Biology and pathogenesis of Acanthamoeba
© Siddiqui and Khan; licensee BioMed Central Ltd. 2012
Received: 22 July 2011
Accepted: 10 January 2012
Published: 10 January 2012
Acanthamoeba is a free-living protist pathogen, capable of causing a blinding keratitis and fatal granulomatous encephalitis. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread and host susceptibility, and are highlighted together with potential therapeutic and preventative measures. The use of Acanthamoeba in the study of cellular differentiation mechanisms, motility and phagocytosis, bacterial pathogenesis and evolutionary processes makes it an attractive model organism. There is a significant emphasis on Acanthamoeba as a Trojan horse of other microbes including viral, bacterial, protists and yeast pathogens.
Discovery of Amoebae
Entamoeba histolytica is a parasitic protist that was discovered in 1873 from a patient suffering from bloody dysentery [7, 8] and named E. histolytica in 1903 [9, 10]. This species was separated into one pathogenic (E. histolytica) and another non-pathogenic (E. dispar) , which also is capable of producing experimental lesions  and questioned by some authors if really it is unable to cause human disease .
Naegleria is a free-living amoebae that was first discovered by Schardinger in 1899, who named it "Amoeba gruberi". In 1912, Alexeieff suggested its genus name as Naegleria, and much later in the 1970, Carter identified Naegleria fowleri as the causative agent of fatal human infections involving the central nervous system (CNS) .
Sappinia diploidea is a free-living amoeba that was isolated from the faeces of lizards and from the soil in 1908-09, and then described as a causative agent of granulomatous amoebic encephalitis in 2001 .
Balamuthia mandrillaris was discovered in 1986, from the brain of a baboon that died of meningoencephalitis and was described as a new genus, i.e., Balamuthia[3, 16]. So far, only one species has been identified, B. mandrillaris. The majority of isolates have been isolated from necropsies while organic-rich soil has been suggested as a potential source. Like Acanthamoeba, it is known to produce infections of the central nervous system, lungs, sinuses and skin. Worryingly, granulomatous encephalitis due to B. mandrillaris has been reported in immunocompetent individuals indicating its potential threat to human and animal health.
In 1930, Acanthamoeba was discovered as a contaminant of yeast culture, Cryptococcus pararoseus and was later placed in the genus Acanthamoeba, and then described as a causative agent of Acanthamoeba granulomatous encephalitis (AGE) in the 1960s and of keratitis in 1970s .
Biology of Acanthamoeba
The term acanth (Greek "acanth" means "spikes") was added to "amoeba" to indicate the presence of spine-like structures (now known as acanthopodia) on its surface. It contains one or more prominent contractile vacuoles, whose function is to expel water for osmotic regulation . Other types of vacuoles in the cytoplasm include lysosomes, digestive vacuoles and a large number of glycogen-containing vacuoles. The plasma membrane consists of proteins (33%), phospholipids (25%), sterols (13%), and lipophosphonoglycan (29%) [19, 20]. The major phospholipids in Acanthamoeba are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), phosphoinositide (6%), and diphosphatidylglycerol (4%). The main fatty acids chains in Acanthamoeba are oleic acids (40-50%), and longer polyunsaturated fatty acids (20-30%) . Acanthamoeba contains low levels of glycolipids. Glucose accounts for about 60% of the sugars of the glycolipids of the whole cells and of the plasma membranes. Among sterols, the non-saponifiable fraction of the total lipids extracted from the trophozoites of pathogenic Acanthamoeba possesses ergosterol and 7-dehydrostigmasterol . Acanthamoeba has been shown to produce prostaglandins .
Acanthamoeba has long been studied as a model eukaryotic cell with special emphasis on the actin cytoskeleton-based motility . Acanthamoeba moves relatively fast compared to other cells, with a locomotory rate of approximately 0.8 μm/second. The movement involves the formation of a hyaline pseudopodium. The manner of Acanthamoeba movement is similar both at solid substrata and water-air interface. Adhesion forces developed between Acanthamoeba and the water-air interface are greater than gravity, and thus amoebae are also transported passively without detachment from the water surface . Actin microfilaments are most concentrated just beneath the plasma membrane, and are responsible for resisting tension and forming cytoplasmic protrusions.
Life cycle of Acanthamoeba
Glycosyl linkage analysis of Acanthamoeba castellanii cyst wall saccharides (reproduced with permission from Dudley et al., 2009).
5 linked Xylofuranose/4XylP
3 linked Galactopyranose
4 linked Glucopyranose
3,4 linked Galactopyranose
3,4 linked Glucopyranose
2,4 linked Gluco or Galactopyranose
4,6 linked Mannopyranose
3,6 Linked Galactopyranose
Distribution in the environment and clinical settings
Acanthamoeba has been isolated from diverse natural environments including sea water, ocean sediments, beaches, pond water, soil, fresh water lakes, hot spring resorts, salt water lakes, Antarctica, water-air interface, and even from the air. They have been isolated from bottled mineral water, distilled water bottles, thermally-polluted factory discharges, cooling towers of the electric and nuclear power plants, Jacuzzi tubs, ventilation ducts, humidifiers, air-conditioning units, shower heads, kitchen sprayers, sewage, compost, vegetables, surgical instruments, contact lenses and their cases, pigeon droppings, fresh water fish, as well as other healthy, diseased, and dead animals. They have been recovered from hospitals, physiotherapeutical swimming pools, dialysis units, portable and stationary eye wash stations, human nasal cavities, throat, pharyngeal swabs, lung tissues, skin lesions, human faeces, corneal biopsies, maxillary sinus, mandibular autografts, stool samples, urine of critically ill patients, cerebrospinal fluids and the brain necropsies. Based on the above, it is accepted that Acanthamoeba is ubiquitously present in the environment and that we commonly encounter this organism in our routine lives as evidenced by the presence of anti-Acanthamoeba antibodies in up to 100% healthy populations in New Zealand and more than 85% in individuals of London that came from different countries [33, 34].
Role in the Ecosystem
In soil, protists such as amoebae, flagellates and ciliates have two major ecological roles: (i) influencing the structure of the microbial community, and (ii) enhancing nutrient recycling. Both of these activities are associated with soil protists feeding on bacteria thus regulating bacterial populations in the soil. Among protists, free-living amoebae are the dominant bacterial consumers and are responsible for up to 60% of the total reduction in bacterial population . The primary decomposers (bacteria) directly decompose organic materials but are inefficient in releasing minerals from their own mass. The secondary decomposers, such as free-living amoebae, consume the primary decomposers and release mineral nutrients as waste products that are tied up in the primary decomposer's biomass. In this way, protists such as Acanthamoeba (as well as other grazers) make nutrients available that would otherwise remain inaccessible for much longer. The soil containing Acanthamoeba and bacteria showed significantly greater mineralization of carbon, nitrogen, and phosphorous compared with the soil containing bacteria but without Acanthamoeba [36, 37]. As well as bacterial consumption, amoebae promote bacterial populations in the soil. The mineral regeneration by the secondary decomposers (protists such as amoebae), relieved nutrient limitation for the primary decomposers. This was demonstrated with the findings that when nitrogen was limiting (but carbon present), nitrogen mineralization by Acanthamoeba permitted continued growth of bacteria (Pseudomonas paucimobilis) resulting in a greater bacterial biomass [36, 37]. And when carbon was limiting, Acanthamoeba was almost entirely responsible for nitrogen mineralization, with bacteria (Pseudomonas paucimobilis) contributing little. Using an experimental model system, the effects of grazing by Acanthamoeba on the composition of bacterial communities in the rhizosphere of Arabidopsis thaliana demonstrated reduction in bacterial populations leading to positive effect on plant growth [35–37]. Overall, Acanthamoeba appears to play an important role in the regulation of bacterial populations in the environment and the nutrient cycling, thus contributing to the functioning of the ecosystems.
Known Acanthamoeba genotypes and their associations with human diseases, i.e., keratitis and granulomatous encephalitis.
Human disease association
^T2b - ccap1501/3c-alike sequences
The diagnosis of AK is problematic and it is often misdiagnosed as bacterial, viral or fungal keratitis. The use of contact lenses by the patient together with excruciating pain is strongly indicative of this infection. The use of in vivo confocal microscopy has emerged as a valuable non-invasive tool for the clinical diagnosis in severe infectious keratitis with high sensitivity [50, 51]. The confirmatory evidence comes from demonstrating parasites using laboratory-based assays. The cultivation of Acanthamoeba from the corneal biopsy or from contact lenses/cases remains the most widely used assay. Immunofluorescence assays and multiplex real-time PCR methods  have also been developed. The multiplex assay is of value for the simultaneous detection of pathogenic free-living amoebae in the same sample. The use of real-time fast-duplex TaqMan PCR for the simultaneous detection of 10 different genotypes of Acanthamoeba can detect 0.1 cyst/μl . In addition, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and 1H NMR spectroscopy has been shown to be of potential value in the rapid identification of Acanthamoeba in the clinical specimens.
Early diagnosis followed by aggressive treatment is essential for the successful prognosis. No single agent is shown to be uniformly effective against all isolates/genotypes of Acanthamoeba. Multiple factors including varied clinical presentation and virulence of Acanthamoeba account for a lack of correlation between in vitro activity and in vivo efficacy. The treatment regimen includes polyhexamethylene biguanide or chlorhexidine digluconate together with propamidine isethionate or hexamidine, is effective. If bacteria are also associated with the infection, addition of antibiotics, i.e., neomycin or chloramphenicol is recommended .
Acanthamoeba granulomatous encephalitis
AGE is a rare infection but it almost always proves fatal. It is of major concern in view of increasing numbers of immunocompromised patients who are susceptible hosts, individuals undergoing immunosuppressive therapy and excessive use of steroids. Individuals with lymphoproliferative or hematologic disorders, diabetes mellitus, pneumonitis, renal failure, liver cirrhosis or other hepatic diseases, gamma-globulinaemia or patients undergoing organ/tissue transplantation with immunosuppressive therapy, steroids and excessive antibiotics are at risk [55, 56]. The gross pathology of the autopsied brains show severe edema and haemorrhagic necrosis. The microscopic findings of the post-mortem necropsies reveal amoebae cysts, predominantly in the perivascular spaces in the parenchyma indicating involvement of the cerebral capillaries as the sites of amoebae entry into the CNS. It is widely accepted that the route of entry for Acanthamoeba include the respiratory tract leading to amoebae invasion of the alveolar blood vessels, followed by the haematogenous spread. Acanthamoeba entry into the CNS most likely occurs through the blood-brain barrier [55, 56]. As AGE is a secondary infection, it is difficult to determine its true burden on human health. The approximate rate of AGE-associated deaths has been suggested as 1.57 deaths per 10,000 HIV/AIDS deaths .
The symptoms of AGE are similar to other CNS infections including virus, bacteria and fungi. The neurological manifestations of AGE may vary and include headache, fever, behavioral changes, hemiparesis, lethargy, stiff neck, agitation, aphasia, ataxia, vomiting, nausea, cranial nerve palsies, increased intracranial pressure, seizures, and coma [55, 56]. The magnetic resonance imaging or computerized tomography of the brain shows ring-enhancing lesions exhibiting a single or multiple space-occupying mass in the cerebral cortex but severely immunocompromised patients may not exhibit such lesions. The CSF findings shows pleocytosis with lymphocytic predominance, increased protein concentrations, decreased glucose concentrations and minimal cloudiness , however the CSF may be devoid of cells in HIV-positive patients. High levels of Acanthamoeba-specific antibodies in patient's serum is indicative of AGE infection. The antibody levels in normal populations may be in the range of 1:20 to 1:60 [33, 57] but patients with severely impaired immune system may not develop a high titre. The confirmatory evidence comes from demonstration of amoebae in the infected tissues.
There is no recommended treatment and the majority of cases are diagnosed at the post-mortem stage. A lack of available antiamoebic compounds together with selectivity of the blood-brain barrier has led to more than 90% mortality rate. Few successful cases involved the use of ketoconazole, fluconazole, sulfadiazine, pentamidine isethionate, amphotericin B, azithromycin, itraconazole, or rifampicin [1, 58, 59] but overall the prognosis remains poor.
Parasite adhesion to the host cell is a primary step and is mediated by a 130 kDa mannose-binding protein (MBP) expressed on the surface of Acanthamoeba . Acanthamoeba mbp consists of 6 exons and 5 introns that spans 3.6 kbp. The 2.5 kbp cDNA codes for an 833 amino acids precursor protein with a signal sequence (residues 1-21aa), an N-terminal extracellular domain (residues 22-733aa) with five N- and three O-glycosylation sites, a transmembrane domain (residues 734-755aa), and a C-terminal intracellular domain (residues 756-833aa). Other adhesins include a laminin-binding protein with a predicted molecular mass of a 28.2 kDa, a 55 kDa laminin-binding protein and a > 207 kDa adhesin [61, 62]. The initial binding leads to secondary events such as phagocytosis and toxin production resulting in the host cell death in a phosphotidylinositol 3-kinasedependent (PI3K) manner . The downstream effectors of PI3K involves activation of proapoptotic molecules, Bak and Bax, loss of mitochondrial membrane potential and release of cytochrome c as well as caspase activation, all well-known mediators of the apoptosis [64–66]. Among host cell receptors, Toll-like receptor 4 (TLR4) showed involvement in Acanthamoeba recognition and exerting an effect through adaptor protein, Myeloid differentiation primary response 88 that led to the activation of transcription factors, nuclear factor-kappa B signalling through extracellular signal-regulated kinases (ERKs) inducing the secretion of cytokines including interleukin-8, tumor necrosis factor-alpha and interferon-beta in human corneal cells . Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, it is shown that Acanthamoeba abolished the HBMEC transendothelial electrical resistance by degrading occludin and zonula occludens-1 tight junction proteins in a Rho kinase-dependent manner leading to increased permeability .
Of contact-independent factors, Acanthamoeba possess hydrolytic enzymes including elastases , phospholipases , glycosidases and a variety of serine, cysteine and metalloproteases (Figure 6) [2, 3, 42, 43]. However, their precise mechanisms of action at the molecular-level are only beginning to emerge. That some of the above proteases are secreted only by the clinical isolates may indicate their role as potent virulence factors and/or diagnostic targets. Future studies in the role of proteases as vaccine targets, search for novel inhibitors by screening of chemical libraries, or rational development of drugs based on structural studies will enhance our ability to target this pathogen. Overall, the mechanism by which Acanthamoeba breaches biological barriers is complex and is likely to involve both parasite (adhesins, proteases, phospholipases) as well as host determinants [interleukin-beta, interleukin-alpha, tumor necrosis factor- alpha, interferon-gamma, host cell apoptosis]. In addition to aforementioned potential virulence factors, the ability of Acanthamoeba to survive harsh environmental conditions and its resistance to chemotherapeutic drugs by differentiating into cysts contributes to its pathogenicity.
Immune response to Acanthamoeba infections
The recurrence of AK infections is common, suggesting that the corneal infection alone does not induce protective immunity against the parasite antigens. Experimental animals immunized orally with Acanthamoeba antigens mixed with the cholera toxin showed significantly lower infection rates compared with the control groups (21.4% versus 72.6% respectively) and protection was associated with higher levels of parasite-specific sIgA. More specifically, oral immunization using recombinant MBP improved AK and protection was associated with the presence of elevated levels of anti-MBP sIgA in tears of the immunized animals . Similarly, oral immunization with a serine protease (~133 kDa) reduced the severity of the corneal infection by modulating MMP-2 and MMP-3 expression . Overall, it is suggested that AK patients show decreased overall levels of sIgA as well as specific anti-Acanthamoeba sIgA, however the role of sIgA was questioned in a recent study in which neither normal tears nor AK tears had any protective effects on Acanthamoeba-mediated corneal epithelial cell cytotoxicity. Tear factors, in addition to sIgA such as lysozyme, lactoferrin, beta-lysins, prostoglandins, and other compounds with antimicrobial and immunological properties were also shown to have no significant effects on Acanthamoeba-mediated binding to and cytotoxicity of human corneal epithelial cells . Tears also contain complement that is composed of serum-borne molecules in a cascade-like manner. Acanthamoeba directly activates the complement system via the alternative pathway, however pathogenic amoebae are resistant to complement-mediated lysis due to expression of complement regulatory proteins including decay accelerating factor . The presence of macrophages in corneas exposed to the parasite-laden contact lenses prevented the development of full-blown AK in vivo by inducing an inflammatory response, in particular secretion of macrophage inflammatory protein-2 .
For AGE, immunization with Acanthamoeba antigens using intranasal, intraperitoneal, intravenous or oral routes of administration had a protective effect validating that AGE is limited to individuals with a weakened immune response. The complement pathway and antibodies in the presence of phagocytes show potent lytic activity against Acanthamoeba in a contact-dependent manner. These interactions also stimulate secretion of pro-inflammatory cytokines including interleukin-1-beta, interleukin-6 and tumor necrosis factor-alpha [76–79]. Other studies in mice have shown significant increased natural killer cell activities in Acanthamoeba-infected animals suggesting that natural killer cells may also play a role in the protective immunity . Overall, a debilitated immune status of the host is a pre-requisite in AGE but the underlying mechanisms together with the role of the host ethnic origin (i.e., genetic predisposition) remain incompletely understood. Pathogenic Acanthamoeba are shown to degrade chemokines and cytokines, antibodies, complement pathway, and macrophages [76, 81, 82].
Future prospects for treatment
A murine monoclonal anti-idiotypic antibody and a synthetic killer mimotope (mimics a yeast killer toxin) showed broad spectrum anti-amoebic activities suggesting their potential use in the prevention and therapy of Acanthamoeba infections . The Fab fragment of a monoclonal antibody specifically reactive to A. castellanii cell surface was covalently linked to the A chain of diphtheria toxin . This immunotoxin inhibited cell division completely, suggesting that specific antibodies coupled with cytotoxic agents could be a useful method in the development of therapeutic interventions or preventative measures. To enhance the potency of available drugs, propamidine isethionate combined with dimethylsulfoxide proved to be highly effective suggesting that use of a carrier for known anti-amoebic drugs may increase their penetration into the cyst form of the organism, which is normally refractory to drug treatment. To this end, the use of liposomes has been shown to improve the potency of pentamidine isethionate in vitro . Similarly, the use of chitosan microspheres improved in vitro anti-amoebic activity of rokitamycin. Such methods will be useful in transporting the drug for either ocular application to treat AK or nasal administration as an alternative route for the administration of the drug to the brain in AGE therapy. Small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase showed promise in the rational development of therapeutic interventions . Photodynamic chemotherapy by linking amoeba-specific antibodies with photosensitizers such as phthalocyanine or Hypocrellins B may be advantageous over conventional methods due to its localized use, in particular for eye infections. In addition, the programmed cell death in protists has emerged as a fascinating field of parasite biology and could serve as a basis of novel anti-Acanthamoebic drugs [87–89].
Acanthamoeba: Trojan Horse of the Microbial World
The majority of Acanthamoeba isolates harbor endosymbionts which may include viruses, yeast, protists and bacteria, some of which are potential human pathogens. The exact nature of symbiosis and the benefit they represent for the amoeba host are unknown. It is suggested that such interactions may help transmit microbial endosymbionts to the susceptible hosts and/or endosymbionts may contribute to the pathogenicity of Acanthamoeba [3, 90]. Future studies in the identification of virulence factors of the endosymbiont and of the host, and their precise role in disease will clarify these issues.
Acanthamoeba is known to host the largest known virus, Mimivirus, initially mistaken for a parasitic bacterium with a particle size of 400 nm and genome size of 1.2 million bp . Among 911 protein coding genes, 10% exhibit similarity to proteins of known functions blurring the established boundaries between viruses and Archea/Bacteria, a finding that may have huge implications in our understanding of the evolutionary processes [3, 91].
Acanthamoeba is shown to harbour a variety of viruses including coxsackieviruses, adenoviruses, poliovirus, echovirus, enterovirus, or vesicular stomatitis virus, and yeast, Cryptococcus neoformans, Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Streptomyces californicus and Exophiala dermatitidis, and protists including Cryptosporidium and Toxoplasma gondii [3, 91].
Among bacterial pathogens, Acanthamoeba are shown to host/reservoir for Aeromonas spp., Bacillus cereus, Bartonella spp., Burkholderia spp., Burkholderia pickettii, Campylobacter jejuni, Candidatus Odyssella thessalonicensis, Chlamydophila pneumoniae, Coxiella burnetii, Cytophaga spp., Escherichia coli O157, neuropathogenic Escherichia coli K1, Flavobacterium spp., Francisella tularensis, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Mycobacteria tuberculosis, M. avium, M. leprae, Parachlamydia Acanthamoebae, Pasteurella multocida, Prevotella intermedia, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rickettsia, Salmonella typhimurium, Shigella dysenteriae, S. sonnei, Simkania negevensis, Vibrio cholerae, V. parahaemolyticus, Waddlia chondrophila as well as novel bacterial endosymbionts that are related to Caedibacter caryophilus, Holospora elegans and Holospora obtuse, which were proposed as 'Candidatus Caedibacter Acanthamoeba e', 'Candidatus Paracaedibacter Acanthamoeba e' and 'Candidatus Paracaedibacter symbiosus' suggesting the usefulness of amoeba co-culture to recover novel chlamydial strains [3, 91]. With the remarkable implications of parasite-parasite interactions, which may contribute to the evolution of one (either bacteria or Acanthamoeba) or both parasites to become successful human and animal pathogens and transmission of microbial pathogens in the environment, this area of research is of particular significance.
Acanthamoeba has gained increasing attention from the scientific community studying cellular microbiology, environmental biology, physiology, cellular interactions, molecular biology, biochemistry and the evolutionary studies. This is due to their versatile roles in the ecosystem and their ability to capture prey by phagocytosis (similar to macrophages), act as vectors, reservoirs and as a Trojan horse for microbial pathogens, and to produce serious human infections including a blinding keratitis and fatal encephalitis. This unicellular organism has been used extensively to understand the molecular biology of cell motility. Being a eukaryote, Acanthamoeba presents an excellent model for cell differentiation studies. The recent availability of the Acanthamoeba genome, together with the development of transfection assays and the RNA interference methods  will undoubtedly increase the pace of our understanding of this complex but fascinating organism.
The authors would like to stress the fact that the work presented in this review is the results of the dedication and insights of many scientists who contributed considerably to our understanding of this fascinating organism. The work is partially supported by the Royal Society, The Nuffield Foundation, British Council for Prevention of Blindness, University of Nottingham and Aga Khan University.
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