Acanthamoeba use of contaminated water (Buehler et al., 2009;

Acanthamoeba
is a microscopic, free-living pathogen which is responsible for causing severe
infections of the eye, skin, and central nervous system. It appears to be one
of the most important protozoa found in natural environment.

Acanthamoeba is responsible for causing
Acanthameba Keratitis (AK), an infection of the eye which can result in visual
impairment or blindness; Granulomatous Amoebic encephalitis, severe infection
of brain and spinal which mainly occurs in immuno-compromised patients and
disseminated infection, which affects the skin, sinuses and lungs.

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The majority of
AK strains are associated with genotype T4, but AK strains belonging to other
genotypes have also been reported worldwide (Ledee et al., 2009; Ertabaklar et
al., 2007). Acanthamoeba belonging to genotype T4 has been primarily
associated with AK; however strains belonging to genotypes T3, T5, T6 and T11
have also been found to cause AK.

The majority of
AK cases are reported from contact lens wearers belonging to developed
countries. However, in patients from developing countries, the causative factor
is majorly corneal trauma or the use of contaminated water (Buehler et al., 2009; Sharma et al., 2000). The chief reason for
developing AK are poor hygienic habits including inappropriate storage of
lenses, use of contact lenses for long durations, formation of biofilm on
contact lenses and use of tap water as a substitute for lens solution (Siddiqui
and Khan, 2012).

Detection of AK
is quite difficult and hence should be suspected in all lens wearers, people
having corneal trauma with soil, use of contaminated water and in patients
recovering from ocular surgeries (Lorenzo-Morales et al., 2013). The diagnosis being problematic, it is often
misdiagnosed as fungal, bacterial or viral keratitis.

A deeper
understanding of the proteins expressed by various Acanthamoeba spp. might help
in the diagnosis and treatment of this vulnerable disease. In reference to this
our study aims to compare the protein profiles of various Acanthamoeba species
which will help in gaining a better understanding about the disease
pathogenesis and will eventually contribute towards the culmination of
effective diagnosis.

 

 

 

 

Review
of Literature

 

Acanthamoeba is
one of the primary causes of keratitis which is a painful sight-threatening
corneal infection. It is an opportunistic amoeba which is found globally. Its
presence has been reported from all kinds of natural environments including sea
water, pond water, beaches, springs and even air. Reportedly, it has been
isolated from packaged mineral water, vegetables, air conditioners, sewage.

It was also
found from hospitals, dialysis units, human nasal cavities, throat, pharyngeal
swabs, lung tissues and human feces (Siddiqui and Khan 2012). The reason for
the wide distribution of Acanthamoeba is due to its ability to withstand all
kinds of harsh weather conditions. The universal presence of Acanthamoeba is
the reason for humans to get easily infected with it and fall prey to AK.

The life cycle
of Acanthamoeba consists of two stages: a vegetative growing trophozoite stage
and an inactive dormant cyst stage. The trophozoite is about 14-40 µm in
diameter whereas the double walled cyst is about 12-16 µm in diameter.

In most of the
developed countries, generally the contact lens wearers report with cases of AK
due to the negligence posed during handling and storage of contacts. In
contrast the cases reported from India and other developing countries come from
non contact lens wearers and those who are working in agricultural fields,
one’s that come in contact with contaminated water and soil. A study in south
India reported an epidemic of AK in rural, non contact lens wearers in 1993 and
2003 (Lalitha et a.,l 2012).

In spite of the
difference in the source of AK, the problems and disease severity posed by
Acanthamoeba are almost similar in developing as well as developed nations. Patients
suffering from AK might suffer ocular pain with photophobia, ring like stromal
infiltrates and lid oedema (Lorenzo-Morales et a.,l 2015).

On the basis of
18S rDNA sequencing Acanthamoeba has been classified into 20 different
genotypes (T1-T18) (Huang et a.,l
2016). Till date the majority of studies have considered T4 as the causative
genotype behind AK, however Siddiqui and Khan in 2012 have reported T2, T3, T4,
T5, T6, T10, T11, T13 and T15 to be related with AK in humans. But there is
limiting knowledge on different Acanthamoeba genotypes based on their
respective protein profiles (Pumidonming et a.,l 2014). These days a large
number of proteomic studies are being used to explore secreted proteins and
study disease pathogenesis. For example, Lourenssen et al., 2010 have reported about a large number of proteins which
cause amoebic colitis by disrupting the mucus layer. Also Lorenzo-Morales et al., 2002 and Mattana et al., 2002 have reported that
Acanthamoeba produces serine, cystein and metalloproteases and extracellular
protease activities are elevated in pathogenic Acanthamoeba strains. This
clearly shows the importance of proteomic based study and its role in studying
disease pathogenesis. 

 

 

 

Aim
of the study

 

To establish a proteomic dataset of
extracellular secretory proteins of 11 Acanthamoeba isolates.Perform a comparative proteomic analysis
of  proteins from all the Acanthamoeba
isolates to be considered

 

 

 

Methodology

The study will
take into consideration 11 Acanthamoeba isolates of five different genotypes
and a comparative proteomic analysis will be carried out which will help in
devising a much more efficient diagnostic method.

An overview of
the methodology:

Acanthamoeba species will be axenically
cultured in a suitable medium.Extracellular secretory proteins will be
isolated and will be subjected to 2D Gel electrophoresis which will help in the
analysis of complex protein mixture.Separated proteins will be identified
using MALD TOF/TOF MS which will help in identifying and genotyping various
Acanthamoeba isolates.

 

 

References

 

Buehler PO, Schein OD, Stamler
JF, Verdier DD, Katz J. The increased risk of ulcerative keratitis among
disposable soft contact lens users. Archives of Ophthalmology. 1992 Nov
1;110(11):1555-8.

 

Ertabaklar H, Türk
M, Dayanir V, et al. Acanthamoeba keratitis due to Acanthamoeba genotype T4 in
a noncontact-lens wearer in Turkey. Parasitol Res 2007; 100(2): 241–246.

 

Huang JM, Lin WC, Li SC, Shih MH, Chan WC, Shin JW, Huang
FC. Comparative proteomic analysis of extracellular secreted proteins expressed
by two pathogenic Acanthamoeba castellanii clinical isolates and a
non-pathogenic ATCC strain. Experimental parasitology. 2016 Jul 31;166:60-7.

 

Lalitha P, Lin CC, Srinivasan M, Mascarenhas J, Prajna
NV, Keenan JD, McLeod SD, Acharya NR, Lietman TM, Porco TC. Acanthamoeba
keratitis in South India: a longitudinal analysis of epidemics. Ophthalmic
epidemiology. 2012 Apr 1;19(2):111-5.

 

Ledee DR,
Iovieno A, Miller D, et al. Molecular identification of T4 and T5 genotypes in
isolates from Acanthamoeba keratitis patients. J Clin Microbiol 2009; 47(5):
1458–1462. ?

Lorenzo-Morales J, Khan NA, Walochnik J. An update on
Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite.
2015;22

Lorenzo-Morales J, Ortega-Rivas A, Foronda P,
Abreu-Acosta N, Ballart D, Martínez E, Valladares B. RNA interference (RNAi)
for the silencing of extracellular serine proteases genes in Acanthamoeba:
molecular analysis and effect on pathogenecity. Molecular and biochemical
parasitology. 2005 Nov 30;144(1):10-5.

 

Lourenssen S, Houpt ER, Chadee K, Blennerhassett MG.
Entamoeba histolytica infection and secreted proteins proteolytically damage
enteric neurons. Infection and immunity. 2010 Dec 1;78(12):5332-40.

 

Mattana A, Cappai V, Alberti L, Serra C, Fiori PL,
Cappuccinelli P. ADP and other metabolites released from Acanthamoeba
castellanii lead to human monocytic cell death through apoptosis and stimulate
the secretion of proinflammatory cytokines. Infection and immunity. 2002 Aug
1;70(8):4424-32.

 

Pumidonming W, Koehsler M, Leitsch D, Walochnik J.
Protein profiles and immunoreactivities of Acanthamoeba morphological groups
and genotypes. Experimental parasitology. 2014 Nov 30;145:S50-6.

 

Sharma S, Garg P, Rao GN.
Patient characteristics, diagnosis, and treatment of non-contact lens related
Acanthamoeba keratitis. British Journal of Ophthalmology. 2000 Oct
1;84(10):1103-8.

 

Siddiqui R, Khan NA. Biology and pathogenesis of
Acanthamoeba. Parasites & vectors. 2012 Jan 10;5(1):6.