Acanthamoeba species have been found to be facultative, opportunistic, protozoan, parasites. They are found ubiquitously in the environment. Acanthamoeba species are emerging pathogens causing a severe corneal infection called Acanthamoeba keratitis (AK) in immunocompetent individuals normally associated with contact lens wearers, which may result in blindness. Many drugs used to treat Acanthamoeba infections are poorly effective because they can induce encystation and/or fail to eliminate cysts, so more reliable and effective therapies are required. To this end, the studies described herein demonstrate the ability of Acanthamoeba unlike its mammalian host to synthesise histidine and lysine. Thus growth of Acanthamoeba spp. was restricted by 3-amino-1, 2, 4-triazole (3AT) (IC50 125μM), an inhibitor of imidazoleglycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19) a key enzyme for de novo histidine biosynthesis. The addition of histidine was able to ablate the effects of 3AT in a dose dependent manner when added to cultures, indicating that 3AT was specifically acting on histidine biosynthesis. 3AT had minimal cytotoxicity to a prostate cancer cell line (PC3-luc) at concentrations that restrict the growth of Acanthamoeba. Bioinformatic analysis demonstrated that Acanthamoeba has the full complement of enzymes necessary for histidine biosynthesis. The complete coding sequence for a novel multifunctional gene, histidinol dehydrogenase (EC 1.1.1.23) and IGPD (EC 4.2.1.19) have both been amplified from A. castellanii and A. polyphaga cDNA, both have been cloned and sequenced. The gene encoding histidinol dehydrogenase from Acanthamoeba has been found to encode several enzymes including imidazoleglycerol phosphate synthase, phosphoribosyl-formimino-5-amino-1-imidazole-carboxyamide ribotide isomerase, phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphatase, histidinol dehydrogenase and ATP Phosphoribosyltransferase. An experimental compound (C348) previously demonstrated to inhibit IGPD of plant species was found to restrict the growth of Acanthamoeba (IC50 305nM). This inhibition was also ablated with the addition of exogenous histidine when added to the medium. C348 showed no cytotoxicity effects to the PC3-luc at concentrations restricting the growth of Acanthamoeba species.

Ubiquitous amoebae from the genus Acanthamoeba are associated with two main serious infections: The more common eye disease acanthamoeba keratitis (AK), which can result in blindness, and the rare and often fatal disease affecting the central nervous system, granulomatous amoebic encephalitis (GAE). The traditional morphological taxonomic system for Acanthamoeba is based on cyst size and shape, and divides the amoebae into three groups (I, II and III). Since the discovery that cyst shape can be modified by culture conditions, the classic system has become largely redundant. A more robust system has been developed, based on the nucleotide sequence of the 18S rRNA (Rns) gene. It types Acanthamoeba into 15 T-groups, with most species including environmental and clinical clumped into three groups T3, T4 and T11, with little resolution between them. Although speciation does not help cure patients directly, it can provide valuable information regarding disease epidemiology and ultimately benefit patient prognosis. Here a system to better resolve strains has been developed, using the mitochondrial cytochrome oxidase subunit 1 and 2 (cox1/2) gene sequence. When used in conjunction with the T-group system, resolution between strains including those with a T3, T4 or T11 genotype is obtained. Additionally the combined approach identified a mixed infection in a patient suffering with AK, and the occurrence of Acanthamoeba strains with multiple alleles of 18S and cox1/2 genes. The combined use of both genotyping systems was used to investigate an unprecedented outbreak of GAE within a Swedish hospital. Results confirmed Acanthamoeba had infected several immunocompromised paediatrics from a single ICU, and the source was from within the unit’s water system. In vitro assays were used to test the strains pathogenic abilities and sensitivities to antimicrobial compounds, identifying if they are more virulent than typical strains of Acanthamoeba.

Acanthamoeba castellanii est une amibe libre ubiquiste de l'environnement. Elle se nourrit principalement de micro-organismes par phagocytose. Seulement, certains micro-organismes ont développé des mécanismes de résistances qui leur permettent d'échapper à la digestion et même de se multiplier à l'intérieur des amibes. C'est le cas de Legionella pneumophila, bactérie responsable de la légionellose. Legionella pneumophila, à travers son système de sécrétion Dot/Icm, injecte plusieurs effecteurs à l'intérieur de son hôte. Ces effecteurs interagissent avec les protéines de l'hôte, et induisent une modification de la physiologie de son hôte, à son avantage. Durant ma thèse, nous nous sommes intéressés aux effets de Legionella pneumophila, sur la prolifération de son hôte amibien. Nous avons montré que Legionella pneumophila arrête la prolifération d'Acanthamoeba castellanii. Ce phénotype était associé une modification de la forme, à une perte d'adhérence et à une baisse de motilité de l'amibe. Sur le plan moléculaire, Legionella pneumophila induit une baisse dans l'expression du gène cdc2b, qui présente des similarités avec le gène cdk1 (cyclin dépendant kinase), codant pour la CDK essentielle au déroulement du cycle cellulaire chez les mammifères. L'arrêt de la prolifération d'Acanthamoeba castellanii, qui passe par une réduction d'expression de cdc2b, est certainement induit par un ou plusieurs effecteur(s) de Legionella pneumophila, car le mutant ΔdotA de L. pneumophila, défectueux au niveau de l'appareil de sécrétion Dot/Icm, n'induit pas l'arrêt de la prolifération d'Acanthamoeba castellanii. / Acanthamoeba castellanii is an ubiquitous free-living amoeba of the environment. This amoeba feeds mainly on micro-organisms by phagocytosis. However, some micro-organisms have acquired resistances that allow them to escape digestion and even multiply inside amoebae. This is the case of Legionella pneumophila, the bacterium responsible for legionellosis. Legionella pneumophila, through its Dot/Icm secretion system, injects several effectors into its host. These effectors interact with the proteins of the host, and induce a modification of the physiology of its host, to its advantage. During my phD, we were interested in the effects of Legionella pneumophila infection on the proliferation of its amoebic host. We showed that Legionella pneumophila prevents the proliferation of Acanthamoeba castellanii. This phenotype was associated with a modification of the shape, a loss of adhesion and a decrease in motility of the amoeba. On the molecular level, Legionella pneumophila induces a decrease in the expression of the cdc2b gene, which share similarities with the cdk1 (cyclin dependent kinase) gene, coding for the major CDK of the mammalian cells cycle. The arrest of proliferation of Acanthamoeba castellanii, which involves a reduction in expression of cdc2b, is certainly induced by one or more effector(s) of Legionella pneumophila, because the mutant ΔdotA of L. pneumophila, defective in the Dot/Icm secretion apparatus, does not induce proliferation arrest of Acanthamoeba castellanii.