Acanthamoeba: Biology and Pathogenesis
Publisher: Caister Academic Press
Author: Naveed Khan University of Nottingham, UK
Publication date: February 2009
The first comprehensive review of Acanthamoeba research to be published. Everything that is known about Acanthamoeba. The current state of research on every aspect of this organism, detailing major advances in areas such as genomics, molecular and cellular biology, life cycles, geographical distribution, role in ecosystem, morphology, motility, phylogenetics, genotyping, metabolism, regulation of morphogenesis, host-parasite interactions, the molecular and immunological basis of pathogenesis, methods of transmission, epidemiology, clinical manifestation, diagnosis, treatment, new target development and drug resistance, as well as its role as a Trojan horse of the microbial world, including viral, bacterial, protozoal and fungal pathogens, and much more. Emphasis on Acanthamoeba infections in the molecular era. Includes a historical perspective on Acanthamoeba research. An essential reference for microbiologists, immunologists, and physicians in the field of basic and medical microbiology, as well as an invaluable reference for new and experienced researchers who wish to gain a better understanding of Acanthamoeba. The definitive guide to current research on this increasingly important organism. read more ...
Figures from the book: Acanthamoeba Biology and PathogenesisPictures and illustrations of Acanthamoeba and related topics.
SECTION A: Biology and PhylogenyFigure 1. Tree of life.
Figure 2. A. The present classification scheme of protists, based largely on their genetic relatedness. B. The traditional classification scheme of protists, based largely on morphological characteristics and it is no longer valid.
Figure 3. The number of published articles in free-living amoebae of major medical importance. Data for Acanthamoeba, Naegleria and Balamuthia were collected from pubmed.
Figure 4. Acanthamoeba spp. The infective form of Acanthamoeba spp., also known as trophozoites, as observed under phase-contrast microscope. They exhibit round or oval shape, and pseudopodia as shown by arrows. Bar = 10 micrometers
Figure 5. Acanthamoeba has been isolated from natural environments, man-made environments and clinical settings.
Figure 6. A simplified view of food chain.
Figure 7. A eukaryotic cell.
Figure 8. An Acanthamoeba trophozoite. Note a nucleus (shown by a white arrow) and large number of mitochondria (some mitochondria are shown by black arrows). Bar = 5 micrometers
Figure 9. Glycogen structure. Glycogen is a polysaccharide of glucose (A). It is highly branched polymer (B). Most of glucose units are linked by alpha-1,4 glycosidic bonds, approximately 1 in 12 glucose residues also make 1,6 glycosidic bond with a second glucose, which results in the creation of a branch.
Figure 10. Structure of ergosterol, 7-dehydrostigmasterol, and cholesterol.
Figure 11. Glycosidic bond is formed when two sugars are joined together via alpha-linkage (A) or beta-linkage (B) and a molecule of water is removed. The two monosaccharides are bonded via a dehydration reaction (also called a condensation reaction) that leads to the loss of a molecule of water and formation of a glycosidic bond.
Figure 12. Steps involved in amoeboid movement including: extension of the cell membrane, formation of focal adhesions, de-adhesion and movement of the cell body.
Figure 13. Microtubule assembly, in vivo.
Figure 14. Role of GTP cap in microtubule assembly. If tubulin concentration is high, tubulin-GTP is added to microtubule tip faster than the incorporated GTP can be hydrolyzed. The resulting GTP cap stabilizes the microtubule tip and promotes further growth. At low concentration, the rate of growth decreases, thereby allowing GTP hydrolysis but creating unstable tip that favours depolymerization.
Figure 15. Structure of kinesin (A) and microtubule-based motility (B).
Figure 16. Steps involved in actin polymerization.
Figure 17. RAS superfamily of GTP-binding protein.
Figure 18. Activation of RhoGTPases (RhoA, Rac1, Cdc42) leads to distinct phenotypes such as stress fibre formation, lamellipodia, or filopodia formation.
SECTION B: Life cycle and GenotypingFigure 1. The life cycle of Acanthamoeba spp. The infective form of Acanthamoeba is known as trophozoites stage, as observed under light microscope. Under harsh conditions such as lack of food, extremes in pH, temperature and osmolarity, or dessication, trophozoites differentiate into double-walled cysts, as indicated by arrows. Bar = 5 micrometers.
Figure 2. Growth phases in asynchronous cultures of Acanthamoeba spp.
Figure 3. The various phases of cell division in asynchronous cultures of Acanthamoeba spp.
Figure 4. A. Acanthamoeba trophozoite under a phase-contrast microscope. B. Transmission electron micrograph of an Acanthamoeba cyst.
Figure 5. Acanthamoeba cyst walls possess glycans. Acanthamoeba cysts were stained with Periodic Acid-Schiff (PAS) and observed under an optical light microscope. The results revealed staining of the inner wall (endocyst) as well as the outer wall layer (ectocyst), suggesting the presence of glycol-containing components. X400.
Figure 6. The synthesis and structure of cellulose.
Figure 7. The structure of starch (polymer of alpha-glucose) and cellulose (polymer of beta-glucose) are similar, i.e., formed by long glucose unit chains. Glycogen (polymer of alpha-glucose) however has a longer chain and is more highly branched as shown in Section A, figure 9B. The orientation of the units in cellulose results in a linear chain, which allows other chains to closely pack by hydrogen bonding. This increases the strength of cellulose (compared with starch) and accounts for its low solubility in water, hence it can be used to make clothes, ropes etc.
Figure 8. Summary of catabolic reactions.
Figure 9. The summary of glucose metabolism.
Figure 10. Summary of Glycolysis. Glucose is cleaved into two molecules of pyruvic acid. Four molecules of ATP are formed and 2 ATP are used, so net gain of 2 ATP. In addition, 2 molecules of NAD+ are reduced to NADH.
Figure 11. Formation of acetyl-CoA.
Figure 12. The Krebs cycle. Acetyl-CoA enters the Krebs cycle by joining with oxaloacetic acid to form citric acid and CoA.
Figure 13. Electron transport chain (A). ATP production is indicated at approx. point in the chain (B) and a possible arrangement of an electron transport chain (B).
Figure 14. A. Acanthamoeba cysts under phase-contrast microscope. i) Non-nutrient agar plates exhibiting Acanthamoeba cysts. ii) Acanthamoeba cysts were collected from non-nutrient agar plates using PBS and observed under the phase-contrast microscope. Note cysts formed clusters in PBS, X400. B. Acanthamoeba trophozoites on non-nutrient agar plates observed under phase-contrast microscope. Note the characteristic contract vacuole in Acanthamoeba trophozoites, X400. C. Acanthamoeba trophozoite binding to glass cover slips observed under scanning electron microscope. Note large number of acanthopodia on the surface of Acanthamoeba trophozoites belonging to the T4 genotype. D. Acanthamoeba binding to corneal epithelial cells. Acanthamoeba castellanii (T4 isolate) were incubated with corneal epithelial cells, followed by several washes and observed under scanning electron microscope. Note that parasites were able to exhibit binding to the host cells and binding was mediated by acanthopodia. A is amoeba, E is corneal epithelial cell. Bar = 10 micrometers.
Figure 15. Trypan blue staining of viable and dead trophozoites and cysts of Acanthamoeba. X400.
Figure 16. Acanthamoeba cysts exhibiting characteristic arm-like structures.
SECTION C: Acanthamoeba InfectionsFigure 1. Acanthamoeba keratitis has become a significant problem in recent years, especially in contact lens wearers exposed to contaminated water.
Figure 2. Anatomy of the eye and cellular structure of the cornea.
Figure 3. Diagrammatic representation of biofilm formation on contact lens surface. Initial bacterial co-aggregation involves protein-saccharide interactions leading to microbial colonization including Acanthamoeba. Note carbohydrate residues (alpha-mannose) on contact lens may directly act as receptors for Acanthamoeba trophozoites.
Figure 4. Normal eye and an Acanthamoeba-infected eyes exhibiting ulcerated epithelium and corneal opacity in acute Acanthamoeba keratitis.
Figure 5. Indirect immunofluorescence assays. Serum or mucosal secretions from a suspected patient is incubated with pathogen (e.g., Acanthamoeba)-coated slides, at various titres. A strong reaction suggests Acanthamoeba as the causative agent. Note that the levels of anti-Acanthamoeba antibodies in normal populations may be in the range of 1:20 to 1:60, however, patients with severely impaired immune system may not develop a high titre, thus other clinical findings should be taken into account for the correct diagnosis.
Figure 6. Antibody fragments isolated from a phage display antibody library and tested using indirect immunofluorescence assays. (A) Under a light microscope. (B) Indirect immunofluorescence reactivities of an antibody fragment with Acanthamoeba.
Figure 7. Flow cytometry analysis of specificities of Acanthamoeba castellanii (T4 genotype)-specific phage clone with (A) A. castellanii (T4 genotype); (B) A. astronyxis (T7 genotype). Acanthamoeba (106) were incubated with phage clone (1012), washed and resuspended in sheep anti-M13 antibody. Following this, cells were washed, resuspended in fluorescein isothiocyanate conjugated anti-sheep IgG (10 microgram/ml) and analyzed by flow cytometry. Note that mean channel fluorescence is higher in pathogenic A. castellanii (T4 genotype) than weak-pathogenic A. astronyxis.
Figure 8. Agarose gel showing PCR amplification of 18S rDNA of Acanthamoeba using genus-specific primers. Note that primers amplified various genotypes of Acanthamoeba but not human DNA, suggesting their use in the diagnosis of Acanthamoeba in the environmental and clinical settings.
Figure 9. The risk factors contributing to Acanthamoeba keratitis. A. swimming, especially while wearing contact lenses; B. washing eyes during or immediately after contact lens wear; C. working with soil and rubbing eyes; D. water-related activities (splashing water), especially during or immediately after contact lens wear; E. handling contact lenses without proper hand washing; F. use of home-made saline (or even chlorine-based disinfectants) for contact lens cleaning.
Figure 10. The likely routes of Acanthamoeba entry into the central nervous system (CNS). Acanthamoeba enter lungs via the nasal route. Next, amoebae traverse the lungs into the bloodstream, followed by haematogenous spread. Finally, amoebae cross the blood-brain barrier and enter into the CNS to produce disease. It is noteworthy that Acanthamoeba may bypass the lower respiratory tract and directly enter into the bloodstream via skin lesions. The olfactory neuroepithelium may provide an alternative route of entry into the CNS.
Figure 11. Acanthamoeba infected brain exhibiting severity of the disease.
SECTION D: PathogenesisFigure 1. A and B. Anatomy of the eye; C. cellular structure of the cornea; and D. in vitro cultures of corneal epithelial cells. Note that in vitro cultures are positive for cytokeratin 3 and cytokeratin 12 indicating their corneal epithelial origin
Figure 2. A. Multilayering of epithelial cells in a 14 day-old construct. Black arrows: epithelial cells; Arrowheads: human extracellular matrix (basement membrane); white arrows: fibrillar collagen bundles.Bar = 30micrometers B. Montage of Transmission Electron Micrograph of a 3 week-old construct. Bar = 2micrometers
Figure 3. Use of locusts as an in vivo model to study Acanthamoeba infection. A. Infection can be studied in the blood (i.e., haemolymph) and B. brains can be isolated to study the involvement of the central nervous system.
Figure 4. A and B. Anatomy and the cellular structures of the microvessels of the human brain tissue; C. in vitro cultures of brain microvascular endothelial cells. Note that in vitro cultures are positive for factor VIII, carbonic anhydrase IV, uptake of fluorescently labelled acetylated low density lipoprotein, and gamma-glutamyl transpeptidase indicating their brain endothelial origin; and D. Ex vivo model of the blood-brain barrier. Transwells can be used to grow brain microvascular endothelial cells into the upper chamber on a permeable support, and astrocytes can be cultured into the bottom chamber.
Figure 5. The pathogenic cascade of Acanthamoeba keratitis.
Figure 6. Schematic illustration of the blood-brain barrier at the cerebral capillary endothelium exhibiting tight junctions. The endothelial cells are surrounded by basement membrane, which is ensheathed by astrocytes and pericytes. The tight junctions are composed of integral proteins including occludin, claudin, junctional adhesion molecule (JAM), and endothelial cell-selective adhesion molecule (ECSAM), that interact with their counterparts on the adjacent endothelial cells. Both tight junctions and adherens junctions are composed of multiple proteins. The cytoplasmic tails of these proteins interact with the actin cytoskeleton via a number of accessory proteins including members of zonula-occludens (ZO).
Figure 7. Central nervous system is protected by the presence of the blood-brain barrier that blocks the entry of pathogens/molecules. Acanthamoeba uses both direct and indirect virulence factors to traverse this biological barrier. In addition, host inflammatory responses also contribute to blood-brain barrier perturbations.
Figure 8. The model of Acanthamoeba granulomatous encephalitis. Acanthamoeba are thought to enter lungs via the nasal route. Next, amoebae traverse the lungs into the bloodstream, followed by haematogenous spread. Finally, Acanthamoeba cross the blood-brain barrier and enter into the central nervous system (CNS) to produce disease. It is noteworthy that Acanthamoeba may bypass the lower respiratory tract and directly enter into the bloodstream via skin lesions. The olfactory neuroepithelium may provide an alternative route of entry into the CNS.
Figure 9. Direct and indirect virulence factors that contribute to Acanthamoeba infections.
Figure 10. Extracellular matrix in relation to epithelium, endothelium and the connective tissue.
Figure 11. Binding of Acanthamoeba to host cells is mediated by acanthopodia. Acanthamoeba castellanii belonging to the T4 genotype were incubated with host cells, followed by several washes and observed under scanning electron microscope. Note that parasites were able to exhibit binding to the host cells and binding was mediated by acanthopodia. A is Acanthamoeba.
Figure 12. Host intracellular signaling in response to Acanthamoeba. Note that Acanthamoeba induces cell cycle arrest in the host cells by i) altering expression of genes as well as by ii) modulating protein retinoblastoma (pRb) phosphorylations. In addition, Acanthamoeba have also been shown to induce host cell death via phosphatidylinositol 3-kinase (PI3K). By secreting proteases, amoebae disrupt tight junctions by targeting zonula-1 and occludin proteins. MBP is mannose-binding protein; E2F is a transcription factor that controls cell proliferation through regulating the expression of essential genes required for cell cycle progression; PIP2 is phosphatidylinositol-4,5-bisphosphate; PIP3 is phosphatidylinositol-3,4,5-trisphosphate; Akt (protein kinase B)-PH domain, a serine/threonine kinase is a critical enzyme in signal transduction pathways involved in cell proliferation, apoptosis, angiogenesis, and diabetes.
Figure 13. Acanthamoeba exhibiting amoebastomes (mouth-like openings). Acanthamoeba incubated with the host cells exhibited the presence of amoebastomes within 30 minutes of incubation. These structures are known be involved in the phagocytosis of Acanthamoeba. A is Acanthamoeba.
Figure 14. Diagrammatic representation of Rho activation leading to the blood-brain barrier permeability changes. RhoA activation induces myosin light-chain phosphorylation (P-MLC) via Rho kinase causing redistribution/alteration of tight junction proteins, zonula occludens-1 (ZO-1) and occludin, resulting in the elevation of barrier permeability.
Figure 15.. Acanthamoeba proteases target tight junctions proteins and the extracellular matrix to induce perturbation of the blood-brain barrier.
Figure 16. Phospholipases and their sites of action.
Figure 17. Schematic illustrates involvement of Acanthamoeba mannose-binding protein (MBP), serine proteases, retinoblastoma protein (pRB), phosphatidylinositol 3-kinase (PI3K), Rho activation, tight junction proteins leading to host cell damage, a pre-requisite in Acanthamoeba translocation of the biological barriers. In addition, host inflammatory responses may also contribute to blood-brain barrier perturbations.
SECTION E: Immune responseFigure 1. An overview of human immune system.
Figure 2. Some of the human innate defences.
Figure 3. Uptake of pathogens by phagocytes leading to their degradation.
Figure 4. Components of blood.
Figure 5. Two pathways by which complement is activated, resulting in the formation of the membrane attack complex (MAC) in the host cell membranes.
Figure 6. Summary of components of the innate / non-specific defences.
Figure 7. Activation of CD4 T-helper cells (Th0) into Type 1 (Th1) and Type 2 (Th2). Microbes or antigens are taken up by antigen presenting cells (APC), processed and presented to lymphocytes resulting in their activation. IFN-gamma is interferon-gamma; IL-2 is interleukin-2; and IL-4 is interleukin-4.
Figure 8. Structure of an antibody.
Figure 9. An overview of parasite immune evasion mechanisms.
SECTION F: Strategies against Acanthamoeba infections
SECTION G: Acanthamoeba: Trojan Horse of the Microbial WorldFigure 1. Acanthamoeba feeds on other microbes, and acts as a host for microbial organisms. Notably, Acanthamoeba acts as a biological host for some microbial pathogens, i.e., pathogen multiplication inside Acanthamoeba.
Figure 2. The 19-storey building of the Bellevue-Stratford Hotel, Philadelphia, USA
Figure 3. Scanning electron micrograph of Legionella pneumophila.
Figure 4. Transmission electron micrograph of an amoeba-filled with Legionella pneumophila.
Figure 5. Bacterial interactions with the clinical isolate of Acanthamoeba. Bacteria invade into and/or taken up by Acanthamoeba. Once intracellular, non-pathogenic bacteria are most likely killed and used as food source, while pathogenic bacteria possess the ability to evade intracellular killing mechanisms, and either survive or multiply within Acanthamoeba. A. Acanthamoeba act as host and/or reservoir for bacteria, B. Acanthamoeba act as Trojan horse for bacteria, and C. Acanthamoeba act as potent predator of bacteria.
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