Acanthamoeba is an opportunistic amphizoic protozoan which causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic mechanism, is used by eukaryotes to control their gene expression. DNA-methyltransferase gene family (dnmt) was studied to determine the possibility of its involvement in programming Acanthamoeba pathogenicity. A virulence-attenuated Acanthamoeba isolate (designation: ATCC) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of dnmt between the ATCC and AC/5 isolates was performed by quantitative reverse transcription PCR (RT-qPCR). Silencing of dnmt in the AC/5 isolate was achieved by small interfering RNA (siRNA). Total RNAs extracted from the ATCC, AC/5 and siRNA-treated (designation: si-146) Acanthamoeba were subjected to RNA sequencing for transcriptomic studies. Pathogenicity assays demonstrated that the AC/5 isolate exhibited greater virulence potential after mouse passages. RT-qPCR revealed that the AC/5 isolate had an almost 15-fold increase in dnmt expression compared to the ATCC isolate (p ≤ 0.001). si-146 duplex significantly reduced the expression of dnmt in the AC/5 isolate by 30%. Comparative transcriptomic analysis identified differentially expressed genes (DEGs), with 1,717 genes in AC/5 versus ATCC; 778 genes in si-146 versus AC/5; and 1,513 genes in si-146 versus ATCC, respectively (log2 fold change of ≥ 2 or ≤ -2 and adjusted p-value of < 0.05). Functional analysis of DEGs in AC/5 versus ATCC and si-146 versus AC/5 using eukaryotic orthologous groups, gene ontology and Kyoto encyclopaedia of genes and genomes pathway revealed that a high percentage of DEGs was related to post-translational modifications, signal transduction, oxidation-reduction and metabolic processes. Of these, a wide range of genes associated with encystation, proteolysis, host-cell adhesion, oxidative stress defence, among others, was identified and could be transcriptionally regulated by dnmt. Decreased in the expression levels of these genes may account in part for Acanthamoeba reduced pathogenicity. These candidate genes could represent potential drug targets for therapeutic interventions. To the best of the author’s knowledge, this is the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba sp. The present data not only provided fundamental understanding of the mechanisms underlying Acanthamoeba virulence, but also identified potential targets for anti-acanthamoebic drugs.

Acanthamoeba parasites as aetiological agents for the sight threatening Acanthamoeba keratitis and the rare granulomatous amoebic encephalitis in humans were officially recognised in the 1970s. Since then, increasing numbers of Acanthamoeba-associated keratitis and encephalitis cases have been reported worldwide. In Malaysia, the first Acanthamoeba keratitis case was reported in 1995, in Hospital Kuala Lumpur. The first case was a female patient contact lens wearer. Subsequently, several more keratitis cases have been diagnosed in patients at the Hospital Kuala Lumpur and Hospital Universiti Kebangsaan Malaysia. However, reports of these cases have not been officially published. Granulomatous amoebic encephalitis has also been diagnosed in Hospital Universiti Sains, Kubang Kerian, Malaysia, but the report has not been officially published. Although Acanthamoeba organisms are ubiquitously distributed in the nature, only a few species are pathogenic to human. Many research groups have carried out studies on parasite characteristics which contribute to Acanthamoeba pathogenicity. Characteristics such as tolerance to high temperature, high osmotic pressure, extreme pH, ability to kill target cells in vitro and to cause pathological lesions in experimental animals, are usually evaluated for clinical isolates of Acanthamoeba. These properties are also useful indicators for predicting the pathogenicity of environmental isolates of Acanthamoeba. In Malaysia, thus far, no pathogenicity study has been conducted on any of the local isolates. The current study is based on isolates of Acanthamoeba spp. from dust samples of air-conditioners in Malaysia. These isolates were assessed for their pathogenic potential based on their physical tolerance to harsh growth condition, presence of molecular markers associated with pathogenicity, the ability to cause glial cell death in vitro and to infect and cause lesions in Balb/c mice. Prior to the pathogenicity characterisation, theidentities of these isolates were determined morphologically and molecularly. The associated mechanism for pathogenesis of Acanthamoeba was investigated using electron microscopy. Acanthamoeba organisms were cultured from dust samples using NNA culture plates incubated at ambient temperature (26C 2C) for up to a month. Twenty-four primary cultures were tested positive for Acanthamoeba spp. based on microscopy and PCR detection. Selected environmental isolates were axenised using HCl acid and gentamicin treatment to eliminate bacteria contaminants. Established axenic Acanthamoeba strains were determined for their clonality using PCR and sequencing. Twenty-one pure-cloned isolates designated as IMU1 to IMU21, were characterised morphologically and molecularly. Two keratitis isolates designated HTH136 and HKL55 were also included in the study. Both clinical isolates were similarly axenised and characterised morphologically and molecularly. Five species were identified according to the morphological criteria of Pussard and Pons (1977) and Page (1988) keys. These species were A. castellanii (IMU1 to IMU3, IMU6, IMU7, IMU9 and IMU18); A. culbertsoni (IMU10 to IMU13); A. griffini (IMU14); A.lenticulata (IMU16 and IMU17), and A. polyphaga (IMU8, IMU19 and HTH136). Species identities for the remaining six isolates (IMU4, IMU5, IMU15, IMU20, IMU21 and HKL55), however, could not be determined morphologically. At molecular characterisation, IMU14 was placed into T3 genotype; IMU16 and IMU17 were clustered in T5 genotype whereas the two clinical isolates and other environmental isolates were placed into T4 genotype. To predict the potential pathogenicity of the Acanthamoeba isolates used in the study, PCR primer pairs which could differentiate the pathogenic from non-pathogenic strains, temperature tolerance tests and osmotic tolerance tests were employed. Many isolates were predicted as potential pathogens based on the results of these tests. The virulence of the potential pathogenic strains was further confirmed by their ability to cause glial cell death in in vitro cytopathogenic assays. Seven environmental isolates (IMU9, IMU10, IMU14, IMU16, IMU17, IMU18 and IMU19) and the two clinical isolates (HTH136 and HKL55) were selected for pathogenicity studies in Balb/c mice. At 30 days post-infection, none of the mice succumbed to the infection with any of the Acanthamoeba isolates tested. However, pathological changes were detected in the liver and lung of mice infected with all the tested Acanthamoeba isolates. The lung was the main organ affected by Acanthamoeba. Infection caused by the more virulent Acanthamoeba isolates resulted in bronchiolitis, bronchopneumonia, and interstitial pneumonia whereas infection by the less virulent isolates provoked mild interstitial lung inflammation. Trophozoites were only occasionally seen within lung tissues. Often, Acanthamoeba organisms were not detected in these lesions; immune complex deposition was probably the predominant cause for the chronic inflammation in affected organ. It has been reported that the clinical and histopathological features of Acanthamoeba infection in humans and mice are essentially the same. Since there are similar pathological changes seen in mice infected with clinical isolates and the environmental isolates tested in the present study, it is concluded that these seven environmental strains can be considered as potential human pathogenic isolates. Electron microscopy was employed to gain some insight into the mechanism of glial cell death caused by one of the experimentally pathogenic strain - IMU17. Electron microscopy showed that the physical contact of Acanthamoeba trophozoites to glial cells could trigger apoptosis and necrosis of the target cells. Phagocytosis of glial cells by trophozoites was also observed. To our knowledge, this is the first report presenting the isolation and molecular characterisation of potential pathogenic Acanthamoeba spp. in indoor air-conditioners in Malaysia. Building occupants therefore should be aware of the risk of acquiring air-borne Acanthamoeba infection through the use of poorly maintained air-conditioners.

Acanthamoeba is ubiquitous free living amoebae in the soil and water. It is recognized as opportunistic pathogens that can cause ulcerative keratitis in contact lens wearer and granulomatous amoebic encephalitis in immune-compromised patients. The present work shows the detection of Acanthamoeba cyst in the mechanical ventilation and air conditioning system could give an indication of the ventilation system faulty. Dust swab on the supplying diffuser is a better approach when assessing the organism in the indoor ambient compared to single stage impactor sampler and dust particulates sampler. A logistic regression model was developed based on field data and human experiences obtained from 107 sampling points from nine industrial indoor air quality assessments. The organism is statistically correlated with total fungi count and respirable particulates, PM10 found within the ambient. It is also positively correlated with the sick building syndrome experienced by the indoor occupants. Efforts should be taken to consider Acanthamoeba as one of the ambient indicator when practicing indoor air quality assessment.