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Comparative in-silico genome analysis of Leishmania (Leishmania) donovani: A step towards its species specificity

Comparative genome analysis of recently sequenced Leishmania (L.) donovani was unexplored so far. The present study deals with the complete scanning of L. (L.) donovani genome revealing its interspecies variations. 60 distinctly present genes in L. (L.) donovani were identified when the whole genome was compared with Leishmania (L.) infantum. Similarly 72, 159, and 265 species specific genes were identified in L. (L.) donovani when compared to Leishmania (L.) major, Leishmania (L.) mexicana and Leishmania (Viannia) braziliensis respectively. The cross comparison of L. (L.) donovani in parallel with the other sequenced species of leishmanial led to the identification of 55 genes which are highly specific and expressed exclusively in L. (L.) donovani. We found mainly the discrepancies of surface proteins such as amastins, proteases, and peptidases. Also 415 repeat containing proteins in L. (L.) donovani and their differential distribution in other leishmanial species were identified which might have a potential role during pathogenesis. The genes identified can be evaluated as drug targets for anti-leishmanial treatment, exploring the scope for extensive future investigations.


Introduction
Leishmaniasis is a vector-borne parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. Leishmaniasis, an endemic disease of tropical and subtropical regions is the second-largest parasitic killer in the world (after malaria), responsible for an estimated 2 million cases each year and 350 million people at risk worldwide clearly imposing a major health problem globally except Australia and Antarctica Meta Gene 2 (2014) 782-798 (Desjeux, 2001). Approximately 88 countries are named to be infected with this sand fly borne disease. Although leishmaniasis is represented by at least 20 leishmanial species, the disease spectrum is generally categorized into 1) cutaneous leishmaniasis, 2) mucocutaneous leishmaniasis and 3) visceral leishmaniasis, depending upon the tropism of the disease and the species causing the infection. Visceral leishmaniasis which is the most severe and deadly form is caused by the old world species Leishmania (L.) donovani and Leishmania (L.) infantum (Africa, Asia, Europe) and the new world species L. (L.) infantum (South America). India, Bangladesh, Nepal, Sudan, and Brazil have been reported to have more than 90% of the world's cases of visceral leishmaniasis with an estimated incidence of 500,000 new cases and 60,000 deaths each year (News, 2006). The severity of the disease is further augmented by drug resistance and drug failure, particularly in L. (L.) donovani strains of India and Nepal, which has been recently documented (Downing et al., 2011). So the raising future risks by this neglected tropical disease among the neglected populations of the world made WHO to include leishmaniasis among the six major diseases targeted for intense research and control.
Genome comparison of Leishmania (L.) major, L. (L.) infantum, Leishmania (Viannia) braziliensis showed great conservation of synteny and identifies only a small number of genes (approx 200) which are differentially distributed (Toledo et al., 2010;Peacock et al., 2007). These species specific genes may be a key factor for difference in pathogenesis between the species. Visceral leishmaniasis poses a fatality rate of greater than 100% within two years if untreated (Peacock et al., 2007). Since the genome information of L. (L.) donovani is very recent, so far, research on visceral leishmaniasis was mostly dependent on L. (L.) infantum genome information (Chappuis et al., 2007). Also most of the drugs commonly used to treat visceral leishmaniasis are toxic and exert unacceptable side effects (Khan et al., 2010). Though many control programs have been organized to control visceral leishmaniasis in Indian sub-continent, the success rate is severely compromised by the developing resistant strains of L. (L.) donovani at least in parts of India, particularly North Bihar and West Bengal. The diverse intra-strain genetic variability and drug resistance developed by the most severe L. (L.) donovani in Indian subcontinent and Nepal imposed the recent concern in the field of visceral leishmaniasis treatments (Pourshafie et al., 2004). So the treatment and control of leishmaniasis caused by L. (L.) donovani indeed require novel drug alternatives.
Although the outcome of infections and presentation of disease depend on many factors such as host immune response, host genetic variability, vector, and protozoan species (Guerin et al., 2002), it is obviously the specific genes of the species which determines the spectrum and severity of the disease. In view of the obvious clinical importance of this human pathogen, a genomic approach is highly desirable and may give insight into the complex mechanisms of pathogenesis. Here we report the comparative genome analysis of L. (L.) donovani with the other sequenced leishmanial species. This study therefore frameworks the experimental verification of few significant genes, consistent with independent existence, to set an avenue of genomic aspect of drug targeting to overcome the current problems in an effective way.

Genome information of L. (L.) donovani
Despite many years of evolution, the genome content of L. (L.) donovani was greatly conserved with the other four leishmanial species except few specific genes. The lack of vast diversity among the leishmanial species over estimated 20-100 Ma of evolution may be due to lack of some machinery that causes diversity in eukaryotes and probably lack of transposable elements in the leishmanial species might be the cause (Khan et al., 2010). Though the presence of retrotransposons and RNAi machinery in Trypanosoma brucei and L. braziliensis was clearly reported, the evolutionary loss of these elements in leishmanial species preserves their genome content (Bringaud et al., 2006;Villanueva et al., 1991). Similarly the L. (L.) donovani genome lacks the retrotransposons and RNAi machinery. L. (L.) donovani contains 36 chromosomes and has a haploid genome size of 32.4 Mb. L. (L.) donovani genome encodes 8032 proteins, out of which 42.84% proteins were found to be functional homologs of other leishmanial species, 56.34% proteins lack functional assignment and the remaining~0.8% proteins were exclusive to L. (L.) donovani. Approximately 5.6% genes were identified to code for repeat containing proteins which were conserved and probably might play a huge role in pathogenesis which was discussed elsewhere. The summary of L. (L.) donovani genome information was given in Supplementary Table S1. Though L. (L.) donovani disease tropism differs greatly from other leishmanial species, till now no specific genes were reported for difference in disease presentation, except for the A2 gene family which was reported to be involved in the survival of the parasite in visceral organs (Zhang et al., 2003). Keeping this gap area in mind, we compared the proteome of L. (L.) donovani with the other four leishmanial species which identifies 55 gene coding proteins which were specific and expressed exclusively in L. (L.) donovani. The list of species specific genes of L. (L.) donovani which has been ascribed putative function was given in Table 1. A total list of L. (L.) donovani species specific genes were given in Supplementary Table S2. Out of the 55 L. (L.) donovani specific genes, only 36 genes were assigned putative function based on homology searches and Gene Ontology. The remaining genes encode for hypothetical proteins with conserved domain or unknown function which requires experimental documentation. Signal peptides were also detected for five specific L. (L.) donovani proteins which may have antigenic role in leishmanial pathogenesis. Among the 36 genes which encode proteins with putative function, few proteins were membrane related proteins, of which the important proteins being amastin like surface protein, lipophosphoglycan biosynthetic protein and phosphoglycan 1,3 galactosyltransferase which might have prime roles in pathogenesis, though the way or the mechanism it Table 1 Species specific L. (L.) donovani proteins. differs from other species was not clearly understood. Also 6 ribosomal genes specific to L. (L.) donovani were reported, though the exact involvement of the product of these genes in pathogenesis was unknown. In addition 2 specific genes (gene IDs: LdBPK_020010, LdBPK_260590) of L. (L.) donovani encode peptidases and heat shock proteins which were well known to have been involved directly or indirectly in pathogenesis. Also 3 other genes (gene IDs: LdBPK_252230, LdBPK_271710, LdBPK_040310) involved in sugar metabolism were identified which might possibly be involved in virulence and pathogenesis (Loughman and Caparon, 2006;Moyrand et al., 2007). Though the relationship between sugar metabolism and virulence remains greatly undefined, the novel proteins encoded by these genes might be involved in sugar metabolism of L. (L.) donovani and may influence the virulence in an unknown manner. Interestingly two novel and specific genes of L. (L.) donovani that encode 1) Apical Membrane Antigen 1 (AMA1) (gene ID: LdBPK_301490) and 2) cathepsin L like protease (gene ID: LdBPK_080950) were identified and its involvement in pathogenesis was discussed as follows.
Apical Membrane Antigen 1 (AMA1), highly suspected protein in parasite interaction and invasion Apical Membrane Antigen 1 was documented as a protein directly involved in invasion of apicomplexon parasites into the host (Tonkin et al., 2011). In plasmodium and toxoplasma, the AMA1 proteins were secreted from microneme of rhoptries and it was targeted to the apical membrane where it gets integrated with the parasite plasma membrane. Earlier studies with plasmodium clearly showed that the integrated AMA1 forms a complex with RON2 which in turn helps the parasite to attach with the host cell to promote invasion. The involvement of AMA1 in signaling and parasite replication was also documented in toxoplasma (Santos et al., 2011). In conjunction, three AMA1 genes were identified in L. (L.) donovani out of which two were conserved in all leishmanial species and the third one was specific to L. (L.) donovani. The homology search showed very less sequence similarity between AMA1 from plasmodium and leishmanial species. The absence of microneme and RON2 in Leishmania species clearly indicates the absence of this mechanism in leishmanial species. Surprisingly the Gene Ontology (GO) studies of specific AMA1 (gene ID: LdBPK_301490) reported in L. (L.) donovani shows cholesterol binding ability. The importance of host membrane cholesterol in L. (L.) donovani infection was already well documented . In L. (L.) donovani, cholesterol is required for binding and internalization of the parasite inside a host cell (Pucadyil et al., 2004;Tewary et al., 2006). Similar phenomenon was also documented in L. (L.) infantum (Rodriguez et al., 2006). Also the L. (L.) donovani specific AMA1 is leucine rich protein and the significance of leucine residues in interacting with host cell membrane was already documented in many organisms including leishmanial species (Kedzierski et al., 2004). Also signal peptide was detected which possibly confers that the protein was secretory. All these clues together made us to hypothesize that in L. (L.) donovani, AMA1 is secreted from an unknown organelle into the apical membrane of L. (L.) donovani where it interacts with the cholesterol of host membrane through leucine residues and helps in parasite internalization. The species specific expression of this protein in L. (L.) donovani indicates the probable role of this protein in visceralization, which needs future experimentations. The ongoing experimental studies in our laboratory regarding AMA1 will be a huge hope in the future for anti-leishmanial therapy.

Cathepsin L like protease
Cathepsin L like protease, a type of lysosomal endopeptidases, has been already identified as class of drug targets which participate in many essential biological processes of the parasite such as embryogenesis, molting, and immune evasion (Lustigman et al., 2004;Dalton et al., 2003). Cathepsin L like protease is expressed in both promastigote and amastigote of leishmanial species (Sakanari et al., 1997), and probably the novel gene (gene ID: LdBPK_080950) from L. (L.) donovani which encodes cathepsin L like protease may assist in evading the host immune system.

Differential gene distribution in leishmanial species
Further the comparative analysis establishes few genes specific between the L. braziliensis. The comparison was depicted in Fig. 1. The lists of few important genes and its protein product from L. (L.) donovani which share homology with some leishmanial species but not conserved with the other leishmanial species were given in Table 2. The full list of such genes including those which encode hypothetical proteins was given in Supplementary Table S3. The Gene Ontology (GO) for the L. (L.) donovani genes which were observed in few leishmanial species (~83 genes which encode functional proteins) showed that these genes mainly encode lipid containing membrane proteins, possibly involved in binding. Also some genes encode proteins which contain the enzymatic activity, like kinase proteins were expected to be involved in signal transduction or sugar metabolism. The Gene Ontology (GO) results were depicted in Fig. 2.
A specific gene (gene ID: LdBPK_290650) found to be conserved between L. (L.) donovani and L. (L.) infantum but was completely absent in other leishmanial species encodes a putative BET1-like protein. Though the exact function of this protein is not known, its close similarity with BET1 shows that the protein might participate in vesicular transport and may function as SNARE in docking of ER-derived vesicles with the cis-golgi membrane. ) infantum but has become a pseudogene or absent in other leishmanial species encodes a putative mitochondrial carrier protein (Agcp2438-like protein) which is a mitochondrial transmembrane protein involved in transport, probably playing a key role inside the oxidative environment of macrophages (Dolezal et al., 2012).
The important surface proteins of leishmanial species amastin, amastin-like surface protein, cysteine protease B (CPB), lipophosphoglycan LPG3 and the leishmanolysin GP63, were clearly reported for their potential role in parasite virulence (Rochette et al., 2005;Mottram et al., 2004;Vinet et al., 2009;Joshi et al., 2002). Though we found no obvious difference in the gene structure of lipophosphoglycan LPG3 (gene IDs: LdBPK_044280 and LdBPK_044281) in L. (L.) donovani in comparison with the other leishmanial species, we do found the discrete gene pattern of amastin, amastin like surface protein, leishmolysin and cysteine   protease in L. (L.) donovani, which was discussed below. The important surface protein of leishmanial species leishmolysin GP63, a metalloprotease ubiquitously distributed in trypanosomatids, plays a myriad of functions and was found conserved in all leishmanial species including Herpetomonas samuelpessoai, an insect trypanosomatid (Pereira et al., 2010).We found that a molecule of GP63(gene ID: LdBPK_100520), leishmanolysin metallo-peptidase, clan MA (M), and family M8 (gene ID: LdBPK_100520) specific in L. (L.) donovani and L. (L.) infantum, released from the surface by proteolysis might participate in different stages of the parasite life cycle (Elias et al., 2006). The enzyme has a physiological function in the promastigote stage of these parasites (Schneider and Glaser, 1993)   The biological process predicted for these proteins is mainly involved in lipid biosynthesis, signal transduction and carbohydrate metabolism. The molecular function shows that these proteins are involved greatly in binding and some proteins contain enzymatic activity and hence involved in signaling pathways. Cellular component shows that these proteins are mainly membrane proteins. NA indicates that the GO predictions were not available.

L. (L.) donovani specific amastins and its gene locations with respect to other leishmanial species
Amastin, encoded by a large gene family was a transmembrane glycoprotein initially documented in amastigote stage of trypanosomatid parasites and subsequently documented as surface proteins expressed more in leishmanial species than trypanosoma species (Jackson, 2010). The exact function of amastin and tuzin, the gene family found to be associated with amastin of unknown functions is yet to be classified. The significance of amastin gene family in pathogenesis of leishmanial species has been partially reported in earlier studies (Rochette et al., 2005). Amastin proteins of leishmanial species were mainly found in chromosome 8, 24, 28, 29, 30 and 34 of which chromosome 34 was most represented and chromosomes 28 and 29 were the least represented. Chromosome 34 contains mainly the amastin genes (6 copies) which were specific for visceralization causing protozoans. One amastin gene was found to be specific for L. (L.) donovani, 2 copies of amastin genes were found conserved in L. The research on L. (L.) donovani till now presents only one specific gene family A2 which was directly involved in disease tropism (Zhang and Matlashewski, 2001). In contrast, L. (L.) major contained only A2 pseudogenes (Zhang et al., 2003) and were completely absent in L. (Viannia) braziliensis. The function of A2 protein in L. (L.) donovani might be to relieve the stress in visceral organs following infection (McCall and Matlashewski, 2010). To study the evolution, A2 protein sequences of L. (L.) donovani were collected from literature (Oliveira et al., 2011). HMMER and Blastp result revealed that the A2 protein had clear homology with Stage Specific S Antigen (SSSA) of other leishmanial species. A2 gene family was specifically expressed in the amastigote stage inside host macrophage, though the exact function of the protein coded by this gene family was unclear (Charest and Matlashewski, 1994). The multiple sequence alignment showed that the A2 protein sequence was conserved in all leishmanial species though the length of the protein was 5 fold less represented in L. (L.) donovani, may be due to deletion events. The multiple sequence alignment was given in (Supplementary Fig. S1). The clear homology and exact length with S-antigen homolog indicates that in course of evolution, part of A2 protein might have got lost in other leishmanial species, leaving the SSSA part to play a role in pathogenesis. So the important role of SSSA in L. (L.) donovani remains to be uncovered in future. Also significant homology found between the A2 protein sequence of L. (L.) donovani species and uncharacterized protein sequences of Streptomyces ambofaciens and Thermobispora bispora indicates that these proteins might have originated from common ancestor and as evolution progresses these proteins diverged depending on the host as well as the environment and formed a significant protein to play a key role in pathogenesis. The evolutionary closeness was further confirmed by phylogenetic analysis (data not shown). Moreover A2 protein from L. (L.) mexicana, yet not declared as non-functional protein coded by a pseudogene, was very much conserved with the uncharacterized protein sequences of Streptomyces ambofaciens and Thermobispora bispora indicating that it was preserved throughout the evolution but the exact role of these protein in L. (L.) mexicana pathogenesis remains unclear. The copy number of A2 gene also plays a significant role in pathogenesis of particular leishmanial species. A2 gene family has become a pseudogene in L. (L.) major genome (Zhang et al., 2003) and was completely absent in L. (Viannia) braziliensis. A2 family genes that are abundantly expressed in amastigote stage of leishmanial species known to be the primary factor for visceralization and virulence. Previous studies demonstrate the crucial role of A2 genes (Zhang and Matlashewski, 2001). A2 gene family present in chromosome 22, consists of a 5′A2 rel, 3′A2 rel, internal A2 rel and A2 genes and organized as 5′A2rel, A2 gene, internal A2 rel and 3′A2 rel (Zhang et al., 2003;Zhang and Matlashewski, 2001). All the available genes in A2 family consisting of 5′A2rel, 3′A2rel, internal A2rel and A2 gene sequences from leishmanial genomes were retrieved from NCBI GenBank and GeneDb. A2 gene sequence was not available for L. (L.) major and L. (Viannia) braziliensis in the databases while 5′A2rel, 3′ A2rel and internal A2rel gene sequences were not available for the species other than L. (L.) donovani and L. (L.) infantum. To understand the organization of A2 gene family within the leishmanial species, A2 genes were searched in the genome of L. (L.) donovani using ACT (Carver et al., 2005). The results of respective A2 gene against respective genome shows that, in L. (L.) infantum single copy of A2 gene is present but in L. (L.) mexicana two copies of A2 gene are present which are adjacent to each other with very little sequence differences. In L. (L.) donovani there are multiple copies of partial A2 gene scattered throughout the chromosome 22. The location of A2 genes on the chromosome 22 of four leishmanial species are depicted in (Fig. 4). But the presence of two A2 genes and its role in L. (L.) mexicana remains unclear. L. (L.) donovani genome evolved with 5 copies of A2 gene as a result of duplication remains the most severe form of visceral leishmaniasis. The copy number difference of A2 gene in different leishmanial species was depicted in (Supplementary Fig. S2).

L. (L.) donovani genes with high-variation
Cysteine peptidases were considered to be the important molecule in leishmanial pathogenesis (Vinet et al., 2009) Table S4).
Repeat analysis identifies huge differences in gene coding for surface proteins between L. (L.) donovani and other leishmanial species Amino acid repeat-containing proteins have a broad range of functions and their identification was of relevance to many experimental biologists. The involvement of these proteins in immune evasion had been shown in protozoan parasites such as the kinetoplastid and Plasmodium species, probably by influencing virulence and pathogenicity (Goto et al., 2010). Leishmanial proteomes were enriched with amino acid repeats, approximately 3-4% proteins were repeat containing proteins and the probable role of these proteins might be to mediate host parasite interaction (Kedzierski et al., 2004;Depledge et al., 2007). Identification of repeatcontaining proteins provides researchers with a defined subset of proteins which can be analyzed by expression profiling and functional characterization, thereby facilitating study of pathogenicity and virulence factors in the parasitic protozoa.
Total repeat containing protein sequences were collected from the nearby species of L. (L.) donovani from Repseq database . The comparative analysis of number and type of repeat containing protein sequences in 4 leishmanial species are illustrated here (Fig. 5). Many virulence factors were already reported in leishmanial species in which surface proteins were of special interest. Comparative analysis of genes encoding the repeat containing surface proteins and other repeat proteins known to be involved in virulence was given in (Table 3). Among the surface proteins, surface antigen proteins and Proteophosphoglycan were well represented repeat proteins of leishmanial species. In L. (L.) donovani, two surface proteins (protein IDs: CBZ33953.1 and CBZ31356.1) from chromosome 12 and 4 were duplicated many times in other leishmanial species. Also calpain like cysteine peptidases from L. (L.) donovani (protein ID: CBZ35211.1), were represented more in other leishmanial species. The genes which encode other repeat containing proteins which were functional homologs or hypothetical proteins of other leishmanial species were also compared and tabulated. The most common type of motif found in repeat proteins was Single Repeat Regions (SRR). The complete list of comparative analysis of genes encoding repeat proteins was given in (Supplementary Tables S5,  S6). The frequency of occurrence of particular amino acid was important for a repeat containing protein to be involved in pathogenesis directly or indirectly. Alanine the common amino acid was occurring more in repeat containing proteins also, though there was no relation reported between alanine and pathogenesis. Leucine was represented more in these proteins and the significance of this amino acid in virulence or pathogenesis of many organisms was well documented in literature (Kedzierski et al., 2004). Also frequency of serine was found more, though the exact involvement of this amino acid in pathogenesis was unknown. The amino acid composition of the repeat containing proteins was given in (Supplementary Fig. S3).

Discussion
The aim of this study is to compare and reveal the species specific differences of L. (L.) donovani with some other important leishmanial species. The difference between L. (L.) donovani and other leishmanial species at the genome level is completely studied. Though genome of leishmanial species are highly conserved as reviewed in the introduction, few important differences were identified in the genome of L. (L.) donovani. The comparative genome analysis of L. (L.) donovani with the other leishmanial species identified 55 species specific genes. Since the proteins encoded by these genes are species specific, the importance of these proteins in disease representation, disease progression, pathogenesis and virulence can be evaluated. To read the functions of these genes, Gene Ontology was done using Amigo tool available at GO database. Major portions of the functionally specific proteins fall into two classes 1) Ribosomal protein and 2) Surface/surface-like proteins. Though the specificity of ribosomal proteins was not urged, the probable involvement of surface proteins in L. (L.) donovani disease spectrum is revealed in our study. In addition some genes involved in sugar metabolism was also identified and the probable role of these proteins in pathogenesis was discussed. Importantly a gene which encodes cathepsin like cysteine protease with its established role in pathogenesis was identified as specific gene of L. (L.) donovani which can be verified as important drug target in future. Also one other gene which encodes Apical Membrane Antigen 1 (AMA1), though less research was done in leishmanial species regarding this protein and the involvement of this protein in pathogenesis is completely undefined, the involvement of this protein in virulence and pathogenesis in organisms such as plasmodium and Toxoplasma gondii attracts the future interest to investigate the importance of this protein in leishmanial pathogenesis. We hypothesize the specific AMA1 of L. (L.) donovani might follow a similar mechanism documented in apicomplexon parasites to invade the host cells though the mediators involved in host-parasite interaction are RON2 in the case of apicomplexon parasites and may be cholesterol in the case of L. (L.) donovani.
Matching our expectation, many genes which are found conserved with L. (L.) infantum showed less homology with remaining three leishmanial species. Apart from this, many genes were identified that are The comparative analysis list contains genes which encode many surface proteins noticeably amastins, amastin like surface proteins, peptidases etc., and these proteins are highly linked with pathogenesis, which may be experimentally verified further for complete understanding of distinct mechanism of L. (L.) donovani pathogenesis. As amastins have significant role in pathogenesis of any leishmanial species, the amastin comparison was done among all the five leishmanial species which identified few L. (L.) donovani specific amastin gene locations. Amastin gene family is represented more in L. (L.) donovani though the exact cause is not known. Also few genes which encode proteins that are not surface proteins but linked to pathogenesis directly or indirectly are discussed to their relevance. In addition, we identified a gene encoding a repeat containing protein, calpain like cysteine peptidases was identified and this particular protein is conserved in all leishmanial    The only specific gene family till now documented to be responsible for visceralization, the A2 gene family was analyzed for its evolutionary divergence. A2 gene family encodes A2 protein which was highly homologous to Stage Specific S Antigen of other leishmanial species, though the exact relevance of this stage specific expression is completely not understood. Also A2 gene copy number difference between leishmanial species was reported which clearly identified high copy number in L. Specific amino acid repeats play a direct role in virulence from prokaryotes to eukaryotes. Complete L. (L.) donovani repeat containing proteins were identified and compared with the other leishmanial species in the present study.
Altogether the current study shows the complete analysis of the recent draft genome of L. (L.) donovani. The genes identified as L. (L.) donovani species specific can be experimented in the future to explore the complexity of L. (L.) donovani genome information and probably if some of these genes are established to be involved in pathogenesis, it can be a clear target for anti-leishmanial therapy.

Data collection
The genome sequence of L.  (Benson et al., 2011;Logan-Klumpler et al., 2012). The total protein sequences of all other four leishmanial species were collected from Trityp database (Aslett et al., 2010).

Comparative analysis
The total protein sequence of L. (L.) donovani was simultaneously searched against the proteomes of other leishmanial species using HMMER package (Eddy, 2009) and Psi-blast (Altschul et al., 1997) and it was further confirmed by Bioedit local blastp package (Hall, 1999). The matches of L. (L.) donovani which was showing greater than 30% homology or e-value lesser than e −05 were eliminated using in-house Perl script and the remaining proteins coded by L. (L.) donovani genes which are completely non-homologous in all leishmanial species were considered species specific genes of L. (L.) donovani. The stringent e value is set to completely eliminate any homologous genes. The identified species specific genes were supposed to length criteria of greater than 90 codons and the genes which code proteins less than 30 amino acids were manually discarded, as it is very difficult to justify the proteins which are coded by less than 90 codons. Further L. (L.) donovani genes were individually compared with the other four leishmanial species and the genes which were present between L. (L.) donovani and any leishmanial species but absent in other leishmanial species were listed. All the possible combinations were done between L. (L.) donovani and other four leishmanial species and compared. In addition, the proteins encoded by these genes were further annotated by transferring functions of already assembled and annotated L. (L.) major, L. (L.) infantum and L. (Viannia) braziliensis proteins using blastp searches. Also annotations of L. (L.) donovani specific genes were done by searching the GO database using Amigo package (Carbon et al., 2009). Functionally homologous genes which show specificity between L. (L.) donovani and other species were also functionally annotated by Gene Ontology using Amigo tool. In addition, genes which were continuously under change (i.e.) genes under positive selection were detected using Psi-blast and blastp matches. The genes meeting these criteria were retrieved using in house Perl scripts. The amastin gene sequences from different leishmanial species were collected and HMMER searches identified the homologous amastin genes in L. (L.) donovani. The identified amastin genes were compared and localized by searching all the leishmanial genome in parallel using ACT software (Carver et al., 2005).

Multiple sequence alignment (MSA)
A2 protein sequences and gene sequences were collected from NCBI. Blastp using NCBI blast (Altschul et al., 1990) and Fasta (Lipman and Pearson, 1985) format of those gene sequences identified the nearby homologous sequences from different species. Multiple sequence alignment was done using MUSCLE (Edgar, 2004). Also the location of the A2 gene family was identified by searching all the leishmanial species using Artemis software (Carver et al., 2005).

Repeat analysis
The entire L. (L.) donovani proteins were analyzed for the presence of amino acid repeats using Repseq database . The entire repeat sequences from L. (L.) infantum, L. (L.) major, and L. (Viannia) braziliensis and Trypanosoma species were retrieved from Repseq database and checking the presence of the homologous sequence in L. (L.) donovani using HMMR, Psi-Blast and Blastp revealed the most probable repeat sequence in L. (L.) donovani. Further the proteins identified as species specific were also checked for the presence of repeats. Altogether this will reveal the complete repeat containing proteins of L. (L.) donovani.