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PubMed1992

A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii.

A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.

Acanthamoeba↗
PubMed1975

Isolation, identification, and biological characterization of Acanthamoeba polyphaga from a human eye.

Trophozoites and cysts of an Acanthamoeba were repeatedly isolated from the corneal scrapings of a patient suffering from acute ulceration of the right eye. The ameba was cloned and cultivated axenically in a proteose peptone-yeast extract-glucose medium. At a later date the organism was identified as A. polyphaga on the basis of its morphologic characteristics, especially those of cysts. Experimental studies on the in vitro interaction of this organism with monkey kidney tissue culture (Vero line) and its pathogenicity to mice indicated that it was a low virulent strain. When large numbers of amebae (25,000+) were inoculated into Vero cell cultures, cytopathic effects (CPE) were noticed within 5 to 6 days. The CPE consisted of cell shrinkage, nuclear pyonosis, and discontinuity of cell sheet, and the cell culture was totally destroyed in 8 to 10 days. When 20,000+ amebae were instilled intranasally into each of 20 2-week-old mice, only 1 mouse died, on the 28th day. Amebae were isolated from the brain of the dead mouse, and trophozoites and cysts were also demonstrated in the brain sections. When amebae isolated from the brain were intranasally instilled into mice, they failed to kill the mice for at least 1 month; however, when 10,000+ amebae were inoculated intracerebrally, the mice died within 5 to 8 days, exhibiting symptoms of primary meningoencephalitis.

Amebiasis↗
PubMed1983

Antigenicity of Entamoeba histolytica strain nih 200: a survey of clinically relevant antigenic components.

The molecular weights of the major polypeptides of Entamoeba histolytica NIH 200 soluble whole extract, and its five fractions eluted from a Sephadex G-200 column, were in the range 150,000-9,000 daltons according to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of SDS-PAGE analysis of 125I surface-labeled amebae revealed more than 12 bands, eight of which corresponded to those recovered in soluble extract. Considering the pronounced phagocytosis and the rapid cell membrane turnover of the ameba, it seems likely that the same antigenic components would be present both on and within the ameba at different stages. The first four gel-filtered fractions were immunogenic and showed non-identical precipitates with sera from 12 Swedish and 12 Ethiopian patients with verified amebic disease. Seven polypeptides in the whole extract reacted with human anti-ameba IgG covalently bound to CNBr activated Sepharose 4B. Immune replica (Western blotting) of SDS-PAGE of whole extract produced by human anti-ameba antisera demonstrated eight bands. The response to the different amebic antigens was not correlated to clinical symptoms, duration of illness or origin of ameba strain. Thus the present results did not support the use of purified fractions rather than the whole soluble extract for diagnostic tests.

Antigen-Antibody Complex↗
PubMed1996

Effect of adhered bacteria on the binding of Acanthamoeba to hydrogel lenses.

OBJECTIVES: To determine the effect of Pseudomonas aeruginosa and Staphylococcus epidermidis on the binding of Acanthamoeba species to hydrogel lenses. METHODS: Cells of amebae and bacteria were incubated with different types of hydrogel lenses. Densities of amebae that were bound to the lenses after rinsing were determined from direct counts with a cell detachment procedure and from scintillation counts of cells, which were radiolabeled with tritiated leucine. RESULTS: With both methods, amebae showed significantly increased binding to hydrogel lenses with attached P aeruginosa. The numbers of amebae that were retained on lenses with attached S epidermidis were not significantly different from those that were retained on lenses without bacteria. The binding of amebae to unworn hydrogel lenses, in contrast to the irreversible adherence of P aeruginosa, was tenuous. CONCLUSIONS: The binding of Acanthamoeba species to unworn hydrogel lenses was tenuous and appeared to be related to water content, surface tensions, and ionic charge. The presence of adhered P aeruginosa on the hydrogel lenses facilitated the binding of Acanthamoeba species. The cocontamination of lens systems with bacteria (eg, P aeruginosa) may be a prime factor in the development of amebic keratitis.

Acanthamoeba↗
PubMed1989

Indirect immunofluorescence localization of ponticulin in motile cells.

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.

Actins↗
PubMed2004

Balamuthia mandrillaris, an opportunistic agent of granulomatous amebic encephalitis, infects the brain via the olfactory nerve pathway.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis (GAE) in humans and other mammals. Its supposed routes of infection have been largely assumed from what is known about Acanthamoeba spp. and Naegleria fowleri, other free-living amebae and opportunistic encephalitis agents. However, formal proof for any migratory pathway, from GAE patients or from animal models, has been lacking. Here, immunodeficient mice were infected with B. mandrillaris amebae by intranasal instillation, the most likely natural portal of entry. By means of classical and immunohistology, the amebae are shown to adhere to the nasal epithelium, progress along the olfactory nerves, traverse the cribriform plate of the ethmoid bone, and finally infect the brain. A similar invasion pathway has been described for N. fowleri. The data suggest that the olfactory nerve pathway is a likely route for natural infection of the brain by B. mandrillaris amebae.

Amebiasis↗
PubMed1995

Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites.

With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes (Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (EmR) clone C2 amebae. The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgp1 (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes. The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%). Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic alpha-helices, which form the putative transmembrane channel. EmR clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgp1 and Eh pgp6 mRNA when compared to wt clone A amebae. In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 micrograms/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe. It appears then that multiple Pgp may contribute to amebic Em resistance in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Breakdown of self/nonself recognition in cannibalistic strains of the predatory slime mold, Dictyostelium caveatum.

Dictyostelium caveatum amebas feed upon both bacteria and the amebas of other cellular slime molds. The capacity to feed extensively upon other cellular slime molds is unique to D. caveatum amebas. They are able to phagocytose amebas larger than themselves by nibbling pieces of the cells until they are small enough to ingest. Here we report the isolation from previously cloned stock cultures of stable, cannibalistic strains of D. caveatum in which self/nonself recognition has broken down. Because of the extensive cannibalism, amebas of these strains do not complete multicellular development, and instead wander about for long periods while feeding upon each other. Although the cannibalistic behavior resembles that exhibited by the presumably diploid giant cells in the sexual cycle of other cellular slime molds, these strains are haploid and do not form macrocysts.

Cell Division↗
PubMedDate not supplied

Immunity to Acanthamoeba.

Human serum contains antibodies, mainly of the IgM and IgG isotypes, to pathogenic species of Acanthamoeba. This, as well as the capacity of these amebas to activate complement via the alternative pathway, may be a first-line defense against acanthamoeba infections in humans. Both antibody and complement appear to be important in promoting recognition of these amebas by phagocytic cells such as neutrophils. However, killing of amebas by neutrophils is dependent on lymphokine/monokine priming of the neutrophil. This priming augments the respiratory-burst activity and release of lysosomal enzymes of neutrophils in their response to the ameba. The products of the oxygen-dependent respiratory burst appear to be of prime importance in the killing of this free-living ameba. Antibodies also may prevent tissue invasion by Acanthamoeba by inhibiting its adherence, phagocytic activity, and migration and by neutralizing cytopathogenic amebic agents. Studies on experimental Acanthamoeba infections in mice showed marked species and strain specificity with regard to induction of protection with amebic antigens. Immune compromise or, alternatively, invasion at unique body sites in healthy individuals may form the basis for human infection with Acanthamoeba.

Acanthamoeba↗
PubMed1993

Attachment and entry of Legionella pneumophila in Hartmannella vermiformis.

Legionella pneumophila is an intracellular parasite of Hartmannella vermiformis. Attachment to the amebae and entry of L. pneumophila were studied by two quantitative assays: One used plate counts to measure the number of bacteria attaching to amebae at 4 degrees C; the other determined the number of intracellular bacteria by use of transmission electron microscopy (TEM). The attachment assay showed that L. pneumophila are inefficient in attachment to amebae. About 0.05% of the bacteria were bound after 1 h with a 10- to 40-fold increase over the next 11 h. Attachment of both virulent and avirulent strains of L. pneumophila occurred at a similar rate. Uptake of L. pneumophila was measured by counting intracellular bacteria using TEM. Limited numbers of virulent L. pneumophila were found intracellularly before 4 h, but the numbers increased logarithmically after this time. The number of amebae containing virulent L. pneumophila increased linearly during the 12-h co-incubation. Avirulent L. pneumophila were rarely detected within amebae throughout the 12-h incubation. Results indicate that entry, not attachment, of virulent L. pneumophila is the limiting step in infection of axenically grown H. vermiformis.

Animals↗
PubMed1983

Scanning electron microscopy of attached trophozoites of pathogenic Entamoeba histolytica.

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2-8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.

Animals↗
PubMed1986

Movement of Naegleria fowleri stimulated by mammalian cells in vitro.

Naegleria fowleri amebae, but not those of N. australiensis, N. gruberi, or N. lovaniensis, demonstrated enhanced motility when placed in proximity to mammalian cells. Amebae of nonpathogenic species of Naegleria, however, were more motile in cell culture medium than the amebae of N. fowleri. The locomotory response of highly pathogenic mouse-passaged N. fowleri amebae to nerve cells was greater than axenically cultured amebae. The enhanced mobility elicited by whole nerve cells or disrupted nerve cells was not directed migration but chemokinetic. Naegleria fowleri responded to disrupted neuroblastoma cells more vigorously than to disrupted African green monkey kidney (Vero) cells.

Animals↗
PubMed1992

Susceptibility of an emetine-resistant mutant of Entamoeba histolytica to multiple drugs and to channel blockers.

Previously a cloned emetine-resistant mutant of the protozoal parasite Entamoeba histolytica was shown to overexpress a gene for an ameba homolog of the mammalian P-glycoprotein, a plasma membrane pump that removes hydrophobic drugs from multidrug-resistant tumor cells. Three sets of experiments were performed to better characterize the multidrug-resistant phenotype of the emetine-resistant amebae. First, the emetine resistance of the mutant amebae was reversed by concentrations of calcium and sodium channel blockers effective in reversing drug resistance by multidrug-resistant tumor cells, but it was reversed only in the presence of very high concentrations of the tricyclic antidepressants. Second, the mutant amebae showed cross-resistance to antiamebic drugs used to treat luminal infection (iodoquinol and diloxanide) but were not cross-resistant to drugs used to treat invasive disease (chloroquine and metronidazole). Third, when amebae were loaded with radiolabeled emetine, the mutant parasites released the drug at approximately 1.6 times the rate of the wild-type organisms. We conclude that the emetine-resistant E. histolytica parasites have some but not all the features of the multidrug-resistant phenotype.

Animals↗
PubMed2002

Entamoeba histolytica-induced dephosphorylation in host cells.

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation.

Animals↗
PubMed2004

Induction of morphological and electrophysiological changes in hamster cornea after in vitro interaction with trophozoites of Acanthamoeba spp.

Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.

Acanthamoeba↗
PubMed2006

Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri.

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

Acanthamoeba↗
PubMed2002

Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples.

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.

Animals↗
PubMed1992

Low risk of invasive amebiasis in cyst carriers. A longitudinal molecular seroepidemiological study.

A seroepidemiological study of a household cohort, using both clinical observational and molecular criteria was conducted in a periurban area endemic for E. histolytica infection. This longitudinal study was undertaken to determine the risk of asymptomatic cyst carriers to develop invasive illness. Zymodeme patterns of strains isolated from these patients were correlated both with the clinical presentation of disease and with the serological response against the M-17 ameba antigen and further compared with that found in 16 proven cases of amebic liver abscess. From a total of 163 housewives screened, 39, (24%) were asymptomatic cyst carriers; 31 of them (index cases) and 114 members of their households remained in the study over an 8 month follow-up period to detect ameba infection and illness. Of the household members at risk, 46 (40%) became infected within 6 weeks. None of the index or secondary cases developed ameba-related symptoms and cyst excretion followed a chronic persistent, intermittent, or transient pattern over the period of the study. Amebas were recovered and zymodemes determined in 19 of 71 (27%) cyst carriers. Ameba shed from each of these 19 carriers exhibited nonpathogenic zymodeme 1, except for one index case where zymodeme 2 was recovered in one sampling, and returned to zymodeme 1 in subsequent samples. Of 48 of 71 cyst carriers studied, antibodies to crude E. histolytica antigen were detected by ELISA in 16 (31%); antibodies to the M-17 fusion protein were found in 8 (16%) by ELISA and in 2 (4%) by Western-Blot (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗