Thesis draft: Acanthamoeba castellanii

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In silico characterization of protein fragment alanine ligaseof
Acanthamoeba culbertsoniT418S rDNAand ofAcanthamoeba
castellaniiT4 rDNA 18S
Thesis presented:ANR
To obtain the degree of: Doctor
thesis supervisors
Mexico City, Mexico
August, 2021

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INDEX
I. General index
II. Table index
III. index of figures
IV. Abbreviations
V. Summary
VI. Acronym
1 General Background
1 Free Living Amoebas (AVL) 4
2 Distribution in Mexico and endemism 4
3 Pathogenic amoebas for humans 4
4 Acanthamoeba spp phylogenetic tree 4
5 Taxonomic location in Mexico: 4
6 Lifecycle 5
7 Acanthamoeba morphology 6
8 acahamoeba castelani 7
9 Acantamoeba cutbersoni 7
10 Acanthamoeba keratitis 8
1 direct virulence factors 8
2 Accession . 8
3 Phagocytosis. 9
4 Ecto-ATPases. 10
5 Neuraminidase activity. 10
6 Superoxide dismutase. 10
7 Induced activation of plasminogen. 11
8 elastase 11
9 proteases 11
10 phospholipases 12
11 glycosidases 13
12 acanthaporin 13
13 Indirect virulence factors 14
14 Cell differentiation 14
15 chemotaxis 15
16 Tolerance to temperature and growth by pH: 15
17 Ubiquity. 16
18 Biofilms 16
19 alginate ligase 16
20 signal transduction. 17
21 Polyhydroxybutyrate depolymerase. 18
22 Host factors. 18
23 Acanthamoeba genotypes. 18
24 Endosymbiosis. 19
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Genomic organization of Acanthamoeba castellanii and A.
curtbersoni 21
26 Acanthamoeba castellanii mitochondrial genome. 22
27 Pan-Genome and Central Genome of Acanthamoeba spp. 25
2 Hypothesis 30
3 Justification 30
4 Goals 30
5 Materials and methods 31
6 Results 40
7 Discussion 50
8 conclusions 55
9 Bibliography 60

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General Background
Free Living Amoebas (AVL):Free Living Amoebas (AVL) are eukaryotic, aerobic,
mitochondrial protozoa found in all kinds of environments. They are heterotrophic protozoa
and have the ability to feed osmotrophically (they consume dissolved organic matter) and
holozoic (they eat bacteria, particulate matter and other organisms) by phagocytosis, thus
controlling bacterial populations. They are amphizoic in their ability to live as free-living
organisms in nature and only occasionally invade hosts and live as a parasite within host
tissue. There are more than 100 species, some of which can potentially cause infection in
humans. They measure from 20 to 200µm and form cysts of resistance to adverse
environmental changes,
Taxonomic location of AVL in Mexico:The species count of the review period carried out
corresponding to 171 years (1841-2012) with 144 contributions yields 315 species, including
some described for the first time. The most reported group corresponded to Amebozoa,
Tubulinea, Testacealobosia (Arcellinida) with 17 genera and 82 species, followed by
Tubulinea, Tubulinida with 8 genera and 37 species. In second place was the group Excavata,
Heterolobosea, Vahlkampfidae, with 8 genera and 44 species. In third place was Rhizaria,
Cercozoa, Silicofilosea (Euglyphida) with 11 genera and 28 species. The fourth place was
Amebozoa, Acanthamoebidae with 2 genera and 23 species, followed by the groups of the
second rank Dactilopodida (3 genera and 19 species) and Thecamoebida (4 genera and 12
species). The rest of the groups show little specific representation and appear in more or less
punctual situations. Among the genera of naked and thecada amoebas with the largest
number of species and that occupy the first 10 places in the record are: Difflugia (26), Arcella
( 25), Acanthamoeba (21), Vahlkampfia (17), Amoeba (16), Naegleria (14), Mayorella (13),
Euglypha (11), Centropyxis (10) and Hartmannella (5). Testate amoebas and acanthamoebas
predominate, perhaps due to the resistance they have to adverse environmental factors
conferred by the theca and the cellulose cyst of Acanthamoeba. Naegleria (14), Mayorella
(13), Euglypha (11), Centropyxis (10) and Hartmannella (5). Testate amoebas and
acanthamoebas predominate, perhaps due to the resistance they have to adverse
environmental factors conferred by the theca and the cellulose cyst of Acanthamoeba.
Naegleria (14), Mayorella (13), Euglypha (11), Centropyxis (10) and Hartmannella (5). Testate
amoebas and acanthamoebas predominate, perhaps due to the resistance they have to
adverse environmental factors conferred by the theca and the cellulose cyst of Acanthamoeba.
Pathogenic amoebas for humans:Members of only 6 genera have an association with
human disease: Naegleria fowleri, Balamuthia mandrillaris, Vermamoeba vermiformis,
Sappinia pedata (diploidea), Paravahlkamfia francinae and Acanthamoeba spp, which have
been described as causing CNS infection causing amoebic keratitis or lesions in the the skin
in humans and animals (Visvesvara Govinda, 2013; Gallegos-Neyra, 2014).

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Phylogenetic tree of Acanthamoeba spp:Molecular phylogeny of the genus Acanthamoeba.
All genotypes include strains with nearly complete 18S rDNA sequences (>2000 bp), except
A. jacobsi T15 (∼1500 bp). A. lenticulata T5 (four strains) and A. jacobsi T15 (three strains)
are collapsed. Morphological groups I to III are also indicated. The new strain recovered in
this study and newly identified genotypes are in bold. For these strains and relatives, the
sources are also reported. CLC Contact Lens Case, Acanthamoeba Keratitis CLC/AK Contact
Lens Case, GAE Granulomatous Amebic Encephalitis. (Corsaro, D, et al., 2015)

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Distribution in Mexico and endemism:Mexico occupies one of the top 5 places in species
richness due to its number of endemic species and is considered to be home to approximately
10% of the world's species. It is calculated that the total number of species of the known
groups in Mexico is approximately 64 878, this is favored by its geographical position in the
intertropical belt between two biogeographic provinces. Based on the above, the study of
amoebid protists in Mexico is promising, because they could provide elements to debate the
two theories about the distribution of protozoa in the world. Therefore, it is important to
increase taxonomic and ecological studies of amoebid protists in our country, since this would
allow to clarify if their species are cosmopolitan or endemic and if they present affinities with
other species found in other parts of the world (Gallegos-Neyra et al., 2008). The largest
number of studies and therefore the largest record of species occurs in the central zone of the
Republic in Mexico City (201) Of the total of the states that make up the Mexican Republic,
only 73% have records of amoebic species and 27% of the states remain without studies on
amoebas (Campeche, Coahuila, Colima, Chiapas, Durango, Tabasco, Tlaxcala, Yucatán and
Zacatecas). The environments with the highest number of amoeba species are: aquifers,
drinking water (cisterns, water tanks, bathrooms, sinks), residual water, atmosphere, irrigation
channels, cave/grotto (water, biofilms, guano and moss), lakes, moss, thermal water pools,
Morphology ofAcanthamoebaspp:The term “acanth” in Greek means spines, it was added
to the term amoeba to indicate the presence of spine-like structures, now known as
acanthopods (Khan et al., 2006). In 1930, Castellani isolated an amoeba that was a
contaminant in a culture plate of the fungus Cryptococcus pararoseus and that was later
Acanthamoeba castellanii. Culbertson in 1958, with an advanced concept, proposed that
AVLs can cause human infection (Marciano-Cabral et al. al., 2003; Khan et al., 2006;
Visvesvara G. 2013). During the production of the polio vaccine they isolated an amoeba from
tissue culture thought to contain an unknown virus, inoculated it into intracerebrally cortisone-
treated mice and monkeys, and demonstrated brain lesions in the animals that died a week
later.
trophozoite: derives from "tropho" which in Greek means "to nourish", measures from 15 to
45μm(Lorenzo-Morales, 2015), is the one that feeds on organic particles, bacteria, algae, fungi
and detritus present in the environment. The absorption of food is carried out by phagocytosis
or by formation of "food cup", which are formed on the surface of the amoeba as temporary
structures. Fine, digital, spine-like pseudopods called acantopodia participate in the feeding
process; the ingested fluid is collected through pinocytosis (Khan et al., 2006). It reproduces
by binary fission (Trabelsi et al., 2012), divides mitotically under optimal conditions of food
supply, neutral pH and approximate temperature of 30ºC. It generally has 1 vesicular nucleus
that is approximately one sixth the size of the trophozoite (Khan et al., 2006), centrally located,
with a large and dense nucleolus. The cytoplasm is finely granular and contains numerous

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mitochondria, ribosomes, lysosomes, food vacuoles, and 1 or more contractile vacuoles,
whose function is to expel water for osmotic regulation. When food becomes scarce or when
faced with desiccation or other environmental pressure, the amoeba rounds up and encysts.
Cyst:The cyst has a wall with 2 layers, measuring between 5 and 25µm (Visvesvara et al.,
2013; Lorenzo Morales et al., 2015). The outer layer, or ectocyst, has folds and undulations
and is made up of proteins, polysaccharides, and lipids. The inner layer, the endocyst,
contains The endocyst varies in shape, being able to be stellate, polygonal, oval, triangular or
spherical. Ecto and endoplasm are connected at various points with pores or ostioles, which
are sealed with mucous plugs, called opercula, which are removed during excystation.
Pussard and Pons in 1977proposed the existence of three groups, based on the size and
morphology of the cysts, presented in Table °1.
Table Nº1 Groups of Pussard and Pons, 24 species of Acanthamoeba spp.
GROUP I
(> 19μm)
GROUP II
(<18μm)
GROUP III
(<18μm)
A. astronyxis A. castellani A. palestinensis
A. commandoni A. mauritaniensis A.culbertsoni
A. echinulata A. polyphaga A. lenticulata
a.tubiashi A. lugdunensis A. pustular
A. byersi A. quina A. royreba
A. rhysodes a healyi
A. divionensis a. jacobsi
A. paradivionensis
A.griffini
A. triangularis
A. hatchetti
A. micheli sp.
Group I)Smooth and rounded ectocyst, separated from the endocyst, with a stellate
appearance.
Group II)Ectocyst a wrinkled appearance and endocyst stellate, polygonal or triangular.
Group III)Smooth ectocyst and a rounded or slightly angular endocyst, with little separation
between them.

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Figure 1. Acanthamoeba cystsin interference contrast microscopy. (A) morphological
group I, (B) morphological group II, (C) morphological group III.
Acanthamoeba castellanii morphology.
Figure 2.Acanthamoeba castellanii, strains were isolated from sediments dating from the late
Pleistocene to early Holocene: A – E Locomotive forms; F Floating form; G multinucleated
trophozoite; H Monolayer; K–N Cysts; J Pseudocysts; O Empty cyst (Stas Malavin, et al,
2020).
>19μm <18μm <18μm

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Acanthamoeba culbertso morphologyneither.
Figure 3.Phase contrast image of a live A. culbertsoni trophozoite with pleomorphic
morphology where numerous irregular thin cytoplasmic projections (asterisks) and a large
hyaline lobopodium (L) are visible. Large, round and centrally located nuclei are also present,
as well as abundant vacuoles of different sizes. Bar = 10µm. B. Scanning electron microscopy
image of an A. culbertsoni trophozoite, whose surface shows numerous cytoplasmic
projections of different sizes, some of which are bifurcated (asterisk). A common finding was
the presence of thin, flat cytoplasmic lamellae (L). Bar = 1µm. C. Thin section of A. culbertsoni
showing an irregular profile bounded by the plasma membrane. The cytoplasm presents a
dense granular appearance where well-known cell organelles are identified, such as a large
nucleus (N) with condensed patches of chromatin bounded by a double-layered nuclear
envelope; Mitochondria (M), rough endoplasmic reticulum (Rer), and Golgi cisternae (G) are
also seen. Bar = 5 µm (González-Robles, et al, 2017).

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Figure 4.Acanthamoeba culbertsoni trophozoite that presents food vessels on the surface
(they are temporary structures that are formed and reformed for the ingestion of bacteria,
yeasts or cellular debris), (Marciano-Cabral, et al, 2003).
Lifecycle:It comprises two stages of biological viability, an active form that feeds and
reproduces, called a trophozoite, and an inactive, resistant form called a cyst. The trophozoite
reproduces asexually by binary fission, giving rise to 2 daughter cells. In some amoebas there
is a temporary ameboflagellar stage or known as precyst or pseudocyst, in which the organism
does not feed or reproduce, it only serves to move to a better microenvironment, the life cycle
can be completed in the environment without the need for a host (Gallegos-Neyra et al., 2014).
Acanthamoeba keratitis (AK):severe, sight-threatening infection of the cornea by this
amoeba. Risk factors are a multifactorial process. The most relevant clinical feature of QA is
the presence of a ring stromal infiltrate. The characteristic ring infiltrate is only seen in
approximately 50% of patients (Lorenzo-Morales, 2015). Trophozoites can infiltrate corneal
nerves, causing neuritis and necrosis. In rare circumstances, Acanthamoeba can spread from
the cornea to the retina, causing chorioretinitis. At the beginning of the infection, a diffuse
superficial keratopathy is found, later multifocal infiltrates are almost always observed in the
stroma. Most QA cases are caused by Pussard's Group II, although group III strains have also
been described as causative agents of QA.
General pathogenesis of Acanthamoeba Keratitis:The pathogenesis of Acanthamoeba is
related to direct and indirect factors.
Direct virulence factors:They are contact dependent mechanisms such as adhesion,
phagocytosis and contact independent mechanisms such as proteases. (Lorenzo-Morales et
al., 2015).
Accession:Adhesion is an important step in the pathogenic cascades of Acanthamoeba
keratitis, leading to secondary events and amoebae crossing biological barriers.They
haveidentified several adhesins in Acanthamoeba, including a mannose-binding protein,
aUnionto laminin with a predicted molecular mass of 28.2 kDa and a laminin-binding protein
of55kDa and the mannose binding protein gene in Acanthamoeba has been shown to contain
six exons and five introns spanning 3.6 kbp. The 2.5 kbp cDNA encodes an 833 amino acid
precursor protein with a signal sequence (residues 1 to 21 aa), an N-terminal extracellular
domain (residues 22 to 733 aa) with five N- and three O-glycosylation sites. , a transmembrane
domain (residues 734-755 aa) and a C-terminal intracellular domain (residues 756-833 aa).
On the host side, parasite binding to specific receptors remains incompletely understood. The
Toll-like receptor-4 (TLR-4) which provides a docking site forAcanthamoeba. Full identification

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of adhesins involved in binding to various cell types, tissues, and surfaces along with specific
receptors is a largely unexplored topic (Garate M, et al., 2004).
Acanthamoeba binding to host cells interferes with host intracellular signaling pathways. For
example, TLR activation leads to TLR4-Myeloid Differentiation Primary Response Gene 88
(MyD88)-Nuclear Factor-Kappa B (NF-kappaB) and extracellular signal-regulated kinase1/2
(ERK1/2) pathways TLR4, it was confirmed using anti-TLR antibodies or specific
pyrrolidinedithiocarbamate (PDTC) inhibitors (for the NF-kappa B pathway) and U0126 (for
the ERK pathway). Using cell cycle microarrays, Acanthamoeba adhesion to host cells has
been shown to regulate the expression of a number of genes important for the cell cycle, such
as GADD45A and p130 Rb, associated with cell cycle arrest, in addition to inhibiting cell cycle
arrest. expression of other genes, such as those for the F cyclins, G1 and cyclin-dependent
kinase 6 encoding proteins important for cell cycle progression.
Acanthamoebainhibitedphosphorylation of pRb (a master cell cycle regulator) in human
corneal epithelial cells, indicating that Acanthamoeba induces cell cycle arrest in host cells.
Acanthamoeba-mediated host cell death is dependent on activation of phosphatidylinositol 3-
kinase. Acanthamoeba-induced host cell apoptosis has been shown to be caspase-
dependent, mediated by the overexpression of pro-apoptotic proteins in the mitochondrial
pathway, and later the role of cytosolic phospholipase A2 (cPLA2a) acting in the pathway was
demonstrated. host cell apoptosis (Ren M, et al., 2010).
Phagocytosis:Acanthamoeba adhesion leads to secondary processes such as phagocytosis
or toxin secretion. The main role of Acanthamoeba in phagocytosis is to take up food particles.
However, the ability of Acanthamoeba to form food cups or amoebastomes during incubations
with host cells suggests that it has a role in Acanthamoeba pathogenesis. Oxidative
metabolism in Acanthamoeba has some similarities to the oxidase respiratory burst of
neutrophils. Cytochalasin D, an inhibitor oftheActin polymerization blocked by Acanthamoeba
mediates host cell death, confirming that actin-mediated cytoskeletal rearrangements play an
important role in Acanthamoeba pathogenesis. Genistein (a protein tyrosine kinase inhibitor)
inhibited, while sodium orthovanadate (protein tyrosine phosphatase inhibitor) stimulated
Acanthamoeba phagocytosis, indicating that tyrosine kinase-induced actin polymerization is
important in phagocytosis. by Acanthamoeba. Ainhibitorof the Rho kinase, Y27632, is partially
blocked by phagocytosis by Acanthamoeba. Y27632 is known to block stress fiber formation
by inhibiting myosin light chain phosphorylation and cofin phosphorylations, but is independent
of the profilin pathway. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase,
inhibited Acanthamoeba phagocytosis. Inhibition of Src kinase using a specific inhibitor, PP2
(4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analogue,

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PP3 (4 -amino- 7-phenylpyrazolo[3,4-d]pyrimidine), hindered the phagocytic ability of
Acanthamoeba castellanii (Siddiqui R, et al., 2012).
Ecto-ATPases: Ecto-ATPases are glycoproteins expressed in plasma membranes with their
active sites against the external environment. Ecto-ATPases hydrolyze extracellular ATP and
other nucleoside triphosphates. The resulting ADP can have toxic effects on host cells. For
example, ADP released by Acanthamoeba has been shown to bind to host cell P2y2
purinergic receptors, causing an increase in intracellular calcium, inducing caspase-3
activation, and eventually resulting in apoptosis. an antagonist ofreceiverP2, suramin,
inhibited through Acanthamoeba host cell death, suggesting thattheecto-ATPases play an
important role in the pathogenesis of Acanthamoeba in a contact-independent mechanism.
Several ectoATPases of approximate molecular weights of 62, 100, 218, 272, and greater
than 300 kDa have been described in Acanthamoeba (Sissons J, et al., 2004).
Neuraminidase activity:Acanthamoeba exhibited neuraminidase activity. Enzyme activity is
optimal at pH 5 and at temperatures of 25° to 30° C. Living amoebae release sialic acid from
human cells. Therefore, the Acanthamoeba neuraminidase could be relevant in the
colonization of amoebas and important in the production of damage to the corneal epithelium
rich in sialic acid. Trypanosoma cruzi and Acanthamoeba neuraminidase are immunologically
related, as demonstrated by antibodies against Trypanosoma cruzi neuraminidase, which
reacted with Acanthamoeba in immunofluorescence, immunoblotting, and immunosorbent
assays.linked toenzymes (Pérez-Serrano J, et al., 2000).
Superoxide dismutase:The enzyme superoxide dismutase catalyzes the dismutation of
superoxide into oxygen and hydrogen peroxide. It is an important antioxidant defense
exposed to oxygen. Superoxide is one of the major reactive oxygen species in the cell, and
as such, superoxide dismutase plays an important antioxidant role. Two superoxide
dismutases have been identified in Acanthamoeba: an iron superoxide dismutase
(approximate molecular weight 50 kDa) and a copper-zinc superoxide dismutase
(approximate molecular weight 38 kDa). These enzymes are presented as cytoplasmic and
detergent extractable fractions. They may be potential virulence factors of Acanthamoeba
acting as antioxidant and anti-inflammatory agents. They may also provide additional targets
for chemotherapy and immunodiagnosis of Acanthamoeba infections. Iron superoxide
dismutase from Acanthamoeba castellanii may play an essential role in amoeba survival by
not only protecting from endogenous oxidative stress, but also detoxifying the oxidative
destruction of amoebas by host effector immune cells (Kim JY, et al ., 2012).
Induced activation of plasminogen:Acanthamoeba showed plasminogen activating activity
by catalyzing the cleavage of host plasminogen to form plasmin, which can activate host

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proteolytic enzymes such as pro-matrix metalloproteases. Once activated, matrix
metalloproteases degrade basement membranes and extracellular matrix components such
as type I and II collagens, fibronectins, and laminin. Thus, matrix metalloproteinases are
involved in tissue remodeling. The pathogen Acanthamoeba showed a positive chemotactic
response to endothelial extracts (Van Klink F, et al., 1997).
elastase: Acanthamoeba is known to produce elastase with a broad specificity. In addition,
elastases are known to degrade a variety of connective tissue proteins such as elastin, an
elastic fiber, fibrinogen, collagen, and proteoglycans, which together determine the
mechanical properties of connective tissue. Tissues altered by elastase pretreatment are
more susceptible to oxygen radical attack, suggesting their involvement in the pathogenesis
and pathophysiology of infections byAcanthamoeba. Elastases were in the region of 70-130
kDa and serine peptidases were found to be possible elastase candidates (Ferreira GA, et
al., 2009).
Proteases:Proteases are degrading enzymes that catalyze the total hydrolysis of proteins.
Acanthamoeba is shown to exhibit proteolytic activities. The main role of Acanthamoeba
proteases is to break down food substances. Acanthamoeba pathogens exhibit increased
extracellular protease activities. The link between pathogenicity and increased levels of
extracellular proteases suggests that Acanthamoeba pathogens use proteases to facilitate
host invasion. Acanthamoeba is known to produce serine, cysteine, and metalloproteases.
Several serine proteases with molecular weights of >20 kDa to 200 kDa have been identified.
It has been shown that they have collagen degradation activity, plasminogen activator and
fibonectin degradation, fibrinogen, IgG, IgA, albumin, hemoglobin, protease inhibitors,
interleukin-1, chemokines and cytokines. Aserineproteaseof 133 kDa, designated MIP133,
has been identified as a crucial component of the Acanthamoeba pathogenic cascade. The
serine protease MIP133 has been shown to induce degradation of keratocytes, iris ciliary
body cells, retinal pigment epithelial cells, corneal epithelial cells, and corneal endothelial
cells, and to induce apoptosis in macrophage-like cells. The properties of serine proteases
facilitate Acanthamoeba invasion of the corneal stroma, which gives rise to secondary
reactions such as edema, necrosis and inflammatory responses, there are studies that
support the idea that extracellular serine proteases are directly involved in the pathogenesis
and virulence of Acanthamoeba . In addition, several cysteine proteases have been identified
in Acanthamoeba, including cysteine proteases 43, 65, 70 and 130 kDa. In addition to serine
and cysteine proteases, there is evidence of metalloprotease activity in Acanthamoeba. An
80 kDa metalloprotease was identified in cocultures of Acanthamoeba and host cells.
Subsequent studies identified a 150 kDa extracellular metalloprotease from Acanthamoeba
isolated from the T1 genotype. This metalloprotease exhibited extracellular matrix
degradation properties, as evidenced by its activity against collagen I and III (main
components of the collagenous extracellular matrix), elastin (elastic fibers of the extracellular

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matrix), plasminogen (involved in the proteolytic degradation of the extracellular matrix), as
well as degradation of casein, gelatin and hemoglobin (Khan NA, et al, 2009; Na BK, et al.,
2001). The complete sequence of a metacaspase type 1 from Acanthamoeba was reported,
comprising 478 amino acids. Subsequent studies revealed that in Acanthamoeba castellanii,
metacaspases associate with the contractile vacuole and have an essential role in cellular
osmoregulation, suggesting their attractiveness as a potential target for treatment therapies
against Acanthamoeba infection.castellani. These studies showed that Acanthamoeba exhibit
various proteases and elastases, which could play an important role in Acanthamoeba
infections (Saheb E, et al., 2013).
Phospholipases:During phagocytosis, there is a large turnover of the plasma membrane in
Acanthamoeba, indicating that there is controlled local degradation of phospholipids leading
to instability of the membrane phospholipid bilayer, which would then reform after acylation of
the membrane. lysophospholipid. All of the enzymes required for this cycle are present in the
Acanthamoeba plasma membrane, including phospholipase A2, acyl CoA:lysolecithin
acyltransferase, and acyl CoA synthetase. Phospholipase A1 and lysophospholipase are also
present in the Acanthamoeba plasma membrane. Plasma membrane lysophospholipase may
also serve to protect the cell from the lytic effect of lysophospholipids of exogenous or
endogenous origin. Plasma membranes have the enzymatic ability to modulate the fatty acyl
composition of phospholipids through deacylation and acylation. Our knowledge of
phospholipases in Acanthamoeba virulence is fragmented, however, several studies have
shown that Acanthamoeba pathogens exhibit cytopathic effects on mammalian cells in vitro
and release more phospholipase, suggesting their possible involvement in Acanthamoeba
infections. Because phospholipases cleave phospholipids, they play a role in membrane
disruptions, host cell penetration, and lysis. Other actions of phospholipases may involve
interference with intracellular signaling pathways.
Glycosidases:Glycoside hydrolases catalyze the hydrolysis of the glycosidic bond to
generate smaller sugars. Glycoside hydrolases are ubiquitous in nature and are involved in
the degradation of biomass such as cellulose and in a variety of cellular functions. Together
with glycosyltransferases, glycosidases form the main catalytic machinery for the synthesis
and cleavage of glycosidic bonds. Acanthamoeba exhibits glycosidase activities including
beta-glycosidase, alpha-glucosidase, beta-galactosidase, beta-N-acetyl-glucosaminidase,
beta-N-acetyl-galactosaminidase, and alpha-mannosidase. Acanthamoeba extracts mediate
enzymatic lysis of the cell walls of several species of bacteria, including Micrococcus
lysodeikticus, Micrococcus roseus, Streptococcus faecalis, Bacillus megaterium, Sarcina
lutea, Micrococcus radiodurans and limited activity against Bacillus subtilis, Bacillus cereus,
but has no effect on cyst walls. Exhaustive digestion of Micrococcus lysodeikticus. Cell walls
released free N-acetyl-glucosamine, N-acetyl-muramic acid, glycine, alanine, glutamic acid

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and lysine, suggesting that Acanthamoeba possess endo- and exo-hexosaminidases and
betaN-acetyl-hexosaminidases. Acanthamoeba is known to use maltose, cellobiose, sucrose
or lactose, some of the glycosidases listed above may suggest the use of these disaccharides
(Henrissat B, et al., 1990). free N-acetyl-muramic acid, glycine, alanine, glutamic acid and
lysine, suggesting that Acanthamoeba possess endo- and exo-hexosaminidases and betaN-
acetyl-hexosaminidases. Acanthamoeba is known to use maltose, cellobiose, sucrose or
lactose, some of the glycosidases listed above may suggest the use of these disaccharides
(Henrissat B, et al., 1990). free N-acetyl-muramic acid, glycine, alanine, glutamic acid and
lysine, suggesting that Acanthamoeba possess endo- and exo-hexosaminidases and betaN-
acetyl-hexosaminidases. Acanthamoeba is known to use maltose, cellobiose, sucrose or
lactose, some of the glycosidases listed above may suggest the use of these disaccharides
(Henrissat B, et al., 1990).
acanthaporin:Acanthamoeba culbertsoni has been isolated from extracts of virulent species
in Acanthamoeba culbertsoni. Acantaporin is cytotoxic to human neuronal cells and exerts
antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes.
The tertiary structures of theshapeactive monomeric and inactive dimeric forms of
acanthaporin, revealed a currently unknown protein fold and pH-dependent activation
mechanism. The tertiary structure reveals a unique protein fold,The structure of acanthaporin
is well defined, composed of fourα-helices (residues 7-24, 32-36, 42-47 and 54-58) connected
by three loop regions and stabilized by five disulfide bridges: Cys5-Cys42, Cys13-Cys45,
Cys24-Cys31, Cys47-Cys55 and Cys51 -Cys61. The membrane permeabilization activity is
initiated by electrostatic interactions. The helix forms a hydrophobic surface that is mainly
composed of alanine residues. In particular, three histidine residues (His50, His52, and His59)
at the C-terminus of the fourth α-helix cluster together and form a putative epitope that is
charged dependent on pH. Acantaporin shows antimicrobial and pore-forming activity in a pH-
dependent manner. Histidine side chains can be chemically modified with
diethylpyrocarbonate (DEPC), thus preventing protonation of these residues below the pK.

15
Figure 5. Three-dimensional structure of monomeric acanthaporin.(a) The ribbon plot
shows the averaged structure of acanthaporin at pH 5.4. α helices are green, loops are gray,
and disulfide bonds are yellow. (b) The charge distribution is shown as the electrostatic surface
potential of acanthaporin. positively charged regions are blue, negatively charged regions are
red, and uncharged regions are white. (c) the positively charged cluster within the c-terminal
helix comprising His50, His52, lys56 and His59 at pH 5.4(Michalek M, Sönnichsen FD, et al.,
2013).
Indirect virulence factors:The ability of Acanthamoeba to cause human disease is a
multifactorial process and depends, among other factors, on its ability to survive outside its
host and under various conditions (high osmolarity, variable temperatures, food deprivation,
and resistance to chemo-drugs). therapeutic), (24. DeJonckheere JF,et al,1980).
Cell differentiation:Cell differentiation is the ability of Acanthamoeba to differentiate into a
morphologically distinct dormant cyst form or a vegetative trophozoite form. This is a reversible
change, depending on environmental conditions. Cysts are resistant to various antimicrobial
agents and adverse conditions such as extreme temperatures, pH, osmolarity, desiccation,
and cysts can become airborne: all of which present a major problem in chemotherapy
because their persistence can lead to disease recurrence . Acanthamoeba cysts can survive
several years while maintaining their pathogenicity. Arecharacteristicssuggested that the main
functions of the cysts lie in resisting adverse conditions and in the propagation of amoebas
through the environment. Furthermore, this may represent the ability of Acanthamoeba to
switch surface protein/glycoprotein expression, in response to changing environments and/or
immune surveillance. Cell differentiation represents an important factor in Acanthamoeba
transmission and recurrence of its infection. However, the molecular mechanisms underlying
these processes remain incompletely understood (Mazur T, Hadas, et al., 1995).

16
Temperature tolerance, osmotolerance and growth at different pH:being afree-living
amoeba, Acanthamoebais exposed to various temperatures, osmolarity, and pH.
Similarly, contact with the tear film exposes Acanthamoeba to high osmolarity (due to the
salinity in the tears), high temperatures as well as alterations in pH. For successful
transmission, Acanthamoeba must withstand such stress and exhibit biological activity.
Acanthamoeba showed high levels of heat shock proteins (ie HSP60 and HSP70)
compared to weak pathogens. Higher levels of heat shock proteins in Acanthamoeba may
indicate their involvement in tolerance to host stressors and/or in virulence of
hosts.thespecies. The ability of Acanthamoeba to grow at high temperatures and high
osmolarity correlates with the pathogenicity of Acanthamoeba isolates and may provide a
good indicator of potential pathogenicity (Podlipaeva IuI, et al., 2006).
Chemotaxis:Chemotaxis directs the movement of amoebas according to certain chemicals
in their environment. This is Acanthamoeba moving towards the highest concentration of food
molecules, or running away from poisons. Acanthamoeba exhibits chemosensory responses
as observed by its response to a variety of bacterial products or potential bacterial products
by actively moving towards the attractant. Acanthamoeba responded to the chemotactic
peptides formylmethionyl-leucyl-phenylalanine, formyl-methionyl-leucyl-
phenylalaninebenzylamide, lipopolysaccharide, and lipid A. In addition, significant responses
were also found to cyclic AMP, lipoteichoic acid, and N-acetylglucosamine.Thissuggests that
Acanthamoeba membranes possess receptors sensitive to bacterial substances, which are
different from mannose-binding protein, involved in binding to host cells to produce cytotoxicity
or involved in binding to bacteria during phagocytosis. The movement rate is relatively
constant (approx. 0.40 µmper second), indicating that the locomotor response to these signals
is a taxi, or possibly aklinokinesis,but not orthokinesis (Schuster FL. 2002).
Ubiquity:Acanthamoeba has been found in a variety of environments, from drinking water to
distilled water wash bottles, on the deep ocean floor, and in Antarctica. It is therefore not
surprising that humans regularly encounter and interact with these organisms, as evidenced
by the findings that in some regions, up to 100% of the population tested possess antibodies
to Acanthamoeba, suggesting that these are one of the most ubiquitous protists and often
comes into contact with humans and can cause serious infections (Maubon D, Dubosson M,
et al., 2012).
Biofilms: Biofilms play an important role in the pathogenesis of Acanthamoeba keratitis.
Biofilms are sessile communities derived from microbes, which can form in aqueous
environments, as well as on any medical material and device, including intravenous catheters,
contact lenses, suture material, and intraocular lenses. In the case of contact lenses, biofilms
form through contamination of the storage case. Once established, biofilms provide attractive

17
niches for Acanthamoeba, satisfying their nutritional requirements and providing resistance
to disinfectants. Furthermore, this allows for greater attachment of Acanthamoeba to contact
lenses.as a biofilm matrix and by Azotobacter species as a component of its cystic wall. A
probable alginate ligase has been identified in the Acanthamoeba castellanii genome
sequence, similar to an alginate ligase from Pseudomonas spp.(Zegans ME, et al., 2002).The
presence of alginate ligase in Acanthamoeba castellanii was unexpected since the only
eukaryotes known to possess this enzyme are marine invertebrates. Alginate ligase may allow
the use of alginate as a food source, but also potentially allows Acanthamoeba castellanii to
break down the matrix of some biofilms and feed on the encased bacteria, as some biofilm-
forming bacteria use alginate ligase to release organisms from the biofilm to allow colonization
of new sites. The presence of alginate ligase also provides an explanation for the observation
that bacterial biofilms support the growth of Acanthamoeba castellanii.
M_degradans_2 gammaproteobacteria
63 pseudomonassp. gammaproteobacteria
67 M_degradans_1 gammaproteobacteria
A_castellanii
Eukaryote:
Amoebozoa
67 M_degradans_3 gammaproteobacteria
s_coelicolor Actinobacteria
P_heparinus sphingobacteria
0.05
Figure 6.Phylogenetic tree of proteins related to alginate ligase from Acanthamoeba
castellani; numbers represent 500 replicate maximum likelihood snatch percentages.
Signal transduction:The ability of Acanthamoeba castellanii to thrive in different
environments requires the ability to sense and interact with its environment and to process
and respond to a variety of external signals. This ability is reflected in a significant repertoire
of genes dedicated to signal transduction. Acanthamoeba castellanii counts
serine/threonine protein kinases among the inferred protein sequences of Acanthamoeba
castellanii, including putative receptor kinases. Receptor kinases are absent from the
genome data of most protists sequenced so far (except E. histolytica). The receptor kinases
are similar to those of Acanthamoeba polyphaga Mimivirus, suggesting a common
origin.(Anderson IJ, Watkins RF, et al, 2005).
A group of 21 histidine kinases and 11 receptor response regulator domains (RRR proteins)
have been identified in Acanthamoeba castellanii. These proteins likely play an important
role in allowing the amoeba to sense and respond to environmental changes. In eukaryotes,
histidine kinases are often involved in development or sensing, encryption, excitation,
and/or detection of stress conditions. Two of the RRR domains are attached to PAS
domains, which in other organisms sense changes in the internal environment, such as
oxygen concentration, light intensity, and redox balance. The presence of histidine kinases

18
may be one of the factors that allow Acanthamoeba castellanii to adapt to many different
environmental niches and resist stress conditions.(Anderson IJ, Watkins RF, et al, 2005).
Polyhydroxybutyrate depolymerase:Acanthamoeba castellanii supposedly possesses an
enzyme to depolymerize bacterial storage compound polyhydroxybutyrate (PHB). PHB is an
energy and carbon storage polymer produced in bacteria as cytoplasmic granules that can
reach up to 80% of the cell mass. PHB is broken down by bacteria to acetyl-CoA, which can
then enter the tricarboxylic acid cycle for energy production. Acanthamoeba castellanii likely
encounters this polymer while feeding on bacteria, and the presence of PHB depolymerase
in Acanthamoeba castellanii suggests that it can also use PHB as an energy source. This is
the first report of this enzyme in a eukaryote, although D. discoideum also appears to have a
copy of this enzyme (Anderson IJ, Watkins RF, et al, 2005).
Host factors: The factors that allow Acanthamoeba to produce the disease are not limited
solely to the pathogen, the findings suggest that factors such as host susceptibility, tissue
specificity, tear factors, IgA, corneal trauma, as well as environmental factors such as
osmolarity (Khan NA, et al, 2001).

19
24 genotypes of Acanthamoeba spp.: Of the 24 genotypes described so far, 12 have been
isolated in human cases, 4 only in keratitis (genotypes T3, T6, T11 and T13), 3 only in
encephalitis (genotypes T1, T12 and T18) and 5 genotypes identified in cases of keratitis and
encephalitis (genotypes T2, T4, T5, T10 and T15). Table Nº2 presents these data.
Table N° 2. T1-T20 genotypes ofAcanthamoeba spp.,disease, species
Pussard and Pons groups
Endosymbiosis:Acanthamoebait can play a significant role in the transmission of some
bacteria in drinking water, especially some pathogenic bacteria by serving as hosts for them,
being described as a "Trojan horse". The intracellular location of these microorganisms,
protected from adverse conditions, also allows the bacteria to evade the host's defenses,
resist the action of antibiotics and increase their virulence (Trabelsi et al., 2012).
Approximately 20 to 24% of clinical and environmental Acanthamoeba isolates were reported
to harbor bacteria intracellularly (Visvesvara et al., 2013). Acanthamoeba feeds on bacteria in
the environment, capturing them within its cytoplasm through phagocytosis. Phagocytosed
bacteria are usually killed and digested by the amoeba, however, some species of bacteria
can grow and reproduce within the cytoplasm and become a symbiont (Nwachuku et al.,
2003). Most Acanthamoeba spp. they host endosymbionts that may include viruses, fungi,
Genotype Disease Species Cluster
T1 Encephalitis A. castellanii
Group III
T2 Keratitis, encephalitis A. palestinensis
T3 keratitis A.griffini
Group II
T4 Keratitis, encephalitis A. castellanii
T5 Keratitis, encephalitis A. lenticulata
Group III
T6 keratitis A palestinensis
T7
not yet described in humans
A. astronyxis
Group I
T8 A. tubiaschi
T9 A. commandoni
T10 Keratitis, encephalitis A.culbertsoni Group III
T11 keratitis A HatchettYo Group II
T12 Encephalitis a healyi Group III
T13 keratitis TO.spp.
Group II
T14 not yet described in humans TO.spp.
T15 Encephalitis-keratitis a. jacobsi Group III
T16 not yet described in humans TO.spp Group II
T17 not yet described in humans TO.spp
Group I
T18 Encephalitis A. byersi
T19 not yet described in humans A. micheli sp. Group II
T20 not yet described in humans OSU-04-023 Group III

20
protists, and bacteria, some of which are potentially pathogenic to humans. It can also
increase the virulence of some bacteria called Amoeba-Resistant Microorganisms (MsRA)
(Coşkun et al., 2013).
Acanthamoebais known to host a variety of viruses: Mimivirus, Marseillevirus, Tupanvirus,
Catovirus, and Pandoravirus (P. quercus, P. inopinatum, P. macleodensis, P. neocaledonia,
and P. salinus), coxsackievirus, adenovirus, poliovirus, echovirus, enterovirus, or virus
vesicular stomatitis. Yeasts such as Cryptococcus neoformans, Blastomyces dermatitidis,
Sporothrix schenckii, Histoplasma capsulatum, and Exophiala dermatitidis. Pathogenic
bacteria include: Aeromonas spp, Bacillus cereus, Bartonella spp, Burkholderia spp,
Burkholderia pickettii, Campylobacter jejuni, Chlamydophila pneumoniae, Chlamydia
pneumoniae, Coxiella burnetti, Escherichia coli 0157, neuropathogenic Escherichia coli K1,
Flavobacterium spp, Francisella tula rensis, Helicobacter pylori , Legionella pneumophila,
Listeria monocytogenes, Staphylococcus aureus, Methicilin-resistant Staphylococcus aureus,
Mycobacteria tuberculosis, M. avium, M. leprae, Pastereulla multocida, Prevotella intermedia,
Porphyromona gingivalis, Pseudomona aeruginosa, Rickettsia, Salmonella typhimurium,
Shigella dysenteriae, S. sonnei, Vibrio cholerae, V. parahaemolyticus, Wadd lia chondrophila
(Winiecka-Krusnell, et al., 2002). Among the protists are Cryptosporidium and Toxoplasma
gondii.
Figure 7. Composition of the Acanthamoeba genome according to sequence
similarity.For each protein the best "BLASTP" analysis is shown in a non-redundant
database. In the central bar, all the annotated genes of Acanthamoeba are represented,
colored according to their similarity with the different kingdoms: Eukaryota (blue), Bacteria
(red), Archaea (green) and virus (purple); orphan genes are represented in yellow (Clarke, et
al., 2013).

21
Acanthamoebathey feed on bacteria, but some of the bacteria can resist the amoeba's
digestion. Acanthamoeba thus act as the "endosymbiont" of the bacterial world and are
considered a training ground for pathogenic bacteria to the extent that passage through
amoebae may be associated with increased bacterial virulence. In its cyst form,
Acanthamoeba can support and protect intracellular bacteria from biocides, leading to
recurrent infections. Some intracellular bacteria have been reported to modulate amoeba
metabolic functions, such as encystment. Francisella tularensis thus induces the
encystment process, unlike Parachlamydiae Acanthamoebae, which inhibits it. Analysis of
Acanthamoeba genomes suggests genetic exchanges with intracellular microorganisms.
The A. castellanii genome contains 2.9% of genes possibly acquired by lateral gene transfer
(LGT). Among them, several are annotated as Acetyltransferase, Gcn5-related N-
acetyltransferase (GNAT) superfamily protein. GNATs are a very large family of enzymes,
comprising more than 10,000 members, that are identified in all kingdoms. of life.GNATs
catalyze the transfer of an acetyl group from acetyl-CoA to the primary amine of a wide
range of substrates, from small molecules to macromolecules.Genes derived from LGT are
differentially expressed in Acanthamoeba castellanii after the growth phase. growth, under
agitation, hypoxia or after bacterial infections, which suggests its participation in the
physiology of Acanthamoeba (Michalek M, et al, 2012). castellanii contains 2.9% of genes
possibly acquired by lateral gene transfer (LGT). Among them, several are annotated as
Acetyltransferase, Gcn5-related N-acetyltransferase (GNAT) superfamily protein. GNATs
are a very large family of enzymes, comprising more than 10,000 members, that are
identified in all kingdoms. of life.GNATs catalyze the transfer of an acetyl group from acetyl-
CoA to the primary amine of a wide range of substrates, from small molecules to
macromolecules.Genes derived from LGT are differentially expressed in Acanthamoeba
castellanii after the growth phase. growth, under agitation, hypoxia or after bacterial
infections, which suggests its participation in the physiology of Acanthamoeba (Michalek
M, et al, 2012). castellanii contains 2.9% of genes possibly acquired by lateral gene transfer
(LGT). Among them, several are annotated as Acetyltransferase, Gcn5-related N-
acetyltransferase (GNAT) superfamily protein. GNATs are a very large family of enzymes,
comprising more than 10,000 members, that are identified in all kingdoms. of life.GNATs
catalyze the transfer of an acetyl group from acetyl-CoA to the primary amine of a wide
range of substrates, from small molecules to macromolecules.Genes derived from LGT are
differentially expressed in Acanthamoeba castellanii after the growth phase. growth, under
agitation, hypoxia or after bacterial infections, which suggests its participation in the
physiology of Acanthamoeba (Michalek M, et al, 2012). 9% of genes possibly acquired by
lateral gene transfer (LGT). Among them, several are annotated as Acetyltransferase,
Gcn5-related N-acetyltransferase (GNAT) superfamily protein. GNATs are a very large
family of enzymes, comprising more than 10,000 members, that are identified in all

22
kingdoms. of life.GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the
primary amine of a wide range of substrates, from small molecules to
macromolecules.Genes derived from LGT are differentially expressed in Acanthamoeba
castellanii after the growth phase. growth, under agitation, hypoxia or after bacterial
infections, which suggests its participation in the physiology of Acanthamoeba (Michalek
M, et al, 2012). 9% of genes possibly acquired by lateral gene transfer (LGT). Among them,
several are annotated as Acetyltransferase, Gcn5-related N-acetyltransferase (GNAT)
superfamily protein. GNATs are a very large family of enzymes, comprising more than
10,000 members, that are identified in all kingdoms. of life.GNATs catalyze the transfer of
an acetyl group from acetyl-CoA to the primary amine of a wide range of substrates, from
small molecules to macromolecules.Genes derived from LGT are differentially expressed
in Acanthamoeba castellanii after the growth phase. growth, under agitation, hypoxia or
after bacterial infections, which suggests its participation in the physiology of
Acanthamoeba (Michalek M, et al, 2012).
Nuclear genomic organization of Acanthamoeba:A wide variety of genes involved in the
synthesis of essential nutrients such as serine, cysteine, tryptophan, arginine, histidine,
purines and pyrimidines, deoxynucleotides and folates, among others, have been identified
from the Acanthamoeba genome, demonstrating its ease of obtaining nutrients from a wide
variety of sources. It also presents a very high number of genes that code for different
kinases as well as for its receptor and signal transduction regulators, which suggests the
ability of Acanthamoeba to interact with the environment and respond to external signals.
The gene for trehalose-6-phosphate synthetase has also been identified, which is an
enzyme closely related to protection against difficult environmental conditions such as
desiccation, osmotic stress, extreme temperatures or pH confirming its high capacity for
survival and adaptability. Other typical metabolism and cell cycle genes have also been
identified, such as those that participate in the formation of the cyst (gene for the formation
of cellulose), or for its lysis (cellubiosidase) when the trophozoite emerges. The
transcriptional unit for Acanthamoeba ribosomal RNA (rRNA) with a length of 12kbp has
also been studied, of which 9.7kbp include the external transcriptional spacer (ETS),
internal transcriptional spacers (ITS), 18S rDNA, 5.8S rDNA and 26S rDNA and the
remaining 2.3 kbp correspond to the intergenic spacer. Although there are only 24 copies
per haploid genome, the number rises to 600 copies per cell due to the polyploid (25n)
genome. Other studies show the importance of lateral gene transfer (TLG) in the
Acanthamoeba genome. They describe 15,455 intron-rich genes of which the majority come
from interkingdom TLGs and which have already been incorporated into the Acanthamoeba
transcriptional machinery. Authors point out that the rich and complex system of signaling
and cellular communication of Acanthamoeba (including the complete chain of signaling by

23
tyrosine kinanases) in addition to the diversity of the extracellular receptors predicted, are
comparable to those of cellular slime molds (Dicthyostellids). As Acanthamoeba is a host
for a wide variety of bacteria and viruses, (Guimaraes AJ, et al, 2016).
Mitochondrial genome of Acanthamoeba castellanii:The complete mitochondrial
genome of Acanthamoeba castellanii genotype T4 is circular and consists of 41,591 bp with
an A+T content of 70.6%. In it, the genes listed in Table 4 have been described: two genes
for rRNA (large and small subunit), 16 genes for transfer RNA, 33 proteins (17 subunits of
the respiratory chain and 16 ribosomal proteins), 8 open frames Open Reading Frame
(ORF) with more than 60 codons and no specific function. Two of these ORFs (orf124 and
orf142) have homologues in other mitochondrial DNAs (orf25 and orfB respectively), 3
ORFs (orf83, orf115 and orf349) are unique to Acanthamoeba castellanii and three are
intronic ORFs. (Burger G, et al., 1995).
Figure8. Physical and genetic map of A. castellanii mtDNA. Identified genes, exons, and non-
intronic ORFs are shown as filled blocks, while intronic ORFs are indicated by hatched blocks.
Intergenic shading denotes overlapping genes. Transfer RNA genes are represented as thin
bars, with corresponding letters indicating their amino acid specificities (see Table 4). X,
Unidentified tRNA having a predicted 8 nt anticodon loop (see text). The arrow shows the
direction of transcription. Gene abbreviations are listed in Table 1 (cox1/2 indicates that a
single continuous ORF specifies COX1 and COX2 polypeptides). A size scale is indicated and
the EcoRI restriction map is shown in the innermost circles(Burger, et al,1995).

24
Table 3.Genes identified in the mtDNA of Acanthamoeba castellanii
A. ribosomal RNA (2)
large subunit (rnl)
small subunit (rns)
B. TransferRNA (16)
C. Electron transport / oxidative phosphorylation
Respiratory chain (13)
NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, nad6,
nad7, nad9, nad11)
apocytochrome b (cob)
cytochrome oxidase (cox1/2, cox3)
ATP synthase complex (3)
F0-ATPase (atp6, atp9)
F1 ATPase (atp1)
D. ribosomal protein(16)
Large subunit (6): rpl2, rpl5, rpl6, rpl11, rpl14, rpl16
Small Subunit (10): rps2, rps3, rps4, rps7, rps8, rps11, rps12, rps13, rps14, rps19
E. ORFs of function unknown(2)
orf142(=ymf19;Aca), homolog to liverwort and angiosperm mtDNA orfB orf124
(=ymf39;Aca), homolog to liverwort and angiosperm mtDNA orf25
F. ORFs unique toA. castellanii mtDNA (3)
orf83(= ymf49), orf115 (= ymf50), orf349 (= ymf51)
G. Group I intronic ORFs (3)
il-orf (=ymf46), i2-orf (=ymf47), i3-orf (=ymf48) (in the first, second, and third
introns, respectively, of rnl)
ORF: Open Reading Frame,(open reading frames), (Burger, et al,1995).

25
Table 4. Genes related to Acanthamoeba spp. keratitis.
gene identification
34934
12288
1047
9969
3258
19397
3757
8789
9969
11390
12288
12995
25924
26649
27916
9026
6737
1586
8645
8337
27752
34693
15075
31764
27487
Function
Accession
mannose binding
metalloproteases
Zinc metalloprotease aminopeptidase I (M18)
metalloenzyme superfamily
metalocarboxypeptidase
metalloenzyme
proteases
peptidase S8 and S53 subtilisin kexin sedolysin
PFAM peptidase T2 asparaginase 2
C19 peptidase family
metalocarboxypeptidase
peptidase S8 and S53, subtilisin, kexin, sedolysin
Aminopeptidase I zinc metalloprotease
(M18)
M17 peptidase family
Serine aminopeptidase, S33
C19 peptidase family protein
temperature tolerance
hsp20/alpha crystallin family
Hsp70 protein
hsp20/alpha crystalline family protein
phospholipases
phospholipase A2 activating activity
phospholipase D
phospholipase D
antioxidant defense
glutathione peroxide
peroxidase
oxidoreductase (Guimaraes AJ, et al, 2016).

26
Table 4. Genes related to Acanthamoeba spp. keratitis:There are 45 genes related to
virulence. The presence of mannose-induced protein (MIP), a gene encoding an important
transmembrane protein involved in adhesion to the corneal surface, is detected. 17 genes
related to the cytoskeleton are found, especially 3 genes that code for actin-binding protein. The
presence of 8 lipases is reported, especially 3 phospholipases that play a potential role in host
cell membrane disruption and lysis. The presence of 11 genes encoding peptidases, which are
enzymes that facilitate host invasion, are shown. . The presence of 1 glycosidase, 1 peroxidase
and 1 glutathione peroxidase which are antioxidant enzymes involved in the defense of
amoebae against reactive oxygen species is reported.(Guimaraes AJ, et al, 2016).
Features of the mitochondrial genoma
Acanthamoeba castellanii: All genes and ORFs are on the same strand and are closely packed;
only 6.8% of the sequences do not have a coding function and the intergenic spacers are small
with a size between 1 and 616 bp with a mean of 64 bp. Ten pairs of protein-coding genes
overlap by 38 bp and two cytochrome oxidase subunits, COX 1 and COX2, are found in a
single ORF. There are only 3 introns, all of them from group I in the gene for the large rRNA
subunit. In the mitochondrial genome there are fewer genes that code for transfer RNA than is
necessary for the synthesis of mitochondrial proteins, for which reason Burger and
cabbage(nineteen ninety five)suggested that blot RNAs not encoded by mitochondria are
imported from the cytosol. The genetic code for Acanthamoeba castellanii mitochondrial DNA
is modified to encode UGA for tryptophan. There are no repeat sequences or motifs associated
with regulatory elements (Lang, BF & Burger, et al, 1995).

27
Pan-Genome and Central Genome of Acanthamoeba spp.
The genome of 8 different Acanthamoeba species was compared in order to analyze the
genetic diversity between the different Acanthamoeba species. Acanthamoeba Pangenome
size reaches 59,450 genes encompassing clusters or single genes(Guimaraes AJ, et al, 2016).
Figure 9. Flower diagram showing core, dispensable, and strain-specific genes of 8
Acanthamoeba species:The flower plot shows the number of core genes (in the center), the
number of dispensable genes (in the ring), and the number of strain-specific genes (in the petals)
for the 8 Acanthamoeba species. The numbers under the strain name indicate the total number
of related genes. Different colors indicate different Acanthamoeba T genotype groups: T4
genotype in blue; T5 strains in yellow; T10 strains in red; T11 strains in green.

28
Of a total of 843 clusters they made up the central genome, which represented 1.5% of the
pangenome. Among the core genome, A. triangularis exhibited 399 clusters (0.7%) with two
representative sequences and 227 clusters (0.4%) composed of three representative
sequences (Table S4). What's more, A. triangularis genes were part of 99 unique gene clusters
(Table S4). The COG analysis showed that the function of "unknown function", "post-
translational modification, protein turnover and chaperones", and the function of "signal
transduction mechanisms" (63) are among the most represented categories for the core
genome andA . triangularis unique sequences (Figures S4 and S5). Surprisingly, we found a
large number of genes unique to Acanthamoeba castellanii (n = 8. 236 genes) and
Acanthamoeba lugdunensis (n = 2,544). These unique genes play a role in signal transduction,
pathway metabolism, and DNA biosynthesis (Figure S6). However, a large part of these genes
is classified as having unknown function (233 and 137 for Acanthamoeba castellanii and
Acanthamoeba lugdunensis, respectively). This high number of unique genes could be
explained by the higher predicted protein numbers of Acanthamoeba castellanii (73,447) and
Acanthamoeba lugdonensis (65,171) compared to the other amoebae used in the analysis
(Acanthamoeba culberstoni, 22,241; Acanthamoeba lenticular, 29,468; Acanthamoeba poly
phaga , 32,524; Acanthamoeba quina, 49,881; Acanthamoeba rhysodes, 47,088 and
Acanthamoeba triangularis, 39,411). Finally,(Guimaraes AJ, et al, 2016).
Genome sequence analysis predicts a broad biosynthetic capacity for Acanthamoeba
castellanii in addition to the ability to utilize complex organic nutrients from its food sources.
This probably frees Acanthamoeba castellanii from having to acquire many nutrients directly
from other organisms, thus increasing the diversity of their food sources and removing a key
obstacle to their ability to adapt and persist in many cases (Guimaraes AJ, et al, 2016). .

29
Table 5. Database matches of Acanthamoeba castellanii readings with metabolic enzymes.
Read intended function CDD Domain E-value
EDCAE55TF homoserine kinase COG0083 - Homoserine kinase 2e-23
EDCA479TR threonine synthase COG0498 - Threonine synthase 5e-35
EDCAL01TF Tryptophan synthase, alpha chain COG0159 - Tryptophan synthase alpha chain 1e-36
EDCA602TF Tryptophan synthase, beta chain
COG0133 - Tryptophan synthase beta
chain 1e-106
EDCA428TR methionine synthase COG0620 - Methionine synthase II 2e-37
EDCCE67TF Phosphoserine aminotransferase COG1932 - Phosphoserine aminotransferase 2e-53
EDCC889TR cysteine synthase COG0031 - Cysteine synthase 6e-18
EDCAD30TR Imidazolglycerol-phosphate dehydratase Pfam00475 - Imidazolglycerol-phosphate 1e-11
dehydratase
EDCB576TR Argininosuccinate ligase COG0165 - Argininosuccinate ligase 7e-51
EDCA320TF 3-dehydroquinate synthetase COG0337 - 3-dehydroquinate synthetase 1e-29
EDCB047TR chorismate synthase Pfam01264 – Chorismate synthase 2e-72
EDCCA12TF 5-aminolevulinate synthase COG0156 - 7-keto-8-aminopelargonate 1e-72
synthetase and related enzymes
EDCAA25TR Uroporphyrinogen decarboxylase COG0407 - Uroporphyrinogen-III 5e-67
decarboxylase
EDCBN30TR Coproporphyrinogen III oxidase Pfam01218 - Coproporphyrinogen III oxidase 4e-84
EDCA547TF GTP cyclohydrolase II COG0807 - GTP cyclohydrolase II 1e-15
EDCAR32TF 6,7-dimethyl-8-ribitylumazine synthase Pfam00885 - 6,7-dimethyl-8-ribitilumazine 3e-21
synthase
EDCBB11TR Panthoate - beta-alanine ligase Pfam02569 - Pantoate: beta-alanine ligase 1e-47
EDCBC12TF Phosphomethylpyrimidine kinase COG0351 - Hydroxymethylpyrimidine / 2e-45
phosphomethylpyrimidine kinase
EDCAS03TF Protein A molybdenum cofactor biosynthesis COG2896 - Molybdenum cofactor 3e-77
biosynthesis enzyme
EDCBP70TF
Formylglycinamide ribonucleotide
amidotransferase COG0047 - FGAM synthase 3e-18
EDCCR39TF phosphoribosylaminoimidazole carboxylase COG0026 - Phosphoribosylaminoimidazole 1e-40
carboxylase
EDCBQ62TF Dihydroorotase COG0044 - Dihydroorotase and related cyclics 2e-39
amidohydrolases
EDCAD04TF thymidylate synthase Pfam00303 - Thymidylate synthase 5e-39
EDCAX65TR ribonucleoside diphosphate reductase COG0209 - Ribonucleotide reductase, alpha 2e-35
subunit
EDCAI59TR NADPH oxidase generating superoxide Pfam01794 - Ferric reductase 5e-25
EDCAM34TF cellulase Pfam00150 - Cellulase 2e-05
EDCAN31TF Cellobiosidase Smart00633 - Glycosyl Hydrolase Family 10 5e-20
EDCAQ93TR Trehalose-6-phosphate synthase COG0380 - Trehalose-6-phosphate synthase 1e-60
(Anderson IJ, Watkins RF, et al, 2005).

30
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