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International Journal of Advances in Pharmaceutical Sciences
1 (2010) 01-14
http://www.arjournals.org/ijoaps.html
Review
ISSN: 0976-1055
Microarray based gene expression: a novel approach for identification
and development of potential drug and effective vaccine against visceral
Leishmaniasis
Awanish Kumar1, Abhik Sen2, Pradeep Das1,2*
*Corresponding author:
Dr. Pradeep Das
1 Department of
Biotechnology,
National Institute of
Pharmaceutical Education and
Research, Hajipur, India
2 Department of Molecular
Biology
Rajendra Memorial Research
Institute of Medical Sciences,
Patna, India
Tel No. 91-0612-2631565
Fax No. 91-0612-2634379
E-mail:
drpradeep.das@gmail.com*
Abstract
Visceral Leishmaniasis (VL) is the well-recognized infectious disease among
the complex of Leishmaniasis (cutaneous, mucocutaneous, visceral) in
tropical and subtropical countries. Treatments for VL are unsatisfactory till
date and alarming rise of drug resistance is a serious problem. Vaccines to
control VL have shown some promise, but none are in current clinical use.
Therefore, urgent needs for new and effective anti-leishmanials are pre-
requisite. To identify the potential factors, DNA microarray an advance, high
throughput technology, has open the possibility of discovering new genes that
can contribute to vaccine initiatives or serve as potential drug targets. It has
been successfully applied to many of the protozoan parasites and identified
some new genes as targets. Target discovery is the key step in the biomarker
and drug discovery pipeline to diagnose. After the completion of genome
sequencing of Leishmania major and L. infantum, advancement in
microarray technologies provide new approaches to study the pattern of gene
expression profile during differentiation and development of parasite. It has
the potential to improve our understanding of pathogenicity, mechanism of
drug resistance and virulence factors by identifying up/down regulated gene
and characterizing the respective gene expression. Keeping these
backgrounds in mind, we reviewed the data obtained from genome-wide wide
expression profiling in Leishmania that focuses on applications of microarray
in novel vaccine/drug targets discovery for VL and discuss the potential
avenues for their future investigation. Ultimately this will be able to translate
the findings into the development of novel therapeutic approaches and targets
for VL.
Keywords: Visceral Leishmaniasis, Microarray, Stage-specific, Gene
expression profiling, Gene discovery, Novel vaccine/drug targets
Introduction
VL a fatal disease is caused by the hemoflagellate
protozoan parasite, (L. donovani, L. infantum
and L. chagasi) through the invasion of the
reticuloendothelial system (spleen, liver and bone
marrow). The Leishmania life cycle consists of two
morphologically distinct stages: intracellular
doi:10.5138/ijaps.2010.0976.1055.01001
©arjournals.org, All rights reserved.
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
2
amastigotes that reside in the phagolysosomes of
mammalian macrophages, and extracellular
promastigotes that reside within the gut of the sand fly
vector. VL is characterized by intermittent fever,
massive hepatosplenomegaly, anemia,
thrombocytopenia, and polyclonal B-cell activation
with hypergammaglobulinemia [1]. The skin becomes
dark gray, corresponding to the name of the disease
Kala-azar or black sickness. In most acute infections
death may occur within a few weeks; in sub acute
cases within a year and in chronic cases within 2 to 3
years [2]. VL is often fatal if not treated properly.
Patients, who recovered from VL, usually have life-
long immunity to re-infection but occasionally relapses
may occur [2, 3]. The incomplete treatment of the
patient may also lead to a condition usually referred to
as post kala-azar dermal leishmaniasis (PKDL) [3, 4].
Although VL is widely distributed throughout the
tropics, it is rampant in the Indian subcontinent and
southwest Asia [5]. Annually, about 500,000 cases of
VL occur worldwide, of which 90% occurs in India,
Sudan, Nepal, Bangladesh and Brazil [6]. In India, VL
is endemic in north-east part especially in Bihar, West
Bengal, Assam and certain pockets of eastern Uttar
Pradesh [7]. Annually, about 100,000 cases of VL are
estimated to occur in India. Of these, the state of Bihar
accounts for more than 90 percent of the cases [8].
State of Bihar and adjoining areas of West Bengal,
Jharkhand and Uttar Pradesh account for about half the
world's burden of VL [7, 8]. Affordable and effective
chemotherapy is still unavailable. Further due to the
lack of fully effective treatment the search for novel
targets is urgently needed. Unfortunately, the
development of vaccines has been hampered due to the
fact that the parasites have a digenetic life cycle in at
least two hosts (sand fly vector and human/animal
reservoir), resulting in significant antigenic diversity.
In the Leishmania life cycle, promastigote forms
develop in the alimentary canal of the sand fly which
differentiate into an infectious, non-dividing form
called metacyclic promastigotes [9]. Macrophages
phagocytized these metacyclics after injection into the
mammalian host. The parasites must differentiate into
the non-motile amastigote form to persist in a
phagolysosome in the macrophage [10]. These
changes have been correlated with changes in gene
expression in several species [11, 12]. It has been
successfully applied to achieve this goal in
Plasmodium and Taxoplasma [13-15]. This review
describes an update on high-throughput microarray
technology, focusing on the gene expression profiling,
advances and novel applications in therapeutic drug
target (DT) discovery for Leishmaniasis. To exemplify
how this tool can be useful, we discussed a broad
overview of some of the past and potential future
aspects of this technology in rapidly growing field of
VL research in the present article. Review provides an
up-to-date look at identified potential vaccine and drug
targets and the application of DNA microarray
technology for VL.
Microarrays: reshaping the molecular parasitology
Microarray is a powerful method to study global gene
expression in terms of quantitation of mRNA levels for
discovery of new drug targets and can be used for
large number of application where high-throughput is
needed [16]. It has been applied in a wide number of
Fig 1: Workflow of Microarray for identification and
development of potential drug target and vaccine candidate for
VL.
organisms to study gene expression profiling under
several physiological and experimental conditions. The
main aim of this review is to focus on the application
of DNA microarray in context with parasitic disease
especially Leishmania for target identification. After
the successful completion of the Leishmania genome
sequencing and the achievement of similar goals in
other parasites have generated a huge amount of free
available information about the genomic sequence of
different parasitic organism. This has opened the door
to a post-genome era where new challenges arise. A
brief and step-by-step protocol of microarray is
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
3
provided for sample preparation, sample labeling and
purification, hybridization and washing, feature
extraction, and data analysis which may provide
unique opportunities for the field of drug/vaccine
target discovery in Leishmaniasis (Figure 1). Targeted
drug or vaccine development is lengthy and expensive,
and it is a major challenge as it precedes through
several decision gates [17] from the identification of a
potential candidate in in vitro and/or animal
experiments to clinical trials for development of more
effective antileishmanial treatments and vaccines
which would also be affordable in the developing
world. Among the different high-throughput
technologies, it is now the first array approach closes
to enter routine diagnostics and used in drug discovery
and clinical applications [18] giving a new direction
for novel drug/vaccine discovery in the field of
parasite biology.
In summary, DNA microarrays are changing the way
of biomedicine and other disciplines that are
addressing the different biological questions and allow
the comparative studies of parasite strains as well as
patients for the study of high level of post-
transcriptional regulation and translation of genome
research to the clinic. Gene-array implementations
combined with the information arising from emerging
genome sequencing projects are expected to be in the
near future a major tool to characterize genes involved
in different processes of parasite biology. So far, gene
expression profile studies in parasites have been
performed in microarrays that use a glass support to
immobilize fragments of genomic DNA followed by
fluorescent detection [19] The gene expression
profiling data led to the identification of sets of tightly
co-regulated genes across all experimental conditions
tested. A comparison of the responses induced by the
individual pathogens by means of microarray study
revealed major differences in the functionally related
gene profiles associated with each infectious agent
[20]. Although the intracellular pathogens induced
responses clearly distinct from the extracellular, they
each displayed a unique pattern of gene expression that
would be predicted clearly on the basis of microarray
study. DNA microarray analysis has been successfully
applied to most of the protozoan parasites that cause
human disease, but progress are primarily at the stage
of validation and new gene discovery [21]. The
outcomes of DNA microarray analysis of protozoan
parasite gene expression indicate towards the
mechanisms of regulation. Due to broad application of
microarray in genomics, it turned into a very popular
tool in the last few years and reshaping the molecular
biology aspect of parasite especially for the study of
transcriptome.
Gene expression profiling and microarray
Microarrays permit the analysis of gene expression,
DNA sequence variation, protein levels, and other
biological and chemical molecules in a massively
parallel format [22]. In this concern, microarrays have
been well utilized in genomics/proteomics approaches
for gene/protein expression profiling and tissue/cell-
scale target validation [23]. The use of basal or
constitutive, comparative or differential gene
expression profiling to understand and predict drug
sensitivity or resistance can described through
microarray. It can be used to identify molecular
biomarkers, pharmacodynamic endpoints and
prognostic markers for predicting outcome and patient
selection [16]. Many different biological questions are
routinely being studied using transcriptional profiling
on microarrays.
Since DNA microarrays allow examination of gene
expression on a genome-wide scale, these studies
revealed substantial new information about the
dynamics of transcript abundance during the
differentiation process [24]. The recent blossoming of
this technology in genome science will improve gene
prediction capabilities and enable the identification of
genes responsible for infectivity. Powerful new
methods, such as expression profiles using
microarrays, have been used to monitor changes in
gene expression levels as a result of a variety of
metabolic, xenobiotic, or pathogenic challenges [25].
This potentially vast quantity of data enables, in
principle, the dissection of the complex genetic
networks that control the patterns and rhythms of stage
specific gene expression. Differentially expressed
genes are associated with pathways involving
cytoskeletal organization, cell adhesion and migration,
coagulation, inflammation, differentiation etc [26]. For
example Bullinger et al, [27] used cDNA microarrays
to determine gene expression in blood and bone
marrow samples from Leukemia patients. A second
study performed by Valk et al, [28] determined gene
expression profiles within blood or bone marrow of
Leukemia patients. It has been successfully and widely
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
applied to most of the protozoan parasites that cause
human disease [21], but has not made equal progress
in case of Leishmania. As a high throughput tool to
discover new genes and to identify coordinate
expression of families of genes and it has already
proven effective in the field of Plasmodium and
Toxoplasma parasites research. The application of
microarray analysis in Toxoplasma gondii gene
expression study has also moved beyond validation
and gene discovery [21]. Using DNA microarray
approach Debnath et al, [29] assessed global variations
in gene expression during collagen interaction with
Entamoeba histolytica to identify cellular mechanisms
of trophozoite activation. MacFarlane et al, [30]
proposed the applications of microarray technology in
expression profiling and strain genotyping. They
advocated the potential future applications of
microarray technology for the study of Entamoeba
biology.
However, results of microarray analysis to till date
have not advanced substantially beyond the stage of
validation and new gene discovery for the
kinetoplastid parasites like Trypanosoma and
Leishmania. In contrast to the above previous gene
expression studies emphasized the role of one or two
genes at a time, but microarrays provide a
revolutionary platform to analyze thousands of genes
at once, instead of studying one or few genes at a time.
Development of such novel techniques and
methodologies allowing us to screen and pin-point a
large number of stage specific gene at a time that is
helpful for identification of promising targets.
Applications and progress of Microarray
technology in Leishmania research
Global gene expression profiling is valuable for
elucidation of biochemical pathways and the
identification of potential targets for novel molecular
therapeutics for Leishmaniasis. As we know that there
are two morphologically distinct stages (Promastigotes
and amastigotes) of Leishmania parasite occur
resulting in significant antigenic diversity. To know
about the antigenic diversity, stage specific gene
expression study, microarray will be helpful for
defining effective targets. Expression (transcript)
profiling allows generating quantitative gene
expression information for many genes at specific
stage of parasite. The morphological and physiological
changes of the Leishmania parasite which occur
following its transfer from the sand fly to a
mammalian host suggest a rapid modulation of the
expression of numerous genes. To identify molecular
events associated only with the amastigote stage of
Leishmania parasites, expression profiling has been
used. The approach was to compare the gene
expression of both forms of the parasite and
characterize transcripts developmentally expressed in
amastigotes. In the last few years some groups have
studied gene expression patterns in Leishmania
through microarray. It revealed the gene expression
between wild type and mutant samples of Leishmania
and this transcript profiling were useful in the study of
resistant parasites to pinpoint several genes linked to
drug resistance [31]. Due to its vast application now a
day it is extensively used in parasite biology to study
stage specific gene expression, drug resistance and is
the foundation of successful drug/vaccine development
[32, 33]. Microarray analysis could compare these
strains to susceptible strains and pinpoint which genes
are activated or suppressed to achieve the drug-
resistant phenotype. Expression profiling through
microarray can be used to compare various forms of
Leishmania like procyclic, metacyclic, promastigote
and amastigote and these studies will be definitely
helpful to examine the changes in gene expression
during the process of differentiation of parasite life
stage. However, such analysis was used to examine the
temporal changes in gene expression as procyclic
promastigotes differentiate into metacyclic in L. major
[24] and in procyclic in L. maxicana [34]. Leifso et al,
[55] reports the use of DNA oligonucleotide genome
microarrays representing 8160 genes to analyze the
mRNA expression profiles of L. major promastigotes
and lesion derived amastigotes. Over 94% of the genes
were expressed in both life stages. Advanced statistical
analysis identified a surprisingly low degree of
differential mRNA expression: 1.4% of the total genes
in amastigotes and 1.5% in promastigotes [55].
Rochette et al,[35] had done microarray analysis of L.
major and L. infantum developmental stages to reveal
the substantial differences in gene expression pattern
between the two species but no more analysis of
transcriptome have been reported in case of VL.
A few microarray based expression profiling have
been done in case of VL. Using promastigote and
amastigote stage of L. donovani [24, 36, 37] and L.
4
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
infantum [38, 39] developmentally regulated genes
was identified as a target in VL. Ettinger et al, [40]
reported the expression of type 1 immune cytokine
activation during Macrophage and T-Cell Gene
Expression study in L. chagasi. Using DNA
microarrays, Rodriguez et al, [41] studied gene
expression in RNAs from BALB/c bone marrow
macrophages with and without L. chagasi infection.
To explore the sodium antimony gluconate (SAG)
resistance mechanism and its effect on macrophage
gene expression, microarray analysis was performed
by El Fadili et al, [42] for compare gene expression
profiling in noninfected and L. donovani infected
THP-1 monocytic cells treated or not treated with
SAG. Nylon macroarrays were employed by Quijada
et al, [19] to search the genes showing increased
mRNA abundance during an axenic differentiation of
L. infantum promastigotes to amastigotes. Gregory et
al, [43] addressed the mechanisms by which different
Leishmania species cause different pathologies by
comparing the gene expression profiles through cDNA
microarray of bone marrow-derived macrophages
infected with either L. donovani or L. major
promastigotes. Buates et al, [44] examined the effect
of infection on the expression of genes in L. donovani
to explore the effect of intracellular infection on
macrophage gene expression and compare gene
expression profiles in non-infected and L. donovani
infected macrophages through cDNA expression array
analysis.
In Leishmania most of the studies have been done
using cDNA microarray approach. Rochette et al, [45]
used a DNA oligonucleotide full-genome array
approach to compare global RNA expression profiling
of L. infantum axenic amastigotes to intracellular
amastigotes derived from infected macrophages. They
found that 40% genes were found upregulated in
axenic amastigotes compared to intracellular
amastigotes. Comparisons in expression profiling
between axenic amastigotes and intracellular
amastigotes revealed substantial differences in
regulated mRNA abundance. The major differences
between axenic and intracellular amastigotes were
observed in metabolic process. These findings were
based on oligonucleotide array (a modern array
approach), which highlighted the importance of the
host macrophage in driving the parasite to specific
adaptations, which consequently result in highly
regulated changes in gene expression. Ubeda et al, [46]
reported the gene amplification, gene deletion and
chromosome aneuploidy i.e. modulation of gene
expression in drug resistant Leishmania using
oligonucleotide microarrays. This microarrays based
study highlighted several mechanisms by which the
copy number of genes involved in resistance was
altered; these include gene deletion, formation of
extra-chromosomal circular or linear amplicons, and
the presence of supernumerary chromosomes.
The genes responsible for infectivity (sand fly- human
infection) and host-seeking behaviors in sand flies can
be major features of interest. Sand flies have evolved
numerous fail-safe activities to combat vertebrate
blood clotting, vasoconstriction and platelet
aggregation. Salivary glands of phlebotomine sand
flies contain a complex array of biologically active
molecules that are both conserved and divergent
among sand fly species; many of these molecules have
immunosuppressive effects. For example, maxadilan,
found only in Lutzomyia species, is the most potent
vasodilatory polypeptide [47] and exhibits a range of
immunomodulatory activities [48]. The presence of an
anti-platelet aggregation enzyme, apyrase, has been
identified in the salivary glands of both Lutzomyia and
Phlebotomus vectors [49]. Microarray has opened a
very active and productive field of research targeted at
the molecular mechanisms responsible for host seeking
and identification of major genes might be involved in
infectivity. In addition, with elucidation of the
molecular events involved in host infection, by sand
fly could prove to be effective targets. Microarray
analysis of human-sand fly infection phase will
provide the knowledge base of sand fly biology that is
so important in initiating the vertebrate infection.
Comparative analysis between the human and sand fly
genome will likely uncover novel human orthologs
that has proven essential for annotation and the
identification of functional genes as targets.
Several investigators have identified the stage-specific
products of Leishmania using conventional approach
of molecular biology. A2 gene was identified as
amastigote specific product in L. donovani, essential
for adaptation in hostile environment of macrophage
and contributes to defining the molecular basis for the
infectivity and the pathogenicity of the parasite [50].
They confirmed that the A2 genes were also expressed
in amastigote-infected macrophages and in
5
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
amastigotes extracted from the spleens of infected
animals and are an important gene trigger for
promastigote- to-amastigote cytodifferentiation.
Boucher et al, [51] identified a developmentally
regulated gene family in Leishmania encoding the
amastin surface proteins and showed that stage-
specific accumulation of the amastin mRNA. Some
studies have characterized heat inducible products like
heat shock proteins (HSPs) in different strains of the
Leishmania parasite [52, 53]. Joshi et al, [54] proposed
genes of ribosomal protein, stress inducing protein that
have been identified by the differential screening of an
L. donovani axenic amastigote cDNA library were
shown to be expressed twofold higher in amastigotes
than in promastigotes. Due to high acridities, DNA
microarray represents an excellent tool to study
regulation of amastigote stage-specific gene
expression in Leishmania cells. Thus, microarray
technology may prove to be an important tool in the
future especially in case of VL although it is currently
not in use in the clinical setting of VL. It can also
reveal the mechanism of drug resistance by identifying
up/down regulated gene and characterizing the
respective gene expression in drug resistant and
sensitive clinical isolates. Although microarrays are
currently not being used in the diagnosis of VL, but it
can provide strong insight into the biology of the
disease. Expression signatures may have predictive
power in classifying VL from patient samples and also
predicting patient response to therapy. So there is
extensive need to explore this technology to pin point
the effective target by discovering new genes in
Leishmania especially in VL diagnosis which will be
the new wave of molecular biology that hold great
promise and ray of hope for progress in potential target
identification.
As we know that DNA microarray is a
multidimensional technology. Therefore, it can be used
for large scale genotyping, gene expression profiling,
comparative genomic hybridization, sequencing and so
many other applications (Figure 2). Although the most
common use of DNA microarrays is gene expression
profiling, scientists have successfully used them for
multiple applications, including genotyping,
sequencing, DNA copy number analysis, and DNA-
protein interactions, among others. Besides being used
as an essential step in analyzing high-throughput
experiments such as those involving microarrays,
bioinformatics can also contribute to the processes of
target identification and validation by providing
functional information about target candidates and
positioning information to biological networks [23]. It
has been put to a number of uses that demonstrates
another application of gene expression analysis by
which pathways, downstream target-genes and genes
up-regulated in response to a specific treatment can be
identified [55], which should be applied to elucidate
various aspects of disease and diagnosis. Since
microarray is a reliable technique for addressing target
identification and validation so it can be applied to the
diagnosis and prognosis of VL; development and
understanding of VL therapies. This review has
highlighted some of the relevant developments of
rapidly emerging microarray technology in
Leishmania with a particular reference to visceral form
and attempt to outline the areas of future research of
for the development of new therapeutic agents (Figure
1).
Fig 2: Possible applications of microarray technology.
Novel Target identification through DNA
Microarray in VL
Novel target identification for the control of VL is a
call for necessary action in concern to discover
potential drug/vaccine. Current advances in molecular
biology techniques and the genome sequencing
projects hold great promise for new target discoveries
that can contribute to diagnosis, treatment and
prevention of parasitic diseases. A deeper
understanding of basic biology and the discovery of
new genes that can contribute toward development of
6
Table 1: Genes identified through microarray study in visceral Leishmaniasis
novel vaccines candidate and new drug targets for VL
is a fundamental goal for the person conducting
research in the field of VL. To accelerate the search
for novel potential stage associated leishmanicidal
targets, microarray-a valuable tools, were being used
to extend the understanding of array of events of
Leishmania infection. In table 1 genes identified
through microarray analysis in case of VL have been
summarized and categorized on the basis of their
ontology. Genes are classified as i) Chaperons/ stress
ii) Cytoskeletal iii) Metabolic iv) Signaling cascade
and v) Surface gene family. The role of some of the
genes identified in microarray study viz., HSP 83,
MAP Kinase, Activated protein kinase c receptor
(LACK), Enolase, gp63, Promastigote surface antigen
(PSA) 2, Calreticulin, Proteophosphoglycan, Kinesin,
Eukaryotic translation initiation factor, Alfa tubulin,
Beta tubulin, Histone H2A, Carboxypeptidase were
reported as strong vaccine candidate (VC). These are
the genes that promote cell growth metabolism, stress,
signal transduction, and transport (Table 1). Glucose
transporter, GTP-binding protein, Leishmanolysin
precursor, Protein Kinase, p-Glycoprotein-like
protein, Proliferative cell nuclear antigen (PCNA),
Glutamate dehydrogenase, Proteasome alpha 1
subunit, ABC1 transporter, Guanine nucleotide
binding protein, Glucose-6-phosphate dehydrogenase,
Adenosylhomocysteinase, Translation elongation
factor 1-beta genes were identified through microarray
study in VL case which was reported as a promising
DT (Table 1). Among metabolic gene family Fructose-
1,6-bisphosphate aldolase, Enolase, Protein disulfide
isomerase (PDI) were proposed as intoxicating DT &
VC.
Although there is not much more microarray study has
been done in case of VL. As we know that microarray
investigations is helpful to examine the transcriptional
differences between Leishmania promastigotes and
amastigotes or from drug-resistant and sensitive
parasites which may improve our knowledge of the
molecular basis of the promastigote-to-amastigote
transformation which is still limited. In VL studies,
Sl.
No. Identified genes Accession no. of gene
( Gen Bank / Gene DB/
EMBL)
Gene ontology/
category Target/
remarks References
1 ABC1 transporter AA741759 Surface gene DT [65]
2 Adenosylhomocysteinase LmjF29.2800 Metabolic gene DT [67]
3 Glucose-6-phosphate dehydrogenase LmjF34.0080 Metabolic gene DT [66]
4 Glucose transporter LmjF36.6300 Metabolic gene DT [60]
5 Glutamate dehydrogenase LmjF15.1010 Metabolic gene DT [64]
6 Guanine nucleotide binding protein AI034761 Surface gene DT [66]
7 Leishmanolysin precursor LmjF10.0460 Metabolic gene DT [61]
8 p-Glycoprotein-like protein AI034703 Surface gene DT [62]
9 Proliferative cell nuclear antigen (PCNA) LmjF13.1480 Signaling cascade DT [63]
10 Protein Kinase LmjF31.1530 Signaling cascade DT [59]
11 Translation elongation factor 1-beta LmjF36.0250 Metabolic gene DT [68]
12 Activated protein kinase c receptor (LACK) LmjF26.1240 Surface genes VC [70, 86]
13 Alfa tubulin LmjF13.0350 Cytoskeletal gene VC [75, 76]
14 Beta tubulin LmjF08.1230 Cytoskeletal gene VC [75, 76]
15 Calreticulin LmjF31.2600 Stress gene VC [67]
16 Carboxypeptidase LmjF30.3520 Metabolic gene VC [81]
17 Enolase LmjF14.1160 Metabolic gene VC [67]
18 Eukaryotic translation initiation factor LmjF36.3880 Metabolic gene VC [74]
19 gp63 M60048.1 Surface gene VC [69]
20 Histone H2A LmjF17.1550 Metabolic gene VC [77, 78]
21 HSP 83 LmjF33.0314 Chaperone/ Stress VC [67]
22 Kinesin LmjF09.0120 Metabolic gene VC [67]
23 MAP Kinase LmjF36.6470 Signaling cascade VC [87]
24 PSA 2 LmjF12.0910 Surface gene VC [71]
25 Proteasome alpha 1 subunit LmjF36.1600 Metabolic gene VC [79]
26 Proteophosphoglycan LmjF35.0520 Surface gene VC [73]
27 Fructose-1,6-bisphosphate aldolase LmjF36.1260 Metabolic gene DT, VC [67]
28 Protein disulfide isomerase (PDI) LmjF36.6940 Metabolic gene DT, VC [67]
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
microarray technology has been used successfully to
identify critical genes expressed during the
development and differentiation of L. donovani [4, 24,
36, 56] and L. infantum [24, 32, 34, 38, 57, 58]. These
studies are helpful to search for genes that may
contribute as strong DT and VC. Transcript profiling
of Leishmania with microarrays pinpointed a number
of genes overexpressed or down regulated. The role of
some of these genes in drug/vaccine targeting was
confirmed. Targets which are described in table 1 were
also proposed as DT or VC or both by so many authors
in different organism.
Till date, cost effective and safe drug treatments are
not available for VL, and the emergence of resistance
are the current problems. Presently, the control of
Leishmania infection relies on some drugs including
Pentavalent Antimonials (SbV), Amphotericin B,
Miltefosine and Pentamidine. But they are toxic, have
serious side effects and are associated with numerous
relapses. On the other hand, in all cases, the evolution
of drug resistance on the part of a parasite determined
to survive is unsurprising and new drug discovery
must stay a step ahead of increasing ineffectiveness of
those in current use. Consequently, discovery of new
genes essential for parasite survival that can be
targeted as new drugs for treatment of these diseases is
crucial. Microarray based screening have been proved
to useful in identifying new and potent drug targets
which could be an efficient ways to make current
treatments of VL more effective. Both cDNA and
genomic microarrays for L. major have identified a
number of new genes that are expressed in a stage
specific fashion and preliminary results from a L.
donovani, and L. infantum genomic microarray
analysis demonstrated some new gene discovery which
may be proved as a novel targets for VL after its
validation and clinical trial (Table 1). Among the
several genes identified through microarray
technology in VL, Cyclin-dependent protein kinase
was proposed as promising drug targets for
antimalarial drug development by Waters et al, [59].
Glucose transporter is known to be involved in the
pathway for transport of glucose to the Plasmodium
falciparum parasite, has attracted increasing interest as
a target for antimalarial chemotherapy [60]. Santos et
al, [61] and TDR targets database
(http://qa.tdrtargets.org/targets/) advocated
Leishmanolysin precursor gene as a DT. According to
Perez-Victoria et al, [62], P-glycoprotein (a Multidrug
resistance transporter gene) was focused as DT in
Leishmania. Proliferative cell nuclear antigen (PCNA)
is an auxiliary protein of DNA polymerase delta and is
involved in the control of eukaryotic DNA replication
by increasing the polymerase's processibility during
elongation of the leading strand. PCNA are involved in
mitogenic signal transduction and cellular proliferation
pathway so its possible role in drug resistance and may
be considered as DT [63]. In P. falciparum glutamate
dehydrogenase, was proposes as DT for novel
antimalarial drugs [64]. ABC transporters are
important mediators of resistance and a detailed
analysis of L. major and L. infantum genomes has
revealed 40 ABC proteins part of 8 different families.
Burnie et al, [65] identified ABC transporters in
vancomycin resistant Enterococcus faecium as
potential drug targets. Glucose-6-phosphate
dehydrogenase and guanine nucleotide-binding protein
are emerging concepts regarding DT by Asensio et al,
[66]. Gupta et al, [67] have strongly advocated
adenosylhomocysteinase as an important DT.
Elongation factor 1-beta was described as DT by
Walker et al, [68] in L. guyansis.
So far, no vaccines exist or at on the verge of
development for VL there is an urgent need to develop
an effective vaccine against VL. Among the identified
genes using microarray approach Enolase was
proposed as a VC by Gupta et al, [67]. According to
Wei et al, [87] MAP kinase was proposed as VC.
Vaccination study was done with gp63, a surface gene
by Jaffe et al, [69] in L. donovani. Gonzales at al, [70]
& Gurunathan et al, [86] advocated LACK as strong
VC in L. infantum and L. major respectively.
Promastigote surface antigen (PSA) a leucine rich
repeats are the main epitopes in L. infantum [71].
Protective vaccination with PSA 2 from L. major is
mediated by a Th1-type of immune response and acts
as a potential VC [72]. Calreticulin, a Th1-stimulatory
stress protein, was identified as VC (67) and involved
in glycoprotein folding. Samant et al, [73] anticipated
proteophosphoglycans (PPGs) as effective VC.
Kinesin have been advocated as strong VC by Gupta et
al, [67]. Eukaryotic translation initiation factor was
proposed as exacebatory antigen in vaccination study
by Stober et al, [74] and also discussed as VC
(http://www.genedb.org). Studies indicated that Alpha-
and beta-tubulin [75] belonging to cytoskeletal
8
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
organization category were reported to be highly
immunogenic [76] so they can also be an effective
VCs. Histone H2A has been identified as
immunogenic protein and reported as VC in the study
done by Molano et al, [77]. Histones are mainly
involved in nucleosomal assembly and have been
reported as vaccine candidate in visceral and
cutaneous Leishmaniasis [78]. Proteasome, a
multicatalytic proteinase complex is characterized by
its ability to cleave peptides with Arg, Phe, Tyr, Leu,
and Glu adjacent to the leaving group at neutral or
slightly basic pH. The proteasome has an ATP-
dependent proteolytic activity that may be a strong VC
[79]. Carboxypeptidase was reported as VC in L.
major [80]. Carboxypeptidases of Anopheles gambiae
had been found to be a blocking vaccine targets in
Plasmodium falciparum [81]. Gupta et al, [67]
proposed Fructose-1,6-bisphosphate aldolase and
Protein disulfide isomerase (PDI) as a potential DT &
VC both and enolase as a VC. Together, these results
provide a framework and emphasize for more
microarray based studies of Leishmania species to
demonstrate that this high throughput tools are
valuable for identification of potential drug/vaccine
targets and also enhance our understanding in drug
resistance mechanisms and factors involved.
Concluding Remarks
Microarrays, a recent development in genomics,
provide a revolutionary platform to analyze thousands
of genes at once, instead of studying one gene at a
time. They have enormous potential in the study of
biological processes involved in health and disease
concern (82) and, obviously, a crucial tool in
diagnostic applications and drug discovery for VL.
Microarray based studies have provided the essential
impetus for biomedical experiments, such as
identification of disease-causing genes and can
identify genes for new and unique potential drug
targets, predict drug responsiveness for individual
patients and, finally, initiate gene therapy and
prevention strategies [83].
It is clear from this review article, that microarray
based gene expression profiling may begin a major
impact on the development of VL therapeutics. Within
recent years, this technology has become a major tool
for the investigation of transcriptional variations in
gene expression. The most obvious benefits have to
enhance our understanding and increased ability to
predict the outcome of VL treatment for individual
patients. Related to this, gene expression profiling of
both the stage of parasite (Promastigote and
amastigote) has now become a standard approach to
the identification and validation of new molecular
targets for therapeutic intervention. As new drugs are
developed, gene expression profiling is increasingly
used to investigate mechanism of action and to
determine on-target versus off-target effects. The
comparison of gene expression changes between
promastigote and amastigote is exceptionally valuable
for therapeutic purpose for VL. Transcriptional
profiling is being used to improve lead optimisation
and to characterise clinical development of drug
targets and vaccine candidates against VL.
Furthermore, there are already several examples of the
use of microarrays to determine global genome
expression changes that are induced in cancer tissues
by drug treatment in cancer patients [16, 84].
Majority of identified genes through microarray are
putative which indicated that they have as yet
unknown functions. It will be interesting to study these
genes (as they might encode proteins with functions
specific to phenotype of interest) which would be the
novel targets for therapeutic intervention and need
their further investigation. Some identified genes
through microarray are under evaluation as vaccine
candidates and some inhibitors of potential drug
targets in different studies of VL. Additional molecular
studies such as cloning and expression of the best
antigenic targets, as determined by their immune-
protective potential, together with their specific
association and definite allocation are needed to
characterize these new proteins. These findings also
offer a possibility of establishing a protein array for
interspecies profiling of Leishmania proteins
Information withdrawn from microarray based study in
Leishmania should be explored from experimental up
to clinical level to develop effective drug/vaccine for
mankind. Microarray is also applicable in the study of
differential expression of Leishmania genes as
biomarkers as a futuristic outlook towards early
diagnosis.
Although microarray has emerged as powerful tools in
the drug/vaccine discovery process, the next challenge
for the future application of these technologies to
Leishmania research is the investigation of genes and
9
Kumar et al. International Journal of Advances in Pharmaceutical Sciences 1(2010) 1-14
proteins in vivo. Further, immune responses to the
vector sand fly saliva have been shown to protect
animals against Leishmania infection [85]. Yet very
little is known about the molecular characteristics of
salivary proteins from different sand flies, particularly
from vectors transmitting VL. Gene expression study
using microarrays and related global technologies are
rapidly taking their place alongside other technologies
as important tools in the discovery, development and
use of new therapeutics. The development of safe and
effective drugs remains challenging. It presents a
methodology that can identify genes or pathways for
new and unique potential drug targets, determine
premorbid diagnosis, predict drug responsiveness for
individual patients, and, eventually, initiate gene
therapy and prevention strategies and proved as
panacea in drug/vaccine discovery and development
for VL.
Acknowledgements
This review is supported by National Institute of
Pharmaceutical Education & Research, Hajipur, India
and Rajendra Memorial Research Institute of Medical
Sciences, Patna, India. Financial support by Ministry
of Chemicals & Fertilizers, Government of India and
Indian Council of Medical Research, New Delhi, India
is gratefully acknowledged.
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