{'fraction_non_alphanumeric': 0.03915213253074034, 'fraction_numerical': 0.0168493080207851, 'mean_word_length': 5.297586262757168, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 2, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Differentially modulated proteins associated with Leishmaniasis—a systematic review of in-vivo and in-vitro studies

High-throughput proteomic technologies are widely used for understanding the disease mechanism, drug-resistant mechanism, and to identify drug targets and markers for diagnostics. Studies with proteomics applications, relating to Leishmaniasis, are being constantly reported in the literature. However, from such studies, a readily accessible knowledge of differentially modulated proteins associated with Leishmaniasis is lacking. Hence, we performed a systematic review concerning differentially modulated proteins (DMP) in Leishmania as well as host infected with Leishmania from the published articles between the years 2000 and 2019. This review is classified into five different sections, namely, DMP in the host after Leishmania infection, DMP between different strains of Leishmania, DMP in drug-resistant Leishmania, DMP in Leishmania under stress, and DMP in different life stages of Leishmania. A lot of consensuses could be observed among the DMP in drug-resistant and stressed Leishmania. In addition to the review, a database was constructed with the data collected in this study (protein accession ID, protein name, gene name, host organism, experimental conditions, fold change, and regulatory data). A total of 2635 records are available in the database. We believe this review and the database will help the researcher in understanding the disease better and provide information for the targeted proteomics study related to Leishmaniasis. Database availability: http://ldepdb.biomedinformri.com/. Electronic supplementary material The online version of this article (10.1007/s11033-020-05936-z) contains supplementary material, which is available to authorized users.


Introduction
Leishmaniasis, a neglected tropical disease, is caused by the protozoa parasite of the genus Leishmania. More than 20 species of Leishmania are responsible for the disease in humans and it is mainly transmitted to humans by the sand flies [1]. Leishmania is a unicellular eukaryote with a well-defined nucleus, kinetoplast, and flagella [2]. It adapts two different structural variants in their life cycle, namely promastigote and amastigote. An infected sandfly injects promastigotes into a human during their blood meal, where they are mostly phagocytized by macrophages and transform themselves into amastigotes. Further, the amastigotes divide and infect other phagocytic cells [3]. Hence, amastigotes are the cause of disease in the mammalian host [4]. Leishmaniasis is mainly associated with the weak immune system, malnutrition, and poor housing [5], thus majorly affecting the poor population of Africa, Asia, and Latin America. Since more than 90 countries are endemic for leishmaniasis [6], it is considered one of the major neglected tropical diseases of serious concern worldwide. There are three main forms of leishmaniasis, cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL). CL, the most common form of the disease, is majorly reported in the new world countries and is caused by L. mexicana, L. amazonensis, L. braziliensis, L. infantum chagasi, and L. major [7]. VL, the most serious form, is mainly caused by Leishmania donovani and Leishmania infantum [8]. While the rare form, MCL, is mostly caused by L. braziliensis complex, L. guyanensis, and L. panamenensis [9]. Countries like India, Bangladesh, Sudan, Ethiopia, and Brazil have reported more VL cases (> 90% in total) than the rest [10]. According to the 2017 World Health Organization report, 75 countries are endemic for visceral leishmaniasis [11]. Also, Post-Kala-Azar Dermal Leishmaniasis (PKDL) [12] is of more serious concern in India and Sudan [13]. Chemotherapy is the preferred choice for treating VL [14]. Drugs available for the treatment of VL include Amphotericin B, Miltefosine, Paromomycin, Pentamidine, and Sodium stibogluconate [15]. Amphotericin B is considered an effective drug for VL in the Indian subcontinent [16][17][18]. However, increased cases of resistance to Amphotericin B in VL patients [19] and the rate of relapse in PKDL patients (after treatment) have increased substantially [20,21]. At the same time, combination therapy proved to be more effective in treating VL than monotherapy [22]. However, there are also reports of resistance to combination therapy [23][24][25]. Efforts were made in the past to identify a lot of small-molecule inhibitors against leishmaniasis [26]. However, the number of well-established drug targets for VL is limited [27]. Hence, identifying new drug target(s) becomes a constant requirement in the process of combating VL. Although several VL vaccine candidates are under different phases of the trial [28,29], none are yet available. These facts indicate the necessity of alternate drugs, drug targets and vaccine candidates to treat VL.
The advancement in proteomic technologies [30] acts as a valuable tool for potential drug target identification, understanding the disease progression, diagnostic biomarker identification [31], and characterization of antigens in vaccine development [32]. Specifically, oncological studies effectively employ the advanced proteomic approaches for the identification of drug targets, the discovery of downstream effectors, protein-protein interaction study, and biomarker discovery [31]. Also, many studies involving highthroughput proteomic techniques (related to leishmaniasis) report the comparison of high-resolution protein data under different experimental conditions. Hence, we conducted a systematic review of such studies involving high-throughput proteomic techniques (mainly focusing on comparable experimental conditions) and provided data that is easy to compare and access. This review is majorly divided into the following sections: (a) data collection, (b) differentially modulated proteins (DMP) in the host after Leishmania infection, (c) DMP between different strains of Leishmania sp., (d) DMP in drug-resistant Leishmania sp., (e) DMP in stressed Leishmania sp., (f) DMP in different life stages of Leishmania sp., and (g) database development. In addition to the information presented in this review, a freely-accessible and searchable database (http://ldepd b.biome dinfo rmri. com/) is also made available. PubMed ID, protein ID, protein name, gene name, fold change, the experimental organism, experimental conditions, and the experimental methods of corresponding studies (collected for this review) are accessible via the database. Hence, we believe this study includes a comprehensive review of differential protein expression studies related to Leishmaniasis and presented the data to the research community in an easy to access way.

Data collection
PubMed and Scopus literature databases are used for searching the articles. The keywords "Leishmania" and "proteome", present in "title" or "abstract", were searched in the literature databases. The search was performed on 24th October 2019 and the search year timeline was between 2000 and 2019. The search resulted in a total of 196 records in PubMed and 318 records in Scopus. The duplicate entries were removed, resulting in a total of 122 unique records. These 122 records were included for the further manual screening process. An overview of the data collection process is depicted in Fig. 1. The inclusion and exclusion criteria for this study are briefed below.

Inclusion and exclusion criteria
Only the published research articles were considered. The full-text of all the 122 unique records were obtained and read manually to identify the relevancy. Two authors independently verified the articles for relevancy. Articles that report DMP and protein identification studies (in addition to 2-Dimentional electrophoresis) relating to Leishmania or leishmaniasis were only considered. Review articles were not considered. Studies that focused only on one protein were not considered. Studies only with genomics or transcriptomics experiments were not considered. Studies involving only the in-silico methods were not considered. Studies that do not involve any proteomic techniques were not considered. Full articles that cannot be obtained through any means (including personal correspondence to the authors of the article) were not considered. Based on the inclusion and exclusion criteria, a total of 44 articles are found relevant and used for this review.

Differentially modulated proteins in the host after Leishmania sp. infection
Comparing the host protein expression levels before and after infection will provide information about the disease mechanism, host-response, and the potential markers for diagnosis [33,34]. Concerning Leishmaniasis, out of articles matching the selection criteria of this review, a total of 13 articles reported differential proteome analysis between infected and healthy states (Tables 1 and 2). Based on the type of host, the 13 articles are further grouped into three categories (Homo sapiens: 3, Mus musculus: 7, and Canis lupus: 3) and discussed below.
Bag et al. [35] conducted the differential proteomic study between VL patients (L. donovani) and healthy human subjects. In this study, with endemic and non-endemic healthy controls, a comparison of proteins and glycoproteins from    protein, phosphodiesterase, and solute carrier family protein.
In another recent study, Montoya et al. [37] conducted a similar study comparing the proteome of L. panamensis infected human skin lesion against normal human skin and reported 12 DMP. The seven up-regulated proteins include keratin type I and II, lysosome-associated membrane glycoprotein, protein S100-A8 and A9, leukocyte elastase inhibitor, and copper-transporting ATPase. The five down-regulated proteins include fatty acid-binding protein, protein S100-A2, peroxiredoxin, hemoglobin subunit-α, and hemoglobin subunit-δ. However, both studies have reported no common DMP. This may be because of differences in the location of the skin biopsies (border and middle of ulcer) and the infectious parasite of corresponding study.
In addition to studies on humans, proteomics studies have been reported on Mus musculus, infected with different Leishmania species. Among such studies (matching the selection criteria), four studies involved L. major and L. amazonensis infection, two studies involved L. infantum infection, and one study involved L. mexicana infection. Menezes et al. [39] reported 15 DMP in infected mice (L. major or L. amazonensis) compared to the control. The four up-regulated proteins of infected mice include phospholipase D1, RAS-related protein, glucuronidase β, and phosphotransferase regulation domain. Negrão et al. [40] performed a proteomic study among mice infected with L. amazonensis, L. major, L. infantum, mice inoculated with lipopolysaccharide (as positive control), and healthy mice (as negative control). The study reported 31 proteins to be differentially regulated only in L. amazonensis, 25 proteins to be differentially regulated only in L. major, and 27 proteins to be differentially regulated only in L. infantum. The study also reported 22 DMP in all three infections compared to healthy. The RAS-related protein was up-regulated in this study as well as in the Menezes et al. [39] study. The same group [41] performed a similar experiment with skin biopsies and reported 17 DMP in infected mice. Further, they [42] adopted the labeled (tandem mass tag) proteomic approach and confirmed their earlier findings. Also, the study reported U6 snRNA-associated Sm-like protein and heat repeat-containing protein 3 to be precisely up-regulated in infection with L. major, while protein tweety homolog 3, phosphatidate cytidylyltransferase, and macrophage metalloelastase to be precisely up-regulated in infection with L. amazonensis. Overall, the studies by Negrão et al. [40,42,41] demonstrated the species-specific protein modulation in Leishmania infection as well as differentiated the host response through LPS inoculation and Leishmania infection. To observe the immune response effects of malnutrition in Leishmania infection, Losada-Barragán et al. [43,44] conducted the differential proteomic studies involving malnourished mice. In both their studies [43,44], they compared the proteomes of protein-malnourished mice (infected with L. infantum) and uninfected well-nourished mice. In the study involving thymic tissue samples [43], 38 and 71 proteins are up-regulated and down-regulated in infected malnourished subjects, respectively. In the study involving splenic interstitial fluid [44], 35 and 60 proteins are up-regulated and down-regulated in infected malnourished subjects, respectively. These studies concluded that the protein malnutrition reduces the pro-inflammatory response towards a Leishmania infection. Hassani and Olivier [45] studied the immunomodulatory effects of L. mexicana infection in macrophages using comparative proteomics and identified 248 proteins. In the infected macrophages, 27 and 7 proteins were up-regulating and down-regulating, respectively. The majority of the up-regulated proteins have involvement in immunological pathways. Moreover, this study claims that it was the first to report surface protease GP63 to be present in exosome released by L. mexicana infected J774 macrophage. The study concluded that the Leishmania parasite has the potential to modulate the host exosome machinery during infection. Three comparative proteomic studies, involving Canis lupus with and without canine leishmaniasis, were reported [46][47][48]. Britti et al. [46] compared the proteins in the serum of naturally infected and uninfected dogs and reported 17 and 5 up-regulated and down-regulated proteins, respectively. Lorena et al. [48] compared salivary proteins in experimentally infected and uninfected dogs and reported 19 up-regulated proteins and 12 down-regulated proteins. More recently, Franco-Martínez et al. [47] compared the serum proteins of the subclinical and clinical stage of infected dogs and reported 15 up-regulated and down-regulated proteins, each. Contrsatingly, the up-regulated apolipoprotein in serum samples [47] was found down-regulating in salivary samples [48]. Similarly, the protein haptoglobin up-regulated in natural infection was found down-regulating in the experimental infection [47].
Among the studies with the different hosts, one could observe consensus as well as contrasting regulation of the same protein. For instance, the transferrin protein downregulated in dogs [47] was up-regulating in mice [43,40]. On the other hand, the amyloid protein is up-regulating in both dogs and humans [35,46].

Differentially modulated proteins between different strains of Leishmania sp.
The severity and virulence of infection may vary widely between the different strains [49]. In this aspect, out of articles matching the selection criteria of this review, a total of five articles were reported involving differential proteome analysis between different infective strains of Leishmania (Tables 1 and 2). Da Fonseca Pires et al. [50] compared two strains of L. infantum BH400 (Canis lupus) and BH46 (human) proteome to observe the virulence factor among the two strains. The study reported 32 and 31 proteins to be upregulated and down-regulated, respectively, in the BH400 strain. Most of the virulence-related proteins like heatshock protein, elongation factor 2, kinetoplastid membrane protein-11, and enolase were up-regulating. However, the confirmatory western blot analysis revealed that the kinetoplastid membrane protein-11 to be up-regulated and elongation factor 1β to be down-regulated in BH400. Despite the presence of a similar abundance level of other virulence-related proteins in both BH400 and BH46, the study reported higher signal intensity in the proteins of BH400. On the other hand, Magalhães et al. [51] compared the protein modulations between severe (day 0 of culture) and less virulent (day 150 of culture) L. amazonensis and reported 56 differentially regulated proteins. Out of them, 19 and 37 proteins were up-regulated and down-regulated, respectively. However, validation by western blot analysis was performed only for four of the proteins. In western blot, the down-regulated α-tubulin and paraflagellar rod proteins were identifiable, while the up-regulated heatshock protein 83 and glucose-regulated protein 78 were not. Also, the up-regulated proteins (enolase and elongation factor 2) in L. infantum BH400 strain [50] are downregulated in virulent L. amazonensis. In another study [52] involving the proteome comparison of virulent strains of L. amazonensis, 12 proteins are up-regulating in the highly virulent strain, which includes the virulent protein GP63. However, in contrast, the GTP-binding protein Rab1 reported to be up-regulated in this study as well as in Da Fonseca Pires et al. [50] study was down-regulated in virulent L. amazonensis [51]. McCall et al. [53] and Hajjaran et al. [54] performed proteomic profiling of L. donovani and L. tropica, respectively. McCall et al. [53] McCall compared the proteomes of L. donovani from CL and VL patients, while Hajjaran et al. [54] compared L. tropica from cutaneous and visceral tissue of the same infected host. McCall et al. [53] reported 38 and 148 proteins to be up-regulated in VL and CL strain, respectively. Proteins involved in stress responses, like mitochondrial peroxidoxin and superoxide dismutase, were up-regulated in VL strain. While proteins involved in transport and trafficking processes, like ABC transporters, kinesin, and dynein, were up-regulated in CL strain. However, the majority of up-regulated proteins in CL strain were uncharacterized protein. Overall, the study hypothesized that the visceral environment could be more stressful for the parasite. Similarly, Hajjaran et al. [54] also reported ubiquitin, co-chaperonin, and tryparedoxin (that aids the parasite survival in stress condition) proteins to be up-regulated in visceral form, indicating the fact that visceral environment could be more stressful.

Differentially modulated proteins in drug-resistant Leishmania sp.
Leishmaniasis, like other infectious diseases, is also prone to drug resistance [55]. Quantitative and comparative proteomic studies are of great tool to understand the drug resistance mechanism of the organism in comparison to drugsensitive organisms [30]. Fourteen such articles, addressing the comparative proteomic study between drug-sensitive and drug resistance in Leishmaniasis, are discussed below.
Nine studies compared the proteomic profiles of antimony resistant Leishmania and antimony sensitive Leishmania (Tables 1 and 2). Vergnes et al. [56] reported only seven DMP between antimony resistant and sensitive L. donovani. Among the resistant strains, 14-3-3 and heat shock 83-1 protein were significantly up-regulated, while the small kinetosome calpain-related protein was significantly downregulated. The study concluded, up-regulated heat shock 83-1 protein increased the antimony resistance and reduced the drug-mediated programmed cell death, on the other hand, the down-regulated small kinetoplastid calpain-related protein promoted the antimonial induced programmed cell death. However, a similar study on antimony resistant L. infantum [57] reported the up-regulation of only one protein (argininosuccinate synthase) in the resistant strain. Since the kinetoplastid membrane protein-11 (KMP-11) was downregulated in both the resistant strains studied, the study concluded that the KMP-11 could be responsible for the drug resistance mechanism. However, the study did not report the implication of KMP-11 in the drug resistance mechanism. Moreover, Kumar et al. [58] reported several proteins to be overexpressed in sodium antimony gluconate (SAG) resistant L. donovani promastigotes. The study reported the up-regulation of more cytosolic proteins than proteins localized in the membrane. The heat shock protein 83 is highly expressed in SAG-sensitive promastigotes (based on fold change). This is in consonance with the study by Vergnes et al. [56]. In a study similar to Kumar et al. [58] (where antimony resistant and antimony sensitive L. donovani promastigotes are studied), Biyani et al. [59] also reported 77 proteins to be up-regulated in antimony resistant promastigotes. The aldose 1-epimerase-like protein was up-regulated highly (based on fold change), while the proteins like betatubulin, heat shock protein, and fructose-1,6-bisphosphate aldolase were up-regulated moderately. These moderately up-regulated proteins are also up-regulating in the Kumar et al. [58] study. In contrast, 60S ribosomal protein L23a was down-regulated in the Biyani et al. [59] study, while it was up-regulated in the Kumar et al. [58] study. In trivalent antimony resistant L. panamensis promastigotes, 12 proteins were reported to be up-regulated [60]. Out of twelve proteins, three proteins had fold change of > 2, which includes ATPase-beta subunit, prostaglandin F2-a synthase, and GTPbinding proteins. Another similar study in promastigotes of L. infantum chagasi and L. braziliensis [61] reported 28 and 27 proteins to be up-regulated in trivalent antimony resistant parasite, respectively. In addition to several up-regulated proteins (like heat shock proteins, enolase, tubulin proteins, etc.), the study reported enzymes of trypanothione synthesis pathway (like treparodoxin and trypanothione reductase) to be up-regulating. The same group [62] performed the phosphoproteomic analysis in wild-type and antimony-resistant L. braziliensis promastigotes. The study reported 48 proteins to be differentially abundant in wild-type and resistant (treated with different concentrations of antimony). Nine of the proteins including proliferating cell nuclear antigen protein, heat shock protein 70, heat shock protein 83-1, α and β-tubulin, tryparedoxin peroxidase, pteridine reductase, iron-sulfur protein, and paraflagellar rod protein are differentially expressed in both total protein and phosphoprotein analysis, indicating the modulation in both protein pools. A combined genomic and proteomic study [63], for the first time, reported the antimony resistant marker (ABC transporter MRPA) to be up-regulated in trivalent antimonial resistant L. infantum promastigotes. They also reported 42 proteins of several important pathways upregulated in antimonial resistant promastigotes, including trypanosome thiol synthesis and sterol biosynthesis. For the first time, Vincent et al. [64] compared the mitochondrial proteomic profile of antimony resistant L. infantum promastigote with the wild type. Out of 28 DMP reported in the drug-resistant parasite, only ten proteins were up-regulating. Most of the up-regulated proteins are hypothetical proteins, while others are flavoprotein subunit like protein, i/6 autoantigen like protein, succinyl co-A, and axoneme central apparatus protein.
Beside, antimonial drug resistance in Leishmania, proteomic studies were also reported on several other drug resistance, including methotrexate, difluoromethylornithine (DMFO), miltefosine, and Amphotericin B (AmB). Drummelsmith et al. [65] conducted the differential proteomic study between methotrexate resistant and sensitive L. major and reported 13 DMP. Enolase, beta-tubulin, and heat shock proteins are overexpressed, in addition to several other proteins. However, the study concluded that methionine adenosyltransferase (AdoMets) overexpression contributed to the methotrexate resistance. The same study confirmed the role of AdoMets in drug resistance through gene transfection and metabolite analysis. Singh et al. [66] studied the differential protein expression between DMFO resistant and sensitive L. donovani promastigotes. Out of 101 DMP, 82 and 19 proteins were up-regulated and down-regulated, respectively. Despite the up-regulation of several proteins from 1 3 different pathways, the study claimed S-adenosylmethionine synthetase and Cystathionine beta-lyase-like protein upregulation playing a major role in DMFO resistance. Two differential proteomic studies compared miltefosine resistant and sensitive parasite. One involves promastigotes of L. infantum chagasi [67] and other L. donovani [68]. A total of 21 and four proteins were up-regulated in drug-resistant L. infantum chagasi and L. donovani, respectively. Carnielli et al. [67] concluded that the up-regulation of proteins NADP-dependent alcohol dehydrogenase and translation elongation factor 1-beta play a key role in redox homeostasis and thereby could aid in miltefosine resistance. Vacchina et al. [69] reported that the mitochondrial HSP70 is involved in miltefosine resistance as well as in the stress response of the parasite. In addition, Veronica et al. [68] reported that the protein iron superoxide dismutase contributes to the miltefosine resistance. The same study reported proteins enolase and tryparedoxin (up-regulated in antimony resistant) to be down-regulating in miltefosine resistant. Garg et al. [70] have recently reported a proteomic study between AmB resistant and sensitive promastigotes of L. donovani secretome. The study reported 54 DMP, among them 32 and 22 proteins were up-regulated and down-regulated, respectively, in AmB resistant promastigotes. The study suggested a majority of up-regulated proteins have a role in parasite adaptiveness to stress condition. The same study also reported proteins tryparedoxin, heat shock protein, and tubulin protein (up-regulated in other drug resistance) to be up-regulated in AmB resistant promastigotes.

Differentially modulated proteins in Leishmania sp. under stress
Stress can be defined as the difference in the biological responses due to intrinsic or extrinsic stimulus. Different types of stress may affect the gene expression, protein expression and metabolism of organisms to varying degrees [71]. The different types of stress experienced by Leishmania includes oxidative, nitrosative, temperature, pH, and drug-induced stress [72][73][74]. The knowledge of protein expression levels during such a condition plays a vital role in understanding the parasite survival under stress. Four such articles (Tables 1 and 2) are discussed below.
Silverman et al. [75] conducted a quantitative proteomic comparison of Leishmania donovani promastigotes (exosomes) under varying temperature (26 ºC and 37 ºC) and pH (5.5 and 7.5). They identified 329 proteins, among which 79, 154, and 96 proteins are differentially expressed in a condition having a change in temperature and pH, the temperature only, and pH only, respectively. The study reported, at 37 ºC and acidic pH, an increase in exosome release. Since the temperature and pH mimic the infection, the study concluded that Leishmania up-regulates and modify exosome production and thereby modulate the regulation and function of macrophages. The 60 s acidic-ribosomal protein and β-fructofuranosidase up-regulated under pH stress are also up-regulating in parasite secretome [76]. Pescher et al. [77] compared the protein expression levels between splenic (nutrition poor condition) and axenic (nutrient-rich) amastigotes and reported the up-regulation of eight proteins in nutrition deprived conditions. Among them, the protein methylthioadenosine phosphorylase was strongly up-regulating. Since methylthioadenosine phosphorylase involves the metabolism of polyamines and purines, they suspected that this enzyme could help in the survival of parasite under nutrition-deprived conditions. The same study confirmed this by culturing the splenic and axenic amastigotes in nutrient-poor medium and found the splenic amastigotes survived while the axenic amastigotes died between 5 and 7 days. Later, Sardar et al. [78] exposed L. donovani promastigotes to three different stress conditions viz., oxidative stress, nitrosative stress, and both oxidative and nitrosative stress, and performed the comparative proteomic study. The study revealed 13 and 3 DMP to be involved in redox homeostasis and reactive oxygen species, respectively. Among those 16 DMP, only four proteins down-regulated in all three stress conditions. The rest of the 12 proteins up-regulated at least by one-fold. The study concluded that the differentially expressed proteins in different stress conditions were mainly involved in trypanothione metabolism, superoxide metabolism, mitochondrial respiration process, and oxidative phosphorylation. In another study, Teixeria et al. [79] compared the protein expression between L. amazonensis amastigotes from BALB/c nude (lack of thymus mediated immunity) and BALB/c mice. The study reported 21 DMP, among which 16 and five proteins were up-regulated and down-regulated, respectively, in amastigotes from BALB/c nude. Proteins like trypanothione reductase, peroxidoxin, tryparedoxin peroxidase, tryparedoxin, and heat shock proteins (associated with oxidative/nitrosative stress condition) were up-regulated at lease by one-fold. From the results it is apparent that the proteins involved in the synthesis and metabolism of trypanothione are differentially modulating during stress. Hence, these proteins can be a potential target for drug development.

Differentially modulated proteins in different life stages of Leishmania sp.
Leishmania parasite has two different forms, namely promastigotes and amastigotes. In general, the amastigotes are infectious in mammals [4], while the promastigotes are mostly present in sand flies [80]. Hence, several proteomic studies have been conducted in the past to study the protein expression levels between the promastigotes and amastigotes. Also, a protein profiling study has reported on promastigotes during different growth phase expression. Seven such articles (Tables 1 and 2) are discussed below.
In L. panamenensis amastigotes, Walker et al. [81] analyzed 51 overexpressed protein spots obtained from 2-Dimentional electrophoresis (2DE) for the differential expression. They identified four proteins, namely betatubulin, cysteine synthase, pyruvate dehydrogenase, and uncharacterized protein, to be present only in amastigotes. In addition, heat-shock protein 83, glucose-6-phosphate dehydrogenase, and ran-binding protein were up-regulated by more than onefold in amastigotes, with respect to promastigotes. Later, Leifso et al. [82] conducted differential mRNA and protein expression studies between the L. infantum promastigotes and amastigotes. They identified 91 proteins (protein expression study), out of which 8 and 35 proteins were up-regulated and down-regulated, respectively, in the amastigotes while the rest of the proteins were neutrally regulated. The up-regulated proteins in amastigotes include hypothetical proteins, chaperonin HSP60, precursor hexokinase, ribosomal protein 13, histone h3, and carboxypeptidase. However, they observed only 1.4% of differentially expressed mRNAs in amastigote. Also, Leifso et al. [82] concluded that the majority of mRNAs are constitutively expressed in both amastigotes as well as promastigotes and thereby emphasizing that the differentiation, virulence, and pathogenesis of Leishmania may not be depending on the regulation of genes or proteins. Brotherton et al. [83] used free-flow electrophoresis to analyze the specific expression of the basic protein in Promastigote and amastigote of Leishmania infantum. A total of 36 and 50 proteins were up-regulated in amastigotes and promastigotes, respectively. Moreover, 37 and 42 proteins are identified only in amastigotes and promastigotes, respectively. Elongation initiation factor-2 alpha subunit, cysteinyl-tRNA synthetase, and glutathione peroxidase-like protein were up-regulated by four-fold in amastigotes than promastigotes. Interestingly, both Leifso et al. and Brotherton et al. [82,83] reported the up-regulation of chaperonin HSP60 protein in L. infantum amastigotes. In contrast, Leifso et al. [82] reported the upregulation of carboxypeptidase in L. infantum amastigotes (with respect to promastigotes) while Brotherton et al. [83] reported the same in L. infantum promastigotes (with respect to amastigotes). Biyani and Madhubala [84] profiled the proteins of three different strains of L. donovani promastigotes and amastigotes for comparison. Among the three strains, two strains (GEI-R and NR3A-R) are resistant to antimony, and the one is sensitive (AG83-S). The study identified 28, 29, and 26 proteins to be up-regulated and 34, 33, and 31 proteins to be down-regulated in amastigotes of AG83-S, GEI-R, and NR3A-R, respectively. Six proteins, namely histone h2b, glucose-regulated protein 78, ADP-ATP carrier protein, GTP-binding protein, and 2 hypothetical proteins, were significantly up-regulated in amastigotes of resistant strains (with respect to sensitive strains). Leifso et al. [82] reported histone h2b to be down-regulating while Biyani et al. [84] reported the same to be up-regulating in L. infantum amastigotes. However, Lynn et al. [85] reported histone h2 protein to be up-regulating in L. infantum amastigotes. In addition, Lynn et al. [85] reported the up-regulation of GTP-binding protein in L. infantum promastigotes, which agrees with the Biyani et al. [84] study. Also, Lynn et al. [85] identified arginase and alpha-adaptin like protein to be up-regulated by two-fold in L. infantum amastigotes (with respect to promastigotes). Alcolea et al. [86] studied the regulation pattern of mRNA and protein in L. infantum promastigotes logarithmic phase and stationary phase. Through differential proteomic techniques, the study identified 16 upregulated and 12 down-regulated proteins in the stationary phase. Among the 28 differentially regulated proteins, ETF and eEF1-alpha were differentially regulated even at the mRNA transcript level. Alcolea et al. [87] also studied the protein expression level of oxidative and nitrosative resistant L. amazonensis promastigotes in the stationary phase and early logarithmic phase. They reported 12 and 14 proteins to be up-regulated and down-regulated, respectively, in the stationary phase. The study concluded that the up-regulation of arginase, tryparedoxin peroxidase, heat shock protein, and protein kinase A (involved in survival of parasite under stress conditions) in the stationary phase favours the preadaptative hypothesis.

Database development and usage
All the differential protein data collected in this study was made available as a database. Supplementary Table 1 describes the structure of the database. The database includes a total of 2635 records from 44 studies. The database includes a dynamic search feature with multiple field filters and includes active links to Uniprot (https ://www. unipr ot.org/) and TriTrypDB [88]. The link in the "Tri-TrypDB Search" column of the database will perform a "Protein Keyword search" in the TriTrypDB for the corresponding Leishmania proteins and will return the genomic information as a ".csv" file (since TriTrypDB is specific to Leishmania, the proteins of host organisms will have "NA" in the corresponding column). The database has two different panels, one for setting the filters and other for the display of records (Supplementary Fig. 1). The filter panel is selfdescriptive. The display panel contains top-section to export data and report the filtered number of records, a table section to display the data and sort data, and bottom-section to adjust the visibility of the records in table and navigation of records. Also, the database includes provisions for exporting the filtered records in multiple formats. PHP, MySQL, CSS, W3CSS, and JavaScript are used to develop the database and the access interface. The database is freely accessible at http://ldepd b.biome dinfo rmri.com/. The following section describes the database usage.
Consider a researcher who is interested in studying a protein (for example, trypanothione reductase in L. donovani) wants to know whether the protein is differentially regulating during stress conditions or not. To query this in the database, a user can select the "STRESS" option in the "Test" filter and "L. donovani" in the "Organism" filter. This will display four records in the display panel containing the information that the protein trypanothione reductase was up-regulating during oxidative stress, nitrosative stress, and temperature stress from two different studies. In addition, for a particular test condition, the database is used to retrieve information like proteins commonly and uniquely up or down-regulated.

Discussion
Advanced proteomics techniques are widely been utilized in Leishmania related studies to understand the disease mechanism, to identify the regulation of proteins or pathways during stress, to study the survival strategy of the drug-resistant parasite, and to study the host response due to the different forms of infection. In this study, to our knowledge, a comprehensive review covering all the above aspects was reported. Understanding drug resistance (14 studies) was the primary focus, while the secondary focus is on understanding the host response during infection (13 studies). Despite the different experimental conditions and setup, heat-shock and Tryparedoxin related proteins were differentially regulated in 17 studies, indicating that those proteins are often modulated in the parasite. Moreover, several proteins were consistently identifiable and also to be similarly modulating in studies with similar experimental conditions [42,40,41]. Only three studies explored the host response (human subjects) during Leishmania infection. However, no consensus results exist among those studies. More studies with similar experimental conditions are required to holistically understand the host (human) response upon Leishmania infection. Studies involving drug-resistant mechanisms and stress reported a lot of consensuses results. However, focus on new studies should be towards targeted proteomics, where the protein of interest is detectable with quantitative accuracy and high sensitivity [89]. This practically assists the researcher in identifying new drug targets and diagnostics. With this in mind, besides the systematic review, this study also collected differentially modulated proteins of different experimental conditions and made them as a database with an easy-to-access interface. In addition, the developed database was also actively linked to Uniprot (https ://www.unipr ot.org/) as well as TriTrypDB [88]. We strongly believe that this review and the database will help the research community in understanding the Leishmania infection, drug resistance, and host-response, and will help provide the information for the future study of targeted-proteomics related to Leishmania.