Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis Survival within Amoebal Cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

Yield Constant for Acanthamoeba polyphaga in Batch and Continuous Culture1

ABSTRACT. A two-stage chemostat modified to accommodate the growth of adhesive organisms was used to determine the yield constant, Y, of a representative soil amoeba, Acanthamoeba polyphaga, utilizing as its prey Pseudomonas paucimobilis. The first stage consisted of a glucose-limited bacterial culture in steady state. The second stage consisted of a simplified predator-prey system, nongrowing bacteria serving as the limiting substrate for amoebae. A refined methodology to more accurately determine Y was developed, and Y for Acanthamoeba polyphaga in batch and continuous culture was determined to be 19.1%.     

Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81–176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.     

Crescent Bodies of Parachlamydia acanthamoeba and Its Life Cycle within Acanthamoeba polyphaga: an Electron Micrograph Study

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.     

Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.     

Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.     

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga

We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DeltaG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation-mediated cytolysis and egress from mammalian cells. In contrast, the icmTDeltaG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore-forming activity. Consistent with their defect in pore formation-mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.     

Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites

Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.     

A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-alpha were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.     

Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection

As part of their strategy for intracellular survival, mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages. The molecular basis for this inhibition is only now beginning to emerge, by way of the molecular characterisation of the phagosome membrane when it encloses virulent mycobacteria. Our own work has shown that at 15 days after the phagocytic uptake of Mycobacterium avium by mouse bone marrow-derived macrophages, the phagosome membrane is depleted about 4-fold for cell surface-derived membrane glycoconjugates, labelled by exogalactosylation, in comparison to the membrane of early endosomes with which it continues to interact. Here we asked whether this depletion occurred at early or late stages after infection. We found that only about half of the depletion had occurred at about 5 hours after the beginning of phagocytic uptake, with the remainder becoming established thereafter, with a half-time of about 2.5 days. Phagosomes became depleted in relation to early endosomes with which they continued to exchange membrane constituents. Early endosomes themselves became gradually depleted by about 30% during the 15-day post-infection period. In contrast, late endosomes/lysosomes remained unchanged, with a concentration of surface-derived glycoconjugates between that of early endosomes and of phagosomes at day 15 post infection. In view of the slowness of the post-infection change of phagosome membrane composition, we proposed that this change did not play a role in preventing maturation immediately after phagosome formation, but rather correlated with the process of maintaining the phagosomes in an immature state.     

Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium

Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis.     

Mycobacterium avium Infections of Acanthamoeba Strains: Host Strain Variability,Grazing-Acquired Infections,and Altered Dynamics of Inactivation with Monochloramine

Stable Mycobacterium avium infections of several Acanthamoeba strains were characterized by increased infection resistance of recent environmental isolates and reduced infectivity in the presence of other bacteria. Exposure of M. avium in coculture with Acanthamoeba castellanii to monochloramine yielded inactivation kinetics markedly similar to those observed for A. castellanii alone.Acanthamoebae are widely distributed in the environment (20) and generally function ecologically as predators of bacteria (23), although numerous types of bacteria resist predation (22). Acanthamoebae are very resistant to a range of disinfectants (5, 6, 8, 28), and bacteria within acanthamoebae are generally afforded extra protection (16). A notable example is the opportunistic pathogen Mycobacterium avium (10), which can survive within Acanthamoeba species trophozoites and cysts (4, 26), resulting in increased resistance to several antimicrobials (22). It has been demonstrated that many Mycobacterium spp. are able to infect the laboratory strain Acanthamoeba polyphaga (1). Acanthamoeba cultures undergo many physiological changes after several passages in the laboratory (15, 17, 21), although it is not known if prolonged cultivation of Acanthamoeba alters their capacity to be infected by M. avium. This knowledge is important for assessing the environmental relevance of associations between Acanthamoeba and M. avium. Therefore, we studied the infectivity and infection stability of M. avium with several laboratory and environmental Acanthamoeba strains for 28 days under high-nutrient (peptone-yeast extract-glucose [PYG] medium) and low-nutrient (Page''s amoeba saline [PAS]) conditions.     

Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks,and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.