Acanthamoeba, a widely distributed free-living amoeba found in various environments, is an opportunistic pathogen responsible for causing Acanthamoeba keratitis, a condition that may lead to blindness. However, identifying the pathogenicity of Acanthamoeba is challenging due to its complex life cycle, ability to adapt to different environments, variable virulence factors, and intricate interactions with the host immune system. Additionally, the development of an effective model for studying Acanthamoeba pathogenicity is limited, hindering a comprehensive understanding of the mechanisms underlying its virulence and host interactions. The aim of this study was to develop an ex vivo model for Acanthamoeba infection using porcine eyeballs and to evaluate the pathogenicity of the Acanthamoeba isolates. Based on slit lamp and biopsy analysis, the developed ex vivo model is capable of successfully infecting Acanthamoeba within 3 days. Histopathological staining revealed that clinical isolates of Acanthamoeba exhibited greater corneal stroma destruction and invasion in this model than environmental isolates. Our results highlight the importance of an ex vivo porcine eye model in elucidating the pathogenesis of Acanthamoeba infection and its potential implications for understanding and managing Acanthamoeba-related ocular diseases.

UNASSIGNED: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood.
UNASSIGNED: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses.
UNASSIGNED: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment.
UNASSIGNED: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.

Pathogenic and free-living Acanthamoeba are widely distributed in the environment and have been reported to cause keratitis and universally fatal encephalitis. Primary cutaneous acanthamoebiasis caused by Acanthamoeba is exceedingly rare and presents as isolated necrotic cutaneous lesions without involvement of the cornea or central nervous system. Cutaneous acanthamoebiasis often occurs in immunocompromised patients and is likely overlooked or even misdiagnosed only by cutaneous biopsy tissue histopathological analysis. Here, we report a HIV-infected 63-year-old female with oral leukoplakia for 4 months and scattered large skin ulcers all over the body for 2 months. The cause of the cutaneous lesions was unclear through cutaneous specimens histopathological analysis, and subsequently Acanthamoeba were detected by metagenomic next-generation sequencing (mNGS), which may be the cause of cutaneous lesions. Based on the mNGS results, a pathologist subsequently reviewed the previous pathological slides and found trophozoites of Acanthamoeba so that the cause was identified, and the skin ulcers improved significantly after treatment with multi-drug combination therapy. Acanthamoeba is also a host of pathogenic microorganisms. The presence of endosymbionts enhances the pathogenicity of Acanthamoeba, and no other pathogens were reported in this case. mNGS is helpful for rapidly diagnosing the etiology of rare skin diseases and can indicate the presence or absence of commensal microorganisms.

Acanthamoeba, a free-living amoeba, is commonly found in various natural environments, such as rivers and soil, as well as in public baths, swimming pools, and sewers. Acanthamoeba can cause severe illness such as granulomatous amoebic encephalitis and Acanthamoeba keratitis (AK) in humans. AK, the most recognized disease, can cause permanent visual impairment or blindness by affecting the cornea. AK commonly affects contact lens wearers who neglect proper cleaning habits. The symptoms of AK include epithelial and stromal destruction, corneal infiltrate, and intense ocular pain, occasionally necessitating surgical removal of the entire eyeball. Current AK treatment involves the hourly application of eye drops containing polyhexamethylene biocide (PHMB). However, studies have revealed their ineffectiveness against drug-resistant strains. Acanthamoeba can form cysts as a survival mechanism in adverse environments, though the exact mechanism remains unknown. Our experiments revealed that sodium P-type ATPase (ACA1_065450) is closely linked to encystation. In addition, various encystation buffers, such as MgCl2 or NaCl, induced the expression of P-type ATPase. Furthermore, we used ouabain, an ATPase inhibitor, to inhibit the Na+/K+ ion pump, consequently decreasing the encystation rate of Acanthamoeba. Our primary objective is to develop an advanced treatment for AK. We anticipate that the combination of ouabain and PHMB may serve as an effective therapeutic approach against AK in the future.

Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth\'s history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth\'s history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.

Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However, the interaction between these microbes and the origin of hypermucoviscous K. pneumoniae have not been reported.
Here, we report that the bacterial capsule is enlarged when co-cultured with Acanthamoeba using India ink staining, and this effect depends on the number of parasites present. This interaction results in an enhancement of capsular polysaccharide production in the subsequent generations of K. pneumoniae, even without co-culturing with Acanthamoeba. The hypermucoviscosity of the capsule was examined using the sedimentation assay and string test. We also screened other K. pneumoniae serotypes, including K1, K2, K5, and K20, for interaction with Acanthamoeba using India ink staining, and found the same interaction effect.
These findings suggest that the interaction between Acanthamoeba and K. pneumoniae could lead to harmful consequences in public health and nosocomial disease control, particularly hypermucoviscous K. pneumoniae infections.

Acanthamoeba is an opportunistic protozoa, which exists widely in nature and is mainly distributed in soil and water. Acanthamoeba usually exists in two forms, trophozoites and cysts. The trophozoite stage is one of growth and reproduction while the cyst stage is characterized by cellular quiescence, commonly resulting in human infection, and the lack of effective monotherapy after initial infection leads to chronic disease. Acanthamoeba can infect several human body tissues such as the skin, cornea, conjunctiva, respiratory tract, and reproductive tract, especially when the tissue barriers are damaged. Furthermore, serious infections can cause Acanthamoeba keratitis, granulomatous amoebic encephalitis, skin, and lung infections. With an increasing number of Acanthamoeba infections in recent years, the pathogenicity of Acanthamoeba is becoming more relevant to mainstream clinical care. This review article will describe the etiological characteristics of Acanthamoeba infection in detail from the aspects of biological characteristic, classification, disease, and pathogenic mechanism in order to provide scientific basis for the diagnosis, treatment, and prevention of Acanthamoeba infection.

Acanthamoeba keratitis is a rare parasitic infection of the cornea that can lead to permanent blindness if not diagnosed and treated promptly. We collected data on the incidences of Acanthamoeba keratitis from 20 countries and calculated an annual incidence of 23,561 cases, with the lowest rates in Tunisia and Belgium, and the highest in India. We analyzed 3755 Acanthamoeba sequences from the GenBank database across Asia, Europe, North America, South America, and Oceania and genotyped them into T1, T2, T3, T4, T5, T10, T11, T12, and T15. Many genotypes possess different characteristics, yet T4 is the most prevalent genotype. As efficient treatment against Acanthamoeba remains lacking, prevention from early diagnosis via staining, PCR, or in vivo confocal microscopy (IVCM) becomes significant for the condition\'s prognosis. IVCM is the most recommended approach for the early detection of Acanthamoeba. If IVCM is unavailable, PCR should be used as an alternative.

BACKGROUND: Acanthamoeba is one of the opportunistic parasites with a global prevalence. Currently, due to the side effects and the emergence of drug resistance to this parasite, much research has been performed on the use of nano-drugs to treat Acanthamoeba-caused diseases. Therefore, this systematic review study aims to evaluate new strategies for treating diseases caused by Acanthamoeba based on nanoparticles (NPs).
METHODS: We designed a systematic review based on the articles published in English between 2000 and 2022. Our search strategy was based on syntax and specific tags for each database, including ScienceDirect, PubMed, Scopus, Ovid, and Cochrane. From the articles, those that had inclusion criteria were selected, and their data were extracted and analyzed.
RESULTS: In this study, 26 studies were selected. Metallic nanoparticles were mostly used against the Acanthamoeba species (80.7%). 19.2% of the studies used polymeric nanoparticles, and 3.8% used emulsion nanoparticles. Most studies (96.1%) were performed in vitro, and only one study (3.8%) was carried out in vivo. Silver NPs were the most used metallic nanoparticles in the studies. The best effect of the anti-Acanthamoeba compound was observed for green synthesized nanoparticles based on stabilization by plant gums, loaded with citrus fruits flavonoids hesperidin (HDN) and naringin (NRG) with a 100% growth inhibition at a concentration of 50 μg/mL.
CONCLUSIONS: This study showed that chlorhexidine and other plant metabolites loaded with silver and gold nanoparticles increase the anti-Acanthambae activity of these nanoparticles. However, green synthesized nanoparticles based on stabilization by plant gums, loaded with citrus fruits flavonoids hesperidin (HDN) and naringin (NRG), showed the best anti-Acanthambae effect. Nevertheless, further studies should be performed to determine their safety for human use.

Acanthamoeba species are among the most ubiquitous protists that are widespread in soil and water and act as both a replicative niche and vectors for dispersal. They are the most important human intracellular pathogens, causing Acanthamoeba keratitis (AK) and severely damaging the human cornea. The sympatric lifestyle within the host and amoeba-resisting microorganisms (ARMs) promotes horizontal gene transfer (HGT). However, the genomic diversity of only A. castellanii and A. polyphaga has been widely studied, and the pathogenic mechanisms remain unknown. Thus, we examined 7 clinically pathogenic strains by comparative genomic, phylogenetic, and rhizome gene mosaicism analyses to explore amoeba-symbiont interactions that possibly contribute to pathogenesis. Genetic characterization and phylogenetic analysis showed differences in functional characteristics between the \"open\" state of T3 and T4 isolates, which may contribute to the differences in virulence and pathogenicity. Through comparative genomic analysis, we identified potential genes related to virulence, such as metalloprotease, laminin-binding protein, and HSP, that were specific to the genus Acanthamoeba. Then, analysis of putative sequence trafficking between Acanthamoeba and Pandoraviruses or Acanthamoeba castellanii medusaviruses provided the best hits with viral genes; among bacteria, Pseudomonas had the most significant numbers. The most parsimonious evolutionary scenarios were between Acanthamoeba and endosymbionts; nevertheless, in most cases, the scenarios are more complex. In addition, the differences in exchanged genes were limited to the same family. In brief, this study provided extensive data to suggest the existence of HGT between Acanthamoeba and ARMs, explaining the occurrence of diseases and challenging Darwin\'s concept of eukaryotic evolution. IMPORTANCE Acanthamoeba has the ability to cause serious blinding keratitis. Although the prevalence of this phenomenon has increased in recent years, our knowledge of the underlying opportunistic pathogenic mechanism maybe remains incomplete. In this study, we highlighted the importance of Pseudomonas in the pathogenesis pathway using comprehensive a whole genomics approach of clinical isolates. The horizontal gene transfer events help to explain how endosymbionts contribute Acanthamoeba to act as an opportunistic pathogen. Our study opens up several potential avenues for future research on the differences in pathogenicity and interactions among clinical strains.