Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa)

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and β-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying "doughnut-shaped" vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity

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Yersinia pseudotuberculosis IP32953 survives and replicates in trophozoites and persists in cysts of Acanthamoeba castellanii.

Yersinia pseudotuberculosis is a foodborne enteric pathogen that causes a mild self-limiting diarrhea in humans. Yersinia pseudotuberculosis is able to persist in soil and water and in association with fresh produce, but the mechanism by which it persists is unknown. It has been shown that Y. pseudotuberculosis co-occurs with protozoans in these environments; therefore, this study investigates if bacterivorous free-living amoeba (FLA) are able to support persistence of Y. pseudotuberculosis. Coculture studies of Y. pseudotuberculosis and the prototype FLA, Acanthamoeba castellanii revealed that bacteria had an enhanced capacity to survive in association with amoeba and in the absence of any cytotoxic effects. Yersinia pseudotuberculosis is able to survive and replicate in trophozoites specifically localized within vacuoles, and persists within cysts over a period of at least a week. These data present the first evidence that Y. pseudotuberculosis is able to resist the bacterivorous nature of FLA and instead exhibits an enhanced ability to replicate and persist in coculture with amoeba. This study sheds light on the potential role of FLA in the ecology of Y. pseudotuberculosis which may have implications for food safety.

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.

Internalization of Mycobacterium shottsii and Mycobacterium pseudoshottsii by Acanthamoeba polyphaga.

Amoebae serve as environmental hosts to a variety of mycobacteria, including Mycobacterium avium and Mycobacterium marinum. Mycobacterium shottsii and Mycobacterium pseudoshottsii are waterborne species isolated from the spleens and dermal lesions of striped bass (Morone saxatilis) from the Chesapeake Bay. The optimal growth temperature for these fish isolates is 25 °C. In the present study, amoebae were examined as a potential environmental reservoir for these fish pathogens. Several studies demonstrated that M. avium bacilli replicate within the trophozoite stage and reside in large numbers within the cytosol of the cyst of the free-living amoeba Acanthamoeba polyphaga. Results from the present study showed that M. shottsii, M. pseudoshottsii, and M. marinum bacilli were internalized by A. polyphaga trophozoites within 6 h but that intracellular viability decreased by 2 to 3 logs over 10 days. While an average of 25 M. marinum bacilli were identified by electron microscopy in the cytosol of the cyst, <5 M. pseudoshottsii and no M. shottsii bacilli were observed in this location. All Mycobacterium species examined remained viable but did not replicate after encystment and subsequent 48 h incubation in 4% HCl. This concentration of HCl will kill mycobacteria but will not enter amoebal cysts. Bacterial viability studies within stages of the amoeba life cycle indicate fewer M. shottsii and M. pseudoshottsii bacilli within the trophozoite and cyst stages relative to M. marinum.

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 mc(2) 155, M. smegmatis ATCC 19420(T) 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.

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

BACKGROUND: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii. METHODOLOGY/PRINCIPAL FINDINGS: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6)) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10(4) CFU/mL) than M. tuberculosis (42×10(1) CFU/mL) and M. bovis (35×10(1) CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde. CONCLUSIONS/SIGNIFICANCE: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts.

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii Trophozoites and Cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.

Evaluation of power ultrasound for disinfection of both Legionella pneumophila and its environmental host Acanthamoeba castellanii.

The objectives of this study were to (1) examine the effect of power ultrasound on the viability of both Legionella pneumophila and Acanthamoeba castellanii trophozoites and cysts, (2) investigate if intracellular Legionella replication in trophozoites positively affects bacterial resistance to ultrasound and (3) study if Legionella renders viable but non-culturable (VBNC) due to ultrasound treatments. Using laboratory scale experiments, microorganisms were exposed for various time periods to power ultrasound at a frequency of 36 kHz and an ultrasound power setting of 50 and 100%. Due to a fast destruction, trophozoite hosts were not able to protect intracellular Legionella from eradication by ultrasound, in contrast to cysts. No significant effects of ultrasound on cyst viability could be detected and power settings of 100% for 30 min only made intracellular Legionella concentrations decrease with 1.3 log units. Due to intracellular replication of Legionella in trophozoites, ultrasound no longer affected bacterial viability. Concerning the VBNC state, ultrasound treatments using a power setting of 50% partly induced Legionella (+/-7%) to transform into VBNC bacteria, in contrast to power settings of 100%. Promising results obtained in this study indicate the possible application of power ultrasound in the control of both Legionella and Acanthamoeba concentrations in anthropogenic water systems.

Effects of a microsporidium from the convergent lady beetle, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), on three non-target coccinellids.

A microsporidium from Hippodamia convergens was transmitted horizontally to three non-target coccinellid hosts (Adalia bipunctata L., Coccinella septempunctata L. and Harmonia axyridis Pallas) under laboratory conditions. For all species examined, microsporidia-infected larvae took significantly longer to develop than did uninfected larvae but the microsporidium had no effect on larval mortality. Adult sex ratios of uninfected and microsporidia-infected adults were about 1:1 (female symbol:male symbol) and did not differ significantly. At the end of a 90-day trial, microsporidia-infected H. convergens produced significantly fewer eggs and did not live as long as uninfected individuals. Differences in fecundity and longevity were not observed for the three non-target coccinellids that were examined. Mean spore counts from smear preparations of microsporidia-infected A. bipunctata did not differ significantly from H. convergens, suggesting that A. bipunctata (a native coccinellid) is a suitable host for the microsporidium but infection was lighter in C. septempunctata and H. axyridis (introduced species). Vertical transmission of the pathogen was observed during the 90-day trial by examining eggs and larvae that were produced by microsporidia-infected adults. For all species examined, 100% vertical transmission of the pathogen was eventually observed. Three eugregarines were found in two adult A. bipunctata: Gregarine A trophozoites are similar in size to those of Gregarina katherina Watson (described earlier from Coccinella spp.), Gregarine B trophozoites are similar in size to those of Gregarine A but are morphology distinct, and Gregarine C trophozoites are similar in size to G. barbarara Watson (described earlier from A. bipunctata).

Nanobacteria are mineralo fetuin complexes.

"Nanobacteria" are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of "nanobacteria" susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of "nanobacteria" as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call "nanons." The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.

An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis.

In the present article, the detection and the development of a parasitic endocytobiont within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis is presented. An otherwise healthy 55-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of a corneal trauma. Acanthamoebae as well as other smaller free-living amoebae could be detected from the fluid of the contact lens storage cases by culture methods. A successful therapy could be provided consequently. Two of these Acanthamoeba strains showed intracellular aggregating organisms. Within 2 to 3 days, the host amoebae ruptured, and numerous microorganisms were released. We succeeded in detecting the mechanism of infection and intrusion of this organisms by using light and electron microscopy. Infection with this endocytobiont is a suitable model for studying the host-parasite relations while the parasites use their hosts as so-called Trojan horses (see Barker, Lambert, Brown, Infect Immun 61:3503-3510, 1992).

Mechanism of intrusion of a microspordian-like organism into the nucleus of host amoebae (Vannella sp.) isolated from a keratitis patient.

Free-living amoebae (FLA) occur ubiquitously in many aquatic habitats and humid soils as well as in "artificial" water samples. In addition to their role as pathogens, FLA are known to serve as natural hosts and vehicles of transmission for various intracellular organisms. An otherwise healthy 24-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of corneal trauma. No acanthamoebae could be determined by culture methods. A Vannella strain (called VanAun0) isolated from corneal scrapings showed intracellular aggregating organisms. Within 1-2 days, the host amoebae ruptured, and numerous coccoid organisms (called Kaun1) were released. We succeeded in detecting the mechanisms of infection and intrusion of this eukaryotic organism, growing within the nucleus of the FLA, by light and electron microscopy. It could be shown that the spores at the cell membrane of strain KAun1 resemble Microsporidia and were taken up into the Amoeba by phagocytosis after adhesion of the spores and food cup formation (infective phase). The spores were transported into the cytoplasm of the vannellae in food vacuoles. Phase contrast microscopy revealed early stages of the parasites moving through the cytoplasm into the nucleus of the host amoeba. Electron microscopy showed the proliferation of polymorphic stages within the karyoplasm. The life cycle of these microsporidian-like organisms ended up with a sporogenic phase in which a terminal differentiation took place and numerous spores were released by rupture of the host cell wall. With the rupture of the host amoeba's cell membrane, the cycle started again from the beginning, the released infectious spores being ingested by other host amoebae. In particular, the morphology of the organelles made visible by electron microscopy finally allowed us to classify the endocytobionts as a microsporidan-like organism. Infection of Vannella sp. with the microsporidia-like organism strain KAun1 is a suitable model for studying the host-parasite relations of organisms using their hosts as so-called Trojan horses.

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii.

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.

[Gimenez staining: a rapid method for initial identification of legionella pneumophila in Amoeba trophozoite].

OBJECTIVE: To establish a rapid staining method for facilitating initial identification of Legionella pneumophila in amoebal trophozoite. METHODS: Acanthamoeba polyphaga and Legionella pneumophila were co-cultured under laboratory condition. At consecutive time points during the culture, smears of the cultured products were made on glass slides for staining purposes. Different types of stainings including Gram's staining, Gimenez staining, Giemsa staining and immunofluorescence were used to determine the best method for the identification of amoebal pathogens. RESULTS: Gimenez staining technique is simpler and yields better results as compared with the other three stainings. Gimenez stain gives the best color and contrast for amoeba and amoebal Legionella Amoeba trophozoites and/or cysts showed a distinct purplish blue with amoebal Legionella in red. Amoebal Legionella can be distinguished clearly at an earlier time of co cul ture, providing a proper sensitivity. It takes only 10 minutes to finish the operation. The other techniques require the use of expensive reagents, are relatively time-consuming, and involve complex staining procedures. CONCLUSION: Gimenez staining is of value for the initial identification of amoebal pathogens, and it is suitable for laboratory diagnosis.