Acanthamoeba differentiation: a two-faced drama of Dr Jekyll and Mr Hyde.

The ability of cyst-forming protists such as Acanthamoeba to escape death by transforming into a cyst form, that is resistant to harsh physiological, environmental and pharmacological conditions, has continued to pose a serious challenge to human and animal health. A complete understanding of the fundamental principles of genome evolution and biochemical pathways of cellular differentiation offers unprecedented opportunities to counter detrimental outcomes. Acanthamoeba can elude inhospitable conditions by forming cysts. Here we unravel the processes involved in the phenotypic switching of Acanthamoeba, which are critical in our efforts to find potential targets for chemotherapy.

Carbohydrate analysis of Acanthamoeba castellanii.

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting "cyst wall" material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.

The role of proteases in the differentiation of Acanthamoeba castellanii.

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

Role of human tear fluid in Acanthamoeba interactions with the human corneal epithelial cells.

Acanthamoeba keratitis is a painful and progressive sight-threatening infection. However, the precise mechanisms associated with the pathogenesis and pathophysiology of Acanthamoeba keratitis remain incompletely understood. Using tears from healthy individuals and an Acanthamoeba keratitis patient, we demonstrated that both subjects exhibited similar levels of Acanthamoeba-specific IgA as demonstrated by Western blotting and enzyme-linked immunosorbent assays. However, normal tears were slightly more potent in reducing Acanthamoeba binding to human corneal epithelial cells, compared with tears from Acanthamoeba keratitis patient (P>0.05 using paired T-test, one-tail distribution). Neither normal tears nor Acanthamoeba keratitis tears had any protective effects on Acanthamoeba-mediated corneal epithelial cell cytotoxicity. Both lysozyme and lactoferrin which are major constituents of the tear film and possess antibacterial properties exhibited no significant effects on Acanthamoeba binding to and cytotoxicity of human corneal epithelial cells. The role of contact lens wear in Acanthamoeba keratitis is discussed further.

Cellulose biosynthesis pathway is a potential target in the improved treatment of Acanthamoeba keratitis.

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.

Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications.

The ability of Acanthamoeba to feed on Gram-negative bacteria, as well as to harbour potential pathogens, such as Legionella pneumophila, Coxiella burnetii, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes and Mycobacterium avium, suggest that both amoebae and bacteria are involved in complex interactions, which may play important roles in the environment and in human health. In this study, Acanthamoeba castellanii (a keratitis isolate belonging to the T4 genotype) was used and its interactions with Escherichia coli (strain K1, a cerebrospinal fluid isolate from a meningitis patient, O18 : K1 : H7, and a K-12 laboratory strain, HB101) were studied. The invasive K1 isolate exhibited a significantly higher association with A. castellanii than the non-invasive K-12 isolate. Similarly, K1 showed significantly increased invasion and/or uptake by A. castellanii in gentamicin protection assays than the non-invasive K-12. Using several mutants derived from K1, it was observed that outer-membrane protein A (OmpA) and LPS were crucial bacterial determinants responsible for E. coli K1 interactions with A. castellanii. Once inside the cell, E. coli K1 remained viable and multiplied within A. castellanii, while E. coli K-12 was killed. Again, OmpA and LPS were crucial for E. coli K1 intracellular survival in A. castellanii. In conclusion, these findings suggest that E. coli K1 interactions with A. castellanii are carefully regulated by the virulence of E. coli.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells.

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Mechanisms associated with Acanthamoeba castellanii (T4) phagocytosis.

Using fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba is studied. This assay is based on the quenching effect of trypan blue on FITC-labelled E. coli. Only intracellular E. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. Acanthamoeba uptake of E. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. In contrast, sodium orthovanadate (protein tyrosine phosphatase inhibitor) increases bacterial uptake by Acanthamoeba. Treatment of Acanthamoeba with cytochalasin D (actin polymerization inhibitor) abolished the ability of Acanthamoeba to phagocytose E. coli suggesting that tyrosine kinase-mediated signaling may play a role in Acanthamoeba phagocytosis. In addition, we showed that phosphatidylinositol 3-kinase (PI3K) plays an important role in Acanthamoeba uptake of E. coli. Role of mannose-binding protein in Acanthamoeba phagocytosis is discussed further.