' for such amoebaresistant mycobacteria. Indeed, intra-amoebal survival has been demonstrated for 37 different Mycobacterium species and intraamoebal surviving became a dogma for amoeba-mycobacteria interactions except for Mycobacterium bovis BCG which is killed by the FLA Acanthamoeba castellanii [8] and Mycobacterium canettii which bypasses amoebal encystement [9].
Amoeba-resistant mycobacteria include both slow-growing mycobacteria, i.e. mycobacteria sub-culturing over more than seven days and fast-growing mycobacteria which produce visible colonies in less than seven days [10]. Whereas fast-growing mycobacteria are comprised of both harmless organisms and opportunistic pathogens, slow-growing mycobacteria are comprised of some of the most successful bacterial human pathogens such as M. tuberculosis complex organisms causing tuberculosis [11], Mycobacterium leprae causing leprosy [12] and Mycobacterium ulcerans causing the Buruli ulcer [13]. Although several experimental studies have demonstrated the interactions of slow-growing mycobacteria, such as Mycobacterium avium complex members, with amoebae [6,8,9,14], the interactions of fast-growing mycobacteria with amoebae remain poorly understood [14,15,16]. For example, conflicting results have been published regarding Mycobacterium smegmatis, ranging from its survival in the amoeba [15,16] to its destruction by amoebae [14,17].
M. smegmatis is the prototypical species of the so-called M. smegmatis group, which also contains Mycobacterium wolinskyi and Mycobacterium goodii [18]. Organisms of this group have seldom been associated with human infection, including orthopedic device infection and bacteremia [19,20]. In the present work, we utilized M. smegmatis as a model organism to study the interactions of fastgrowing mycobacteria with Acanthamoeba polyphaga which, together with Acanthamoeba castellanii, is one of two FLA routinely used to probe bacteria-FLA interactions [21] at large and more specifically mycobacteria-FLA interactions [22].

Microscopic detection of A. polyphaga infected with mycobacteria
A. polyphaga Linc-AP1 strain (a gift from T. J. Rowbotham, Public Health Laboratory, Leeds, United Kingdom) was grown at 28uC for 4 days in 150-cm 3 culture flasks (Corning, New York, USA) containing 30 mL of peptone-yeast extract-glucose (PYG) broth. When average amoeba concentration reached 5610 5 cells/ mL, amoebae were centrifuged at 500 g for 10 min and the pellet was suspended twice in 30 mL Page's modified Neff's Ameoba Saline (PAS) (Solution A-NaCl 1.20 g; MgSO 4 .7H 2 O 0.04 g; Na 2 HPO 4 1.42 g; KH 2 PO 4 1.36 g/100 mL of glass distilled water. Solution B-CaCl 2 .2H 2 O 0.04 g/100 mL of distilled water. Amoeba saline, 10 mL of solution A+10 mL of solution B+980 mL distilled water). Liquid medium-cultured M. smegmatis organisms were washed twice with PBS and the pellet was suspended in PAS. This inoculum was strongly vortexed to minimize mycobacterial clumping and the inoculum was determined by optic microscopy counting after Ziehl-Neelsen staining. Ten milliliters of the amoebal suspension in PAS (10 5 amoeba/ mL) were inoculated with 10 6 mycobacteria/mL to achieve a MOI of 10 mycobacteria/amoeba. As controls, A. polyphaga and M. smegmatis were cultured separately in PAS. After incubation for 6 h at 32uC, the co-culture was washed three times with PAS to remove any remaining extracellular or adherent mycobacteria, and it was incubated in 10 mL PAS for 5 days at 32uC. After gentle shaking and cytocentrifugation at 100 g for 10 min, mycobacteria were detected inside amoebal trophozoites by Ziehl-Neelsen staining. Also, the presence of viable mycobacteria inside amoebal trophozoites was documented by sub-culturing. At 0, 24, 48, 72, 96 and 120 h time points, A. polyphaga monolayer were lysed with 0.1% Sodium dodecyl sulfate (SDS) (Sigma-Aldrich Logistic Gmbh) for 30 min and passed through a 26-gauge needle to ensure complete lysis of the amoebae. The lysate (100 mL) was plated onto 7H10 agar and incubated for four days at 37uC to determine the number of colonies (CFU) of intracellular M. smegmatis. The viability of amoeba, with and without bacteria, was done using Trypan Bleu coloration 0.4% (Sigma-Aldrich, Taufkirchen, Germany) and counting in the Glasstic slide chamber (HycoR, Garden Grove, California USA). Experiments were done in triplicate.

Encystment of infected amoeba
Fifty milliliters of a 48-hour amoebal co-culture (concentration, 5610 5 amoebal cells/mL of PAS) were put in a 175-cm 3 culture flask (Corning) and infected with 5 mL (concentration, 10 7 mycobacteria cells/mL of PAS) of M. smegmatis suspension in PAS for 6 hours (time point, 0). The co-culture was washed twice with PAS to remove any remaining extracellular or adherent mycobacteria and it was incubated in 50 mL PAS for 5 days. In parallel, at different time points after infection (each 24 hours), ten milliliters of co-culture was taken, the supernatant was discarded and the amoebal monolayer was rinsed twice with encystment buffer (0.1 M KCl, 0.02 M Tris, 8 mM MgSO 4 , 0.4 mM CaCl 2 , 1 mM NaHCO 3 ) before being incubated (at 32uC for 3 days) in fresh encystment buffer (0.1 M KCl, 0.02 M Tris, 8 mM MgSO 4 , 0.4 mM CaCl 2 , 1 mM NaHCO 3 ). As control, A. polyphaga was cultured in encystment buffer. The process of excystment was verified by light microscopic examination of Ziehl-Neelsen smears. After 3 days, the number of cysts and trophozoites at different time points was determined by microscopic observation.
Moreover, the cysts corresponding to the time point 0 were then centrifuged at 1,000 g for 10 min and washed three times with PAS before using it for electron microscopic observation. Experiments were done in triplicate.

Culture of M. smegmatis with amoeba debris
A. polyphaga and M. smegmatis were prepared as described before. After washing with PAS, 10 mL of A. polyphaga cells suspension (,5610 5 amoeba/mL) were lysed (1 min at liquid nitrogen and 1 min at 37uC for three times) and centrifuged at 800 g for 10 min. 10 3 mycobacteria/mL was separately incubated with amoeba lysis pellet and supernatant for 5 days at 32uC. M. smegmatis were observed in the culture at each time point by Ziehl-

Ultrastructural study
Amoebal cysts and monolayers inoculated with mycobacteria were washed twice with sterile PAS to eliminate non-ingested mycobacteria. Samples were fixed in 2% glutaraldehyde and 0.1 M cacodylate buffer overnight, then in 2% glutaraldehyde and 0.33% acroleine in 0.07 M cacodylate buffer for one hour. After washing in 0.2 M cacodylate buffer, the preparation was post-fixed in 1% osmium tetraoxide in 0.1 M potassium ferrycyanure for one hour and dehydrated in an ascending

M. smegmatis -A. polyphaga trophozoites co-culture
We first observed that the number of both non-infected and infected A. polyphaga trophozoites incubated into PAS at 32uC decreased over the time with the number of infected-amoeba decreasing significantly more than the number of non-infected amoeba (p#0.05) at day four of co-culture for M. smegmatis ATCC 27204 and at day five of co-culture for M. smegmatis mc 2 155 and M. smegmatis ATCC 19420 T , in triplicate experiment (Figure 1). In parallel, we observed that the three tested M. smegmatis strains survived but did not multiply over five-day incubation in PAS at 32uC (Figure 2). At six-hour incubation, M. smegmatis mc 2 155-A. polyphaga co-culture yielded 72% infected amoeba presenting at least one vacuole containing mycobacteria (Figure 3). Such vacuoles were surrounded by several mitochondria and displayed morphological features consistent with mycobacterial division, i.e. two organisms tightly attached by one extremity into a single vacuole (Figure 3). For the three tested M. smegmatis strains, quantification of colony forming units (CFU) co-cultured with amoeba indicated a significant increase (p,0.05) in the number of mycobacteria organisms starting at day 2 ( Figure 2). To understand whether this significant increase in the growth of M. smegmatis co-cultured with A. polyphaga necessitated viable amoeba, we further cultured each one of the three tested M. smegmatis strains in PAS enriched in an amoeba lysis pellet (ALP) or an amoeba lysis supernatant (ALS). Regardless of the M. smegmatis strain, we observed that growth of M. smegmatis organisms was significantly increased (p#0.05) by the addition of ALP or the addition of ALS to the PAS (Figure 4).

Interaction of M. smegmatis mc 2 155 with A. polyphaga cysts
We further infected A. polyphaga trophozoites with M. smegmatis mc 2 155 organisms for 6 hours, and then incubated in encystement buffer for 3 days noted as days 0-3. A sample was then taken every 24 hours and microscopic examination disclosed cystic formation in 43% of M. smegmatis-infected amoebae at day 0 (6 hours of infection); 38% at day 1; 19% at day 2 and 8% at day 3. Non-infected, negative control amoeba yielded 46% encystment at day 0; 52% at day 1; 71% at day 2 and 78% at day 3. This difference in the percentage of encysted amoeba was statistically significant from day 0 to day 3 in triplicate experiment (p#0.05). Electron microscopy further identified mature cysts by the presence of condensation of indistinct components implicated in the metabolism and replication in the middle of this form ( Figure 5A), and pre-cysts identified by the presence of the nucleus and mitochondria scattered into the cytoplasm ( Figure 5B). Careful electron microscopy observation of 500 cysts formed at day 3 failed to reveal any M. smegmatis organisms into A. polyphaga cysts ( Figure 5C). In one case only the M. smegmatis organism was observed to have moved from the endocyst of a pre-cyst present in the earlier phase of encystation after three-day encystment ( Figure 5B). Experimental encystment of A. polyphaga co-culture yielded no intracystic mycobacteria after a three-day encystement.

Discussion
The data presented in this study were interpreted as authentic because negative controls remained negative in each experimental step. In this work, two model organisms have been used in order to set-up a standardized co-culture system. Moreover, similar results were obtained when testing three different strains of M. smegmatis, including one type strain as well as M. smegmatis mc 2 155 (ATCC 700084). Indeed, M. smegmatis mc 2 155 strain, the only M. smegmatis strain with available genome sequence, has particular parietal features which may not be found in other M. smegmatis strains. This could have biased results. We herein show that this was not the case. Moreover, M. smegmatis mc 2 155 has known genetics and it has been previously used in 37/46 (80%) studies dealing with M. smegmatis -macrophage/amoeba interactions (Table S1). In addition, M. smegmatis mc 2 155 is commonly used as a model strain for the cloning genes from harmful mycobacteria [17]. Likewise, A. polyphaga has been extensively used for studying amoeba-mycobacteria interactions [6]. The co-culture system herein reported is therefore a standardized system which could be Table 1. Described interactions of rapid and slow-growing mycobacteria with FLA.

Bacterial species
Growing mycobacteria Described interaction with protozoa References
Interestingly, we further observed that pelleted debris of lysed amoeba and the supernatant of such lysed amoeba also significantly enhanced the growth M. smegmatis mycobacteria, regardless of the strain under study. This observation reminds recent observations made when co-culturing Salmonella enterica Typhi with A. castellanii [21] and suggests that amoeba contain one or several currently uncharacterized growth-promoting factors or nutriments for M. smegmatis. Determining such factors was beyond the scope of present study, but further culture-based experiments incorporating fractions of amoeba supernatant are warranted to precise the nature of these factors.
We further observed that M. smegmatis moved out of the A. polyphaga pre-cyst before its maturation; this observation extended previous data found for other rapidly growing mycobacteria such as Mycobacterium septicum [6]. This observation contrasts with previous observations that slowly growing mycobacteria survived within the amoebal exocyst [26]. It was observed that 92% of M. avium-infected trophozoites evolved into mature cysts whereas we observed that only 8% of M. smegmatis-infected trophozoites produced mature cysts at the same time [26]. Accordingly, forced encystment of M. smegmatis-infected A. polyphaga amoeba yielded no mycobacteria in the cysts. Taken together, these data suggest that fast-growing mycobacteria rapidly escape the encystment to infect new amoebal trophozoites. Interestingly, we recently observed that M. canettii was the only tested M. tuberculosis complex member to by-pass the A. polyphaga encystement [9]. Exactly as for M. smegmatis, M. canettii also massively invaded the amoeba host [9].
We previously proposed that amoeba are a training field for macrophage resistance of mycobacteria [28]. Several studies used amoeba to investigate the phagocytosis and intracellular survival mechanisms of pathogens including Legionella pneumophila [29], Yersinia pseudotuberculosis [23] and P. aeruginosa [30]. M. smegmatis has been used to develop genetic engineering of mycobacteria and the M. smegmatis-amoeba co-culture developed here could therefore be used as a simple and rapid first-line system to scan mycobacterial factors implicated in the intracellular growth of mycobacteria.
In conclusion, the spectrum of interactions between amoeba and environmental mycobacteria may be wider than previously appreciated. It includes mycobacteria such as M. leprae surviving in amoeba [12,27], mycobacteria such as M. avium and M. tuberculosis multiplying in amoeba as opportunistic organisms [9,7,14] and mycobacteria such as M. chelonae [23] and M. smegmatis killing the amoeba ( Figure 6).