Prosopis juliflora (Sw.) DC.induces apoptotic-like programmed cell death in Leishmania donovani via over production of oxidative stress, mitochondrial dysfunction and ATP depletion.

Background: Leishmaniasis is endemic in more than 60 countries with a large number of mortality cases. The current chemotherapy approaches employed for managing the leishmaniasis is associated with severe side effects. Therefore there is a need to develop effective, safe, and cost affordable antileishmanial drug candidates. Purpose of the study: This study was designed to evaluate the in vitro antileishmanial activity of a Prosopis juliflora leaves extract (PJLME) towards the Leishmania donovani parasites. Material and methods: PJLME was evaluated for its cytotoxicity against the L. donovani parasites and the mouse macrophage cells. Further, various in vitro experiments like ROS assay, mitochondrial membrane potential assay, annexin v assay, cell cycle assay, and caspase 3/7 assay were performed to understand the mechanism of cell death. Phytochemical profiling of P. juliflorawas performed by utilizing HPTLC and GC-MS analysis. Results: PJLME demonstrated antileishmanial activity at a remarkably lower concentration of IC50 6.5 µg/mL. Of note, interestingly PJLME IC50 concentration has not demonstrated cytotoxicity against the mouse macrophage cell line. Performed experiments confirmed ROS inducing potential of PJLME which adversely affected the mitochondrial membrane potential and caused loss of mitochondrial membrane potential and thereby ATP levels. PJLME also arrested the cell cycle and induced apoptotic-like cell death in PJLME treated L. donovani promastigotes. Conclusion: The results clearly established the significance of Prosopis juliflora as an effective and safe natural resource for managing visceral leishmaniasis. The findings can be used as a baseline reference for developing novel leads/formulations for effective management of visceral leishmaniasis.

Structural aspects of lesional and non-lesional skin microbiota reveal key community changes in leprosy patients from India.

Although skin is the primary affected organ in Leprosy, the role of the skin microbiome in its pathogenesis is not well understood. Recent reports have shown that skin of leprosy patients (LP) harbours perturbed microbiota which grants inflammation and disease progression. Herein, we present the results of nested Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) which was initially performed for investigating the diversity of bacterial communities from lesional skin (LS) and non-lesional skin (NLS) sites of LP (n = 11). Further, we performed comprehensive analysis of 16S rRNA profiles corresponding to skin samples from participants (n = 90) located in two geographical locations i.e. Hyderabad and Miraj in India. The genus Staphylococcus was observed to be one of the representative bacteria characterizing healthy controls (HC; n = 30), which in contrast was underrepresented in skin microbiota of LP. Taxa affiliated to phyla Firmicutes and Proteobacteria were found to be signatures of HC and LS, respectively. Observed diversity level changes, shifts in core microbiota, and community network structure support the evident dysbiosis in normal skin microbiota due to leprosy. Insights obtained indicate the need for exploring skin microbiota modulation as a potential therapeutic option for leprosy.

Distribution of Pathogenic Yeasts in Different Clinical Samples: Their Identification, Antifungal Susceptibility Pattern, and Cell Invasion Assays.

INTRODUCTION: Species of genus Candida are part of the common microbiota of humans; however, some of the Candida species are known opportunistic pathogens. Formation of biofilms, resistance to antifungal drugs, and increase in asymptomatic infections demands more studies on isolation, identification and characterization of Candida from clinical samples. METHODS: The present manuscript deals with assessment of authentic yeast identification by three methods viz., DNA sequencing of 28S rRNA gene, protein profiles using MALDI-TOF MS, and colony coloration on chromogenic media. Antifungal susceptibility and in vitro cell invasion assays were performed to further characterize these isolates. RESULTS: Comparison of three methods showed that DNA sequence analysis correctly identified more than 99.4% of the isolates up to species level as compared to 89% by MALDI-TOF MS. In this study, we isolated a total of 176 yeasts from clinical samples and preliminary morphological characters indicated that these yeast isolates belong to the genus Candida. The species distribution of isolates was as follows: 75 isolates of Candida albicans (42.61%), 50 of C. tropicalis (28.40%), 22 of C. glabrata (12.5%), 14 of C. parapsilosis (7.95%) and 4 of Clavispora lusitaniae (2.27%). Other species like Cyberlindnera fabianii, Issatchenkia orientalis, Kluyveromyces marxianus, Kodamaea ohmeri, Lodderomyces sp., and Trichosporon asahii were less than 2%. Antifungal susceptibility assay performed with 157 isolates showed that most of the isolates were resistant to the four azoles viz., clotrimazole, fluconazole, itraconazole, and ketoconazole, and the frequency of resistance was more in non-albicans Candida isolates. The susceptibility to azole drugs ranged from 7% to 48%, while 75% of the tested yeasts were susceptible to nystatin. Moreover, 88 isolates were also tested for their capacity to invade human cells using HeLa cells. In vitro invasion assay showed that most of the C. albicans isolates showed epithelial cell invasion as compared to isolates belonging to C. glabrata, C. parapsilosis and C. tropicalis. DISCUSSION: The identification of yeasts of clinical origin by sequencing of 28S rRNA gene performed better than MALDI-TOF MS. The present study reiterates the world scenario wherein there is a shift from Candida strains to emerging opportunistic pathogens which were earlier regarded as environmental strains. The present study enlightens the current understanding of identification methods for clinical yeast isolates, increased antifungal drug resistance, epithelial cell invasion as a virulence factor, and diversity of yeasts in Indian clinical samples.

Genome-Wide Proteomics and Phosphoproteomics Analysis of Leishmania spp. During Differentiation.

Determining variations in protein abundance and/or posttranslational modification as a function of time or upon induction by a signal in a particular cell type is central to quantitative proteomics. Isobaric labeling methodologies now allow for parallel quantification of proteins at various conditions concurrently or multiplexing in relatively quantitative proteomics workflows. Hence, mapping the protein expression profiles of various developmental stages of Leishmania parasites is possible with high-resolution mass spectrometry. To analyze global changes in protein expression and cellular signaling pathways during Leishmania differentiation and development is possible with a quantitative proteomics approach. The tandem mass tags (TMT) approach provides a chemical labeling method based on the principle of amine reactive tags; the maximum number of conditions that can be multiplexed is 10-plex. We describe herein a detailed method for sample preparation, TMT-labeling, mass spectrometry and data analysis of different developmental stages of Leishmania donovani parasites. This quantitative proteomic approach is useful to study dynamic changes in protein expression levels during L. donovani differentiation, and also allows in-depth analysis of signaling pathways via phosphoproteomics.

16S rDNA based skin microbiome data of healthy individuals and leprosy patients from India.

Leprosy is an infectious disease that has predilection in skin and peripheral nerves. Skin has its own microbiome, however it is not extensively studied in Indian leprosy patients. Here, by using next-generation 16S rDNA sequencing, we have attempted to assess the skin associated microbial diversity pertaining to affected and unaffected skin of Indian leprosy patients. A total of 90 skin swab samples were collected from 60 individuals (30 healthy controls, 30 patients) residing in Hyderabad and Miraj, two distinct geographical locations in India to assess the homo/heterogeneity of skin microbial signatures. While a large increase in genus Methylobacterium and Pseudomonas was seen in patients from Miraj and Hyderabad respectively, a considerable decrease in genus Staphylococcus in the leprosy patients (as compared to controls) from both geographical locations was also observed. We expect that, these datasets can not-only provide further interesting insights, but will also help to observe dynamics of microbiome in the diseased state and generate hypotheses to test for skin microbiome transplantation studies in leprosy.

Diversity and Succession of Microbiota during Fermentation of the Traditional Indian Food Idli.

Idli, a naturally fermented Indian food, is prepared from a mixture of rice and black gram (lentil). To understand its microbial community during fermentation, detailed analysis of the structural and functional dynamics of the idli microbiome was performed by culture-dependent and -independent approaches. The bacterial diversity and microbial succession were assessed at different times of fermentation by 16S rRNA amplicon sequencing. Results highlighted that most microbiota belonged to phylum Firmicutes (70%) and Proteobacteria (22%). Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) analysis confirmed the diversity and succession involved therein. A culture-dependent approach revealed that the microbially diverse populations were conserved across different geographical locations. The fermentation was primarily driven by lactic acid bacteria as they constitute 86% of the total bacterial population, and genus Weissella emerged as the most important organism in fermentation. The natural microbiota of the grains mainly drives the fermentation, as surface sterilized grains did not show any fermentation. Growth kinetics of idli microbiota and physicochemical parameters corroborated the changes in microbial dynamics, acid production, and leavening occurring during fermentation. Using a metagenomic prediction tool, we found that the major metabolic activities of these microbial fermenters were augmented during the important phase of fermentation. The involvement of the heterofermentative hexose monophosphate (HMP) pathway in batter leavening was substantiated by radiolabeled carbon dioxide generated from d-[1-14C]-glucose. Hydrolases degrading starch and phytins and the production of B vitamins were reported. Moreover, culturable isolates showing beneficial attributes, such as acid and bile tolerance, hydrophobicity, antibiotic sensitivity, and antimicrobial activity, suggest idli to be a potential dietary supplement.IMPORTANCE This is a comprehensive analysis of idli fermentation employing modern molecular tools which provided valuable information about the bacterial diversity enabling its fermentation. The study has demonstrated the relationship between the bacterial population and its functional role in the process. The nature of idli fermentation was found to be more complex than other food fermentations due to the succession of the bacterial population. Further studies using metatranscriptomics and metabolomics may enhance the understanding of this complex fermentation process. Moreover, the presence of microorganisms with beneficial properties plausibly makes idli a suitable functional food.

Correction: Prosopis juliflora (Sw.), DC induces apoptosis and cell cycle arrest in triple negative breast cancer cells: in vitro and in vivo investigations.

[This corrects the article DOI: 10.18632/oncotarget.25717.].

Prosopis juliflora (Sw.), DC induces apoptosis and cell cycle arrest in triple negative breast cancer cells: in vitro and in vivo investigations.

Plant originated drugs/formulations are extensively prescribed by the physicians as a complementary therapy for treating various human ailments including cancer. In this study Prosopis juliflora leaves methanol extract was prepared and exposed to human breast cancer cell lines i.e. MDA-MB-231 and MCF-7 and human keratinocytes HaCaT as a representative of normal cells. Initially, a series of in vitro experiments like cell proliferation, migration, colony formation, cell cycle arrest and inhibition of angiogenesis. After confirmation of the efficient and selective activity against triple negative breast cancer cell line, we further evaluated the possible mechanism of inducing cell death and experiments like detection of reactive oxygen species, caspases and poly (ADP-ribose) polymerase cleavage study and Annexin V assay were performed. We also evaluated in vivo anti tumorigenic activity of the P. juliflora leaves by using 4T1 cells (a triple negative mouse origin breast cancer cell line) and BALB/c xenograft mouse model. In vitro experiments revealed that methanol extract of Prosopis juliflora leaves possess impressive anti-breast cancer activity more specifically against triple negative breast cancer cells, while the in vivo studies demonstrated that P. juliflora leaves extract significantly suppressed the 4T1 induced tumor growth. Present investigations clearly focus the significance of P. juliflora as an important resource for finding novel leads against triple negative breast cancer. The results may also act as a ready reference towards developing P. juliflora based formulation as an alternative and complementary medicine for the management of breast cancer.

Bioengineered mannan sulphate capped silver nanoparticles for accelerated and targeted wound healing: Physicochemical and biological investigations.

In the present study, surface functionalized mannan sulphate silver nanoparticles (MS-AgNPs) were prepared and assessed for their wound healing potential. 20nm sized, spherical MS-AgNPs were prepared by one pot synthesis approach wherein the sulphated polysaccharide mannan sulphate (MS) played dual role of reducing as well as capping agent. The crystalline MS-AgNPs exhibited surface plasmon resonance centered at 400nm along with -32.40mV zeta potential. These stable MS-AgNPs showed enhanced cytocompatibility, targeting potential and cellular uptake in murine macrophages, human skin fibroblasts and human keratinocytes as compared to citrate reduced silver nanoparticles (C-AgNPs). In the in vivo excision and incision wound models, MS-AgNPs as hydrogel formulations indicated better efficacy than the conventional and marketed silver formulations. Thus, the synthesized MS-AgNPs depicted a promising potential for site-specific topical delivery in accelerated wound therapy.

Multilocus sequence typing of Ochrobactrum spp. isolated from gastric niche.

The human stomach is colonized by diverse bacterial species. The presence of non-Helicobacter pylori bacteria in urease-positive biopsies of individuals has been reported. Bacteria belonging to the Ochrobactrum genus have been documented in the human gastric niche. The co-occurrence of Ochrobactrum spp. with H. pylori was previously reported in an antral biopsy of a non-ulcer dyspeptic (NUD) subject from Northern India. There is no information on the genetic diversity of Ochrobactrum spp. isolated from the gastric niche in the stomach. We aimed to study the species distribution and diversity of Ochrobactrum spp. with and without H. pylori in urease-positive biopsies across three different geographical regions in India. Sixty-two Ochrobactrum isolates recovered from patients with an upper gastric disorder (n=218) were subjected to molecular identification and multilocus sequence typing. H. pylori DNA was found in the majority of biopsies, which had a variable degree of Ochrobactrum spp present. Interestingly, some of the urease-positive biopsies only had Ochrobactrum without any H. pylori DNA. Based on phylogenetic analysis, the Ochrobactrum isolates were distributed into the O. intermedium, O. anthropi and O. oryzae groups. This indicates there are multiple species in the gastric niche irrespective of the presence or absence of H. pylori. Antibiotyping based on colistin and polymyxin B could differentiate between O. intermedium and O. anthropi without revealing the resistance-driven diversity. Considering the prevalence of multiple Ochrobactrum spp. in the human gastric niche, it is important to evaluate the commensal and/or pathogenic nature of non-H. pylori bacteria with respect to their geographical distribution, lifestyle and nutrition needs.

A novel protein coding potential of long intergenic non-coding RNAs (lincRNAs) in the kinetoplastid protozoan parasite Leishmania major.

Cutaneous leishmaniasis (CL) is caused by a kinetoplastid protozoan parasite Leishmania major, as a skin ulcer at the site of the sandfly bite. CL is curable and in most cases ulcers heal spontaneously within three to six months leaving a scar and disfiguration. Complete genome of L. major was reported in 2005 at the very initial phase of kinetoplastid parasite genome sequencing project. Presently, L. major genome is most studied and comprehensively annotated genome and therefore, it is being used as a reference genome for annotating recently sequenced Leishmanial genomes. A recent study reporting global transcriptome of L. major promastigotes, identified 1884 uniquely expressed non-coding RNAs (ncRNA) in L. major. In the current study, an in-depth analysis of the 1884 novel ncRNAs was carried out using a proteogenomic approach to identify their protein coding potential. Our analysis resulted in identification of eight novel protein coding genes based on mass spectrometry data. We have analyzed each of these eight novel CDS and in the process have improved the genome annotation of L. major on the basis of mass spectrometry derived peptide data. Although sequenced a decade ago, the improvement in the L. major genome annotation thus is an ongoing process.

A proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi using cell line.

The debilitating disease kala-azar or visceral leishmaniasis is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sand fly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with the transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. Understanding the molecular interaction of the sand fly and Leishmania, during the development of parasite within the sand fly gut is crucial to the understanding of the parasite life cycle. The complete genome sequences of sand flies (Phlebotomus and Lutzomyia) are currently not available and this hinders identification of proteins in the sand fly vector. The current study utilizes a three frame translated transcriptomic data of P. papatasi in the absence of genomic sequences to analyze the mass spectrometry data of P. papatasi cell line using a proteogenomic approach. Additionally, we have carried out the proteogenomic analysis of P. papatasi by comparative homology-based searches using related sequenced dipteran protein data. This study resulted in the identification of 1313 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms.

The crux and crust of ebolavirus: Analysis of genome sequences and glycoprotein gene.

The recent 2013-15 epidemic of Ebola virus disease (EVD) has initiated extensive sequencing and analysis of ebolavirus genomes. All ebolavirus genomes available until December 2014 have been collated and analyzed in this study to obtain phylogenetic relationship and uncover the variations amongst them. The terminal 'leader' and 'trailer' nucleotide sequences of the genomes were omitted and analysis of the intermediate region accommodating the sole seven genes (hepta-CDS region) of the virus showed relative stability of the genome, including the ones isolated from the current epidemic. The genome information was scrutinized to detect the variation in the surface glycoprotein gene and annotate its three protein products, resulting from its atypical transcription. This study will make an easy understanding of the genomes for those who desire to exploit the genome sequences for different investigations in EVD.

Comprehensive proteomics analysis of glycosomes from Leishmania donovani.

Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes.

Prevalence and subtype analysis of Blastocystis in healthy Indian individuals.

There is a growing interest in subtype (ST) analysis of the intestinal parasite Blastocystis due to its extensive genetic diversity that might reflect differences in pathogenicity. Although essential for reference, few studies are available on Blastocystis in healthy individuals. Moreover, molecular epidemiology data on Blastocystis in India still remain to emerge. In the present study we identified the prevalence and ST distribution of Blastocystis in healthy Indian individuals. A total of 220 stool samples were obtained; four of 100 samples from 100 adults were chosen randomly for construction of small subunit (SSU) rRNA gene clone libraries in order to elucidate micro-eukaryotic diversity in the human gut. From the SSU rDNA library, 64 sequences annotated to Blastocystis were used for ST analysis along with sequences obtained by direct sequencing of SSU rDNA PCR products amplified from the remaining samples and generated using primers targeting Blastocystis. Of 220 stool samples collected, 120 samples from 30 infants (aged 1week to 1year) were PCR-negative. Of the remaining 100 samples from 100 adults, 27 resulted in specific amplification. Out of these 27, four samples were suspected of mixed ST infection and so these samples were further analyzed by construction of clone libraries. Analysis of cloned sequences revealed that indeed 2 samples had mixed ST infection (ST1 and ST3) while the remaining two showed infection with two separate ST3 strains. ST3 was the most common ST present in our study group (100%) followed by ST1 (7.4%); ST1 was seen only in mixed infections. SSU rDNA clone library sequences generated by processing of pooled samples were identified as ST3. The majority of ST3 sequences exhibited allele 34 commonly found in the European population.

Cellular interactions and photoprotective effects of idebenone-loaded nanostructured lipid carriers stabilized using PEG-free surfactant.

In past years, nanostructured lipid carriers (NLCs) have emerged as novel topical antioxidant delivery systems because of combined positive features of liposomes and polymeric nanoparticles. Here, we seek to unlock the possibility of idebenone (IDB; an antioxidant)-loaded NLCs (IDB-NLCs) cellular interactions such as, viability and uptake, and its photoprotective effects against Ultraviolet-B (UVB)-mediated oxidative stress in immortal human keratinocyte cell line (HaCaT). The two-step preformulation strategy followed by three-level, three-variable, L9 (3(3)) Taguchi robust orthogonal design employed was important in improving IDB-NLCs key physicochemical aspects such as, entrapment efficiency, drug release (sustained), occlusion, skin deposition and physical stability. UV crosslinker, confocal microscopy and flow cytometry techniques were used to (1) mediate oxidative stress in HaCaT cells, (2) study a qualitative cellular uptake, (3) measure intracellular reactive oxygen species (ROS), and mitochondrial membrane potential, respectively. NLCs markedly improved biocompatibility of IDB under normal as well as stress conditions. Quantitative and qualitative cell uptake studies demonstrated a significant uptake of IDB-NLCs (3-fold increase) and nile red-labeled IDB-NLCs (NR-IDB-NLCs) at 2 h, respectively, hence exerted improved photoprotective effects.

A bioinformatics approach to reanalyze the genome annotation of kinetoplastid protozoan parasite Leishmania donovani.

Leishmania donovani is a kinetoplastid protozoan parasite which causes the fatal disease visceral leishmaniasis in humans. Genome sequencing of L. donovani revealed information about the arrangement of genes and genome architecture. After curation of the genome sequence, many genes in L. donovani were assigned as truncated or "partial" genes by the genome sequencing group. In the present study, we have carried out an extensive analysis and attempted to improve the gene models of these partial genes. Our analysis resulted in the identification of 308 partial genes in L. donovani, which were further categorized as C-terminal extensions, joining of genes, tandemly repeated paralogs and wrong chromosomal assignments. We have analyzed each of these genes from these categories and have improved the annotation of existing gene models in L. donovani. Some of these corrections have been confirmed by mass spectrometry derived peptide data from our previous comparative proteogenomics study in L. donovani.

Montelukast-loaded nanostructured lipid carriers: part II pulmonary drug delivery and in vitro-in vivo aerosol performance.

The aim of the present study was to establish the potential of montelukast loaded nanostructured lipid carrier (MNLC) for pulmonary application. The formulated nanoparticles were evaluated in vitro for aerodynamic characterization and in vivo for pulmokinetics in Wistar rats. The in vitro cytotoxicity was performed on A549 cell line and compared with montelukast-aqueous solution. MNLC was prepared with montelukast (0.2%), Precirol ATO5 (solid lipid), and Capryol-90 (liquid lipid) in the ratio of 7:3 using melt-emulsification-homogenization method. dl-Pyrrolidonecarboxylic acid salt of l-cocyl arginine ethyl ester (CAE), a biodegradable surfactant in the concentration of 1% was used to stabilize the nanoparticles. The particle size and encapsulation efficiency (EE) were 184.6 ± 2.7 nm and >95%, respectively. MNLC-Dry powder for inhalation (DPI) was prepared by lyophilization using 3% mannitol as cryoprotectant and carrier. MNLC-DPI was evaluated for flow, crystallographic and thermal properties. Mass median diameters (MMD) and density for MNLC-DPI were found to be 15.1 ± 1.4 µm and 0.051 ± 0.002 g/cc, respectively. In vitro aerosol performance study indicated more than 95% of the emitted dose (ED) at both the flow rates studied. Mass median aerodynamic diameters (MMAD) of 3.24 ± 0.67 µm with 69.98 ± 1.9% fine particle fraction (FPF) were obtained at 30 L/min flow rate, whereas at 60 L/min MMAD and FPF were found to be 2.83 ± 0.46 µm and 90.22 ± 2.6%, respectively. In vitro cytotoxicity study on A549 cells revealed higher safety of MNLC than pure drug. The pulmonary pharmacokinetic study demonstrated improved bioavailability, longer residence of drug in the lung and targeting factor of 11.76 for MNLC as compared to montelukast-aqueous solution. Thus, the results of the study demonstrated the potential of montelukast lipidic nanoparticulate formulation to improve the efficacy with reduced toxicity leading to better performance of drug as MNLC-DPI for inhalation use.

Neglected tropical diseases and omics science: proteogenomics analysis of the promastigote stage of Leishmania major parasite.

Among the neglected tropical diseases, leishmaniasis is one of the most devastating, resulting in significant mortality and contributing to nearly 2 million disability-adjusted life years. Cutaneous leishmaniasis is a debilitating disorder caused by the kinetoplastid protozoan parasite Leishmania major, which results in disfiguration and scars. L. major genome was the first to be sequenced within the genus Leishmania. Use of proteomic data for annotating genomes is a complementary approach to conventional genome annotation approaches and is referred to as proteogenomics. We have used a proteogenomics-based approach to map the proteome of L. major and also annotate its genome. In this study, we searched L. major promastigote proteomic data against the annotated L. major protein database. Additionally, we searched the proteomic data against six-frame translated L. major genome. In all, we identified 3613 proteins in L. major promastigotes, which covered 43% of its proteome. We also identified 26 genome search-specific peptides, which led to the identification of three novel genes previously not identified in L. major. We also corrected the annotation of N-termini of 15 genes, which resulted in extension of their protein products. We have validated our proteogenomics findings by RT-PCR and sequencing. In addition, our study resulted in identification of 266 N-terminally acetylated peptides in L. major, one of the largest acetylated peptide datasets thus far in Leishmania. This dataset should be a valuable resource to researchers focusing on neglected tropical diseases.

Moving from unsequenced to sequenced genome: reanalysis of the proteome of Leishmania donovani.

The kinetoplastid protozoan parasite, Leishmania donovani, is the causative agent of kala azar or visceral leishmaniasis. Kala azar is a severe form of leishmaniasis that is fatal in the majority of untreated cases. Studies on proteomic analysis of L. donovani thus far have been carried out using homology-based identification based on related Leishmania species (L. infantum, L. major and L. braziliensis) whose genomes have been sequenced. Recently, the genome of L. donovani was fully sequenced and the data became publicly available. We took advantage of the availability of its genomic sequence to carry out a more accurate proteogenomic analysis of L. donovani proteome using our previously generated dataset. This resulted in identification of 17,504 unique peptides upon database-dependent search against the annotated proteins in L. donovani. These peptides were assigned to 3999 unique proteins in L. donovani. 2296 proteins were identified in both the life stages of L. donovani, while 613 and 1090 proteins were identified only from amastigote and promastigote stages, respectively. The proteomic data was also searched against six-frame translated L. donovani genome, which led to 255 genome search-specific peptides (GSSPs) resulting in identification of 20 novel genes and correction of 40 existing gene models in L. donovani. BIOLOGICAL SIGNIFICANCE: Leishmania donovani genome sequencing was recently completed, which permitted us to use a proteogenomic approach to map its proteome and to carry out annotation of it genome. This resulted in mapping of 50% (3999 proteins) of L. donovani proteome. Our study identified 20 novel genes previously not predicted from the L. donovani genome in addition to correcting annotations of 40 existing gene models. The identified proteins may help in better understanding of stage-specific protein expression profiles in L. donovani and to identify novel stage-specific drug targets in L. donovani which could be used in the treatment of leishmaniasis. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.