Mapping the Hidden Secretome in Leishmania Parasites Using a Proteogenomics Approach

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Clinical outcomes range from self-healing cutaneous lesions to fatal visceral disease across twenty recognised human-pathogenic species. Although Leishmania genomes are remarkably conserved, substantial phenotypic diversity arises through post-transcriptional and post-translational regulatory mechanisms, highlighting the essential role of proteomic investigations. The Leishmania secretome contains virulence factors crucial for establishing infection, modulating host immunity, and promoting parasite survival within macrophages. Previous proteomic studies have characterised secreted proteins across multiple species, identifying a conserved core secretome predominantly released via extracellular vesicles through non-classical pathways. However, large portions of the predicted proteome remain experimentally unvalidated. Materials and Methods To uncover overlooked proteins, we conducted a comprehensive proteogenomic analysis using publicly available mass spectrometry datasets. Custom six-frame translated genome databases were generated for seven reference Leishmania species, enabling the identification of genome search-specific peptides. This approach helps to detect the novel secreted proteins that have been missed in previous secretome researches together with low-abundance features, hypothetical proteins and the proteins that are from the canonical translation procedures. Results This proteogenomic study identified 469 secreted proteins in the seven Leishmania species that were not reported previously. The proteins were significantly explored and validated in the Leishmania proteome and disclose a higher level of functional diversity. The visceralising species were abundant in ATP-binding proteins and oxidoreductases, while the cutaneous species were represented with proteasome-rich secretome profiles. Discussion The identification of these further secreted proteins highlights the boundaries of previous studies and emphasises the higher resolution provided by the proteogenomic approaches. The species-specific categorical and functional differences explored in this study can contribute to diverse tissue tropisms, host-pathogen interactions and the clinical indicators of leishmaniasis. The newly identified proteins in this study likely include previously unidentified virulence factors with potential significance for parasite variations, pathogenesis, and therapeutic interference. Conclusion This study reveals that the proteogenomic analysis is a prevailing approach for exploring the hidden secretome of Leishmania species. These findings advance the understanding of parasite evolution and biology by identifying 469 novel secreted proteins and detecting the significant species-specific functional diversity; also highlight potential indicators for future functional research and therapeutic interventions. Leishmania Secretome Proteogenomic Mass spectrometry Novel protein-coding regions Comparative proteomics Figures Figure 1 Figure 2 Figure 3 Introduction Leishmania , which is a kinetoplastid protozoan parasite, is the causative agent of a wide range of infectious diseases mentioned to as leishmaniasis. This signifies one of the most significant neglected tropical diseases worldwide, infecting annually around 12 million people and threatening over 1 billion individuals in endemic regions (Alvar et al., 2012 ). This disease composite is caused by the obligate intracellular protozoan parasites belonging to the genus Leishmania , transmitted to mammalian hosts by the bite of the infected female Phlebotomine sand flies (WHO, 2023 ). The clinical manifestations of leishmaniasis are strangely diverse, ranging from cutaneous lesions to life-threatening visceral disease, depending on the infecting species and complex host-parasite interactions (Burza et al., 2018 ). Presently, 20 different Leishmania species are known as human pathogens, identified into three major subgenera: Leishmania ( Leishmania ), Leishmania ( Viannia ), and Sauroleishmania , based on the developmental features within the sand fly vector and phylogenetic relationships (Akhoundi et al., 2016 ). The heterogeneity in clinical demonstrations reflects not only species-specific virulence factors but also differences in tissue tropism, immune evasion mechanisms, and metabolic adaptations (Peacock et al., 2007 ; Rogers et al., 2011). Cutaneous leishmaniasis (CL), the most common form, is caused by species such as Leishmania (L.) major , Leishmania (L.) tropica , Leishmania (L.) mexicana , Leishmania (L.) amazonensis , and Leishmania (V.) braziliensis , resulting in skin lesions that can lead to permanent scarring and social stigmatisation (Akhoundi et al., 2016 ). Visceral leishmaniasis (VL) is caused by Leishmania (L.) donovani and Leishmania (L.) infantum , representing the most severe form, with parasites disseminating to the liver, spleen, and bone marrow, proving fatal if left untreated (Lypaczewski et al., 2018 ; Zijlstra, 2016 ). Mucocutaneous leishmaniasis (MCL), mostly associated with L. (V.) braziliensis infection, affects the mucous membranes of the nose, mouth, and throat, causing severe tissue destruction and functional damage (Burza et al., 2018 ). The first Leishmania genome sequence was published in 2005 (Ivens et al., 2005 ). Comparative genomic analyses have represented an unexpectedly high level of genetic conservation among species, with over 7,000 genes shared across the genus (Peacock et al., 2007 ). Despite this distinguished genomic similarity, important variation exists in chromosome ploidy, gene copy number, and chromosomal rearrangements, which contribute to phenotypic diversity and clinical outcomes (Rogers et al., 2011; Teixeira et al., 2012 ). Interestingly, the inadequate variation observed at the transcriptional level between different life cycle stages, promastigotes in the insect vector and amastigotes in mammalian hosts, proposes that post-transcriptional and post-translational regulatory mechanisms play predominant roles in Leishmania differentiation, virulence, and pathogenesis. This unusual gene regulation model, distinctive of trypanosomatids, highlights the key importance of proteomic approaches to understand the molecular basis of Leishmania biology and disease manifestation (Alcolea et al., 2014 ; Cohen-Freue et al., 2007 ; Leifso et al., 2007 ; Pawar et al., 2012 ; Rochette et al., 2009 ; Rosenzweig et al., 2008 ). Proteomics has developed as an essential tool for explaining the functional biology of Leishmania parasites, providing insights into protein expression profiles that cannot be inferred from genomic or transcriptomic data alone (de Jesus et al., 2014 ; Tsigankov et al., 2012 ). Previously, the relative proteomic research between promastigote and amastigote forms represented the stage-specific protein expression profiles in several species, including L. (L.) infantum (El Fakhry et al., 2002 ), L. (L.) donovani (Bentel et al., 2003 ; Pawar et al., 2012 ), L. (L.) mexicana (Nugent et al., 2004 )d (V.) panamensis (Walker et al., 2006 ). Moreover, the comparison between cutaneous and visceral clinical isolates at the proteomic level has identified protein signatures linked with tissue tropism and disease severity, with visceral strains showing enhanced expression of proteins involved in translation, biosynthetic processes, antioxidant defence, and cell signalling (McCall et al., 2015 ). The Leishmania secretome is the full set of proteins released into the extracellular environment, which is essential to parasite survival and pathogenesis. These excreted-secreted proteins help begin infection, control host immunity, and support survival within macrophage phagolysosomes (Corrales et al., 2010 ; Lambertz et al., 2012 ; Markikou-Ouni et al., 2015 ; Rosa et al., 2005 ; J. M. Silverman et al., 2010 ; J. M. Silverman et al., 2011 ). This secreted “molecular arsenal” includes virulence factors that suppress host signalling, inhibit macrophage activation, and initiate an anti-inflammatory response (Lemesre et al., 2005 ; Tonui et al., 2004 ). Proteomic profiling of the secretome from various species, including L. (L.) donovani (Maxwell et al., 2008 ), L. (L.) mexicana (Hassani et al., 2011 ), L. (V.) braziliensis (Cuervo et al., 2009 )d (L.) major (Atayde et al., 2015 ), has verified that the primary secretion mechanism involves extracellular vesicles, particularly exosomes and microvesicles, which are important for parasite virulence and host-pathogen communication (Atayde et al., 2016 ; J. M. Silverman et al., 2011 ). The small fraction of secreted proteins expressed classical N-terminal signal peptides, with the common being released through unusual, non-classical secretory pathways (J. M. Silverman et al., 2010 ). Proteomic analysis of promastigote secretomes from seven Leishmania species shows that nearly one-third of secreted proteins form a conserved core set, reflecting shared mechanisms of host adaptation across diverse lineages (Pissarra et al., 2022 ). The analysis of the gene ontology designates that most secreted proteins have catalytic functions crucial for host interaction, and bioinformatic predictions represent that over 42% are exported via non-classical secretion pathways in all species (Pissarra et al., 2022 ). However, despite advances in Leishmania proteomics, a large percentage of the predicted proteome remains experimentally unvalidated and is still annotated as hypothetical (Ivens et al., 2005 ). This “hidden proteome” may represent novel virulence factors, stage-specific regulators, metabolic enzymes, immunomodulators, and proteins arising from non-canonical translation (alternative ORFs, uORFs, smORFs) that escape detection due to low abundance and unusual properties (Castellana et al., 2008 ; Nesvizhskii, 2014 ). The exploration of these uncharacterised proteins in the Leishmania secretome, including several limited to human-pathogenic species, highlights their potential roles in virulence (Pissarra et al., 2022 ). Proteogenomic approaches in protozoan parasites have generated insights into genome annotation refinement and the discovery of novel protein-coding genes. In L. donovani , proteogenomics has confirmed predicted genes, identified new ORFs, and refined gene models (Nirujogi et al., 2014 ; Pawar et al., 2012 ). Similar analyses in L. major and L. braziliensis have uncovered previously unannotated coding sequences, corrected incorrect predictions, and identified novel proteins important for parasite adaptation and biology (Pawar et al., 2014 , 2017 ; Shenoy and Chowdhury et al., 2025 ). These findings underscore the appropriateness of proteogenomics for trypanosomatids, whose compressed genomes, polycistronic transcription, and extensive post-transcriptional regulation complicate conventional gene prediction. In this study, we used a comprehensive proteogenomic approach to identify and characterise the hidden proteome of Leishmania parasites systematically. To accomplish this, we utilised publicly available mass spectrometry data and searched it against a custom-generated six-frame translated genome database of seven Leishmania species reference strains. Our current study identified 693 novel secreted proteins that were missed in the previous large-scale secretome study of seven Leishmania species. Our findings significantly expand the experimentally validated Leishmania proteome, reveal novel potential virulence factors and drug targets, and provide insights into species-specific adaptations underlying clinical diversity. Overall, these results emphasise the value of GSSP-informed proteogenomics in uncovering novel hidden secreted proteins in Leishmania parasites. Materials and Methods 2.1 Generation of protein database and mass spectrometry data analysis The whole genome sequence fasta files (Version 68) for the following seven Leishmania species ( L. amazonensis MHOM/BR/71973/M2269, L. braziliensis MHOM/BR/75/M2904, Leishmania donovani CL-SL, L. infantum JPCM5, L. major strain Friedlin, Leishmania tarentolae Parrot-TarII, and L. tropica L590) were downloaded from TriTrypDB ( https://tritrypdb.org/tritrypdb/app/ ). Using in-house Python scripts, a custom-built six-frame translated genome database was generated, with a minimum length of each translated protein entry being 10 amino acids or above. An unbiased approach was used to generate the six-frame translated protein database as previously described (Nirujogi et al., 2014 ; Pawar et al., 2012 ). The publicly available mass spectrometry data (PXD023228) that was previously published by another group was downloaded from the PRIDE proteomics server (Pissarra et al., 2022 ). 2.2 Proteogenomic analysis : The Leishmania species secretome mass spectrometry data were searched against the custom six-frame translated seven Leishmania species genome databases. The database-dependent searches were analysed using the MaxQuant environment and Andromeda. The following search parameters were applied: trypsin digestion specified as the proteolytic enzymes, allowing up to one missed cleavage. Peptide mass tolerance was set to 20 ppm, and fragment mass tolerance was set to 0.1 Da. Carbamidomethylation of cysteine was designated as a fixed modification, while oxidation of methionine and acetylation of protein N-termini were included as variable modifications. Peptide data from MaxQuant searches were filtered using a 1% false discovery rate (FDR) threshold. The resulting unique peptides identified through both search algorithms, following database-dependent searches, were subsequently used for downstream analysis (Shenoy and Chowdhury et al., 2025 ). The total unique peptides identified from each Leishmania species secretome searches were initially mapped to the seven Leishmania species-specific protein database (version 68) downloaded from TriTrypDB. Those peptides that mapped to the respective Leishmania species protein database were discarded. While the peptides that did not map to the respective Leishmania species protein database but mapped to the six-frame translated genome database were referred to as genome search-specific peptides (GSSPs) and were further used for analysis. We defined genome search-specific peptides (GSSPs) through mass spectrometry (MS) data analysis that maps specifically to a translated genome database but is not present in the currently annotated protein databases. Hence, these peptides represent experimental evidence of novel or unannotated protein-coding regions within a genome, or indicate inaccuracies in existing genome annotations. These GSSPs were subsequently mapped to the genome to identify novel coding regions and to refine existing gene models, based on their genomic coordinates relative to current annotations (Shenoy and Chowdhury et al., 2025 ). 2.3 Bioinformatics analysis: The GSSPs that mapped to the six-frame translated genome database of respective Leishmania species were further analysed by undertaking BLAST analysis (protein) using UniProt and TriTrypDB databases to identify the genomic region to which these GSSPs map and the conserved orthologous proteins in other related Leishmania in which these GSSPs map. Using this approach, we could categorise our proteogenomic identifications into novel secreted proteins previously not identified in the seven Leishmania species. An overview of the proteogenomic workflow used in this study is shown in Fig. 1 . Results 3.1. Overall Composition of the Secretome Across Leishmania Species The analysis of the seven Leishmania species secretome MS/MS datasets yielded 7,146 unique peptides following searches against the species-specific six-frame translated genome databases. Peptides mapping to the annotated protein databases were excluded, and the remaining unmapped peptides were classified as genome search-specific peptides (GSSPs), resulting in 693 unique GSSPs. Subsequent genomic mapping of these GSSPs enabled the identification of 469 novel open reading frames (ORFs) across the seven species. These ORFs represent previously unannotated, hidden secreted proteins that were not captured in existing genome annotations, thereby expanding the catalogue of secreted proteins in Leishmania . An overview of the proteogenomic workflow used in this study is shown in Fig. 1 . Comparative analysis of the promastigote secretomes of L. amazonensis, L. braziliensis, L. donovani, L. infantum, L. major, L. tarentolae , and L. tropica revealed substantial qualitative and quantitative variations across species, extending well beyond what was previously reported in Pissarra et al. (Pissarra et al., 2022 ). Although Pissarra and colleagues identified a conserved core secretome comprising 306 proteins, our reanalysis of the dataset demonstrated that each species possesses a wider range and more functionally diverse secretome than earlier recognised. Several additional secreted proteins detected in the present proteogenomics study were not missed in Pissarra’s reported dataset, suggesting that prior analyses underestimated species-specific and niche-dependent secreted proteins. This inconsistency is likely due to differences in peptide identification thresholds, protein database versions, annotation pipelines, and species-specific proteomic depth. The extended number of secreted proteins identified in this study highlights the complication of secretome biology across pathogenic and non-pathogenic Leishmania species. The overlap and species-specific indicates to the secretome reveals a wide range of diversity, highlighting the heterogeneity of protein secretion across the genus and pathways that may underlie species-specific virulence and clinical manifestations, as represented in Fig. 2 . Further comparison with previously reported secretomes (Pissarra et al., 2022 ) is shown in Fig. 3 . 3.2. Molecular Function Variability Among Species The catalytic and binding features of the most predominant molecular functional categories have been identified across all species, which are in line with the previous studies; though this dataset allows a more granular understanding of functional specialisation. In L. donovani , the distinct abundance of the ATP and nucleotide-binding and helicase-related proteins was identified. These functions align with its visceral disease phenotype and support enhanced intracellular survival strategies involving nucleic-acid modulation, metabolic flexibility, and host immune evasion. Although Pissarra et al. had noted nucleic-acid binding enrichment in visceralising species, the recent findings extended this range by highlighting additional transport-associated enzymes and regulatory proteins involved in RNA processing. In L. infantum , ion-binding activities, particularly Zn-binding oxidoreductases and structural molecule categories, were more abundant than previously described. These findings emphasise the importance of redox regulation, ion homeostasis, and metabolic control mechanisms in visceral infection biology. In contrast, L. braziliensis revealed a uniquely protease-rich secretome dominated by serine-type endopeptidase and extracellular matrix-modifying enzymes, reflecting its capacity to drive tissue destruction and mucosal pathology. These proteolytic signatures extend beyond what Pissarra et al. reported and reveal additional peptidases previously unrecognised. The species-wise distributions of molecular functions and biological processes are shown in Supplementary Figs. 1 and 2 , where bar plots represent the variation in functional categories across the six Leishmania secretomes. The distinguishing enzymatic profiles were also detected in L. amazonensis , which showed increased levels of lipid-binding proteins, lyases, and isomerases. This pattern supports its dependence on lipid-based metabolic pathways and membrane remodelling mechanisms, characteristic of diffuse cutaneous leishmaniasis. L. tropica presented molecular functional diversity but included several enzymes connected with redox regulation, signal transduction, and protein refolding, which may reflect adaptive responses to environmental stress encountered within the cutaneous niche. Surprisingly, L. tarentolae shared several functional categories traditionally associated with pathogenic species, including structural molecule activities, biosynthetic enzymes, and redox regulators. This overlap suggests conservation of key physiological functions even in non-pathogenic species and implies that some secreted pathways may serve essential biological roles beyond virulence. 3.3. Species-Specific Differences in Biological Processes Biological process analysis discovered both shared and species-specific features across the Leishmania secretomes. Core biological processes, including metabolic processes, cellular regulation, and responses to environmental stimuli, were conserved across all species, consistent with their shared need to resist oxidative stress, adapt to nutrient fluctuations, and maintain homeostasis in both vector and host environments. However, species-level determination highlighted several previously unreported patterns. In the visceralising species ( L. donovani and L. infantum ), proteins involved in metabolism, RNA processing, stress adaptation, and macromolecule biosynthesis were more dominant than in cutaneous species. These processes likely support the metabolic plasticity required for survival in visceral organs, particularly within macrophages, where parasites encounter high oxidative pressure and nutrient limitation. In L. braziliensis , biological processes associated with proteolysis and extracellular matrix organisation were dominant, reflecting its pathogenic role in mucocutaneous tissue destruction. L. amazonensis exhibited enrichment in lipid metabolic processes, fatty acid turnover, and vesicle-mediated transport, consistent with its unique pathology characterised by parasitophorous vacuoles enriched in host lipids. L. tropica was characterised by enhanced stress-response pathways, including protein refolding and cellular response mechanisms, indicating heightened sensitivity to environmental fluctuations. Meanwhile, L. tarentolae showed surprisingly high levels of biological processes associated with cellular homeostasis and biosynthesis, suggesting evolutionary conservation of core survival mechanisms regardless of pathogenicity. 3.4. Highly Expressed Functional Groups Relevant to Leishmania Biology Across all species, the secretome was dominated by several highly expressed protein groups central to Leishmania survival and virulence. These included heat-shock proteins such as HSP70, HSP83, and HSP60, which are involved in thermotolerance, immune modulation, and the transition between vector and mammalian environments. Protein disulfide isomerases, peroxiredoxins, and trypanothione-dependent redox enzymes were also abundant, consistent with their established roles in antioxidant defence and parasite resilience in oxidative environments. Tubulins, actins, and associated motor proteins were frequently detected, supporting their role in vesicle-mediated secretion and host-parasite interactions. Moreover, numerous metabolic enzymes known to exert moonlighting functions, such as glycolytic enzymes and dehydrogenases at high abundance, underscoring their dual roles in both metabolism and immunomodulation. Compared with prior reports, these proteins were detected with greater species-specific resolution, revealing new insights into their differential contributions to parasite pathogenicity. A summary of representative functionally characterised proteins is provided in Table 1 . Table 1 Key functionally characterised proteins selected across six Leishmania species and their predicted biological roles (full list is presented in the supplementary file). This table represents the key proteins from each Leishmania species analysed in this study ( L. braziliensis , L. donovani , L. infantum , L. amazonensis , L. tarentolae , and L. tropica ). For each protein, the UniProt accession number, corresponding gene identifier, functional description, and experimentally supported or literature-based biological role are provided. The listed proteins represent diverse molecular functions, including proteases, metabolic enzymes, mitochondrial translocases, ubiquitination machinery, antioxidant defence proteins, and RNA-binding regulators, highlighting essential processes such as parasite survival, metabolism, stress response, differentiation, host–pathogen interactions, and virulence. The complete list of identified proteins is provided in the supplementary data. Sl No. Organisms Accession number Gene Description Biological role 1. L. braziliensis A0A3S7WZY5 LdCL_260021200 Metallopeptidase belonging to the thimet oligopeptidase (TOP) family, a zinc-dependent endopeptidase. TOP enzymes are involved in peptide processing, protein turnover and regulation of intracellular signalling peptides (Besteiro et al., 2007 ). In trypanosomatids, TOP-like enzymes contribute to parasite survival, proteolysis, stress response, and may modulate host–parasite interactions (Sundar & Singh, 2018 ). 2. A0A640KD91 LtaPh_1412600 Core component of the TIM23 mitochondrial translocase complex, which imports preproteins into the inner mitochondrial membrane. TIM50 is essential for mitochondrial protein import, organelle biogenesis, and parasite viability (Roberts et al., 2022 ). In kinetoplastids, TIM complex proteins are critical due to their single, specialised mitochondrion (kinetoplast) (Mani et al., 2015 ). 3. L. donovani A0A3S7X395 LdCL_300014700 Adenosine kinase (AK). Catalyses the phosphorylation of adenosine to AMP, part of the purine-nucleoside salvage pathway critical for parasite survival. Has been biochemically purified and characterised in L. donovani , showing unique kinetic and regulatory properties (Boitz et al., 2012 ) 4. A0A3S7WT53 LdCL_140017800 Kinesin K39, putative. Member of the kinesin-motor protein family; the “K39” repeat region is antigenic and widely used in serodiagnosis of visceral leishmaniasis. It likely plays a role in microtubule-based motility or cytoskeletal organisation in the parasite (Gerald et al., 2007 ). 5. L. infantum A4I1S9 LINJ_25_2400 RING-type E3 ubiquitin transferase. RING-type E3 ubiquitin ligases are key regulators of the ubiquitin–proteasome system (UPS) in Leishmania , directing the ubiquitination and turnover of specific proteins. They contribute to promastigote–amastigote differentiation, stress adaptation, and intracellular survival within macrophages. UPS components, including RING E3 ligases, are upregulated during oxidative and heat stress and help maintain protein quality. Studies in kinetoplastids also show that RING-type E3 ligases are required for proper cell-cycle progression and DNA replication. E3 ligase consider as potential drug target as the UPS-associated proteins effect the parasite virulence and inhibit the pathway lethal to Leishmania (Bulatov et al., 2018 ; Burge et al., 2020 ). 6. A4HX10 LINJ_16_1170 Tyrosyl or methionyl-tRNA synthetase-like protein. Tyrosol- and methionyl- tRNA synthetases in aminoscyl-tRNA synthetases (aaRS), are useful enzymes in Leishmania that help to catalyse the amino acid attachment to their corresponding tRNAs throughout protein synthesis. Mostly, these enzymes in kinetoplastids indicate the unique structural adaptations that differ from mammalian homologues, making them crucial for the parasite's survival and potential drug target (Burge et al., 2020 ). Several aaRS, including TyrRS and MetRS in L. donovani , are essential for mitochondrial and cytosolic translation, parasite growth, and survival under stress (Abhishek et al., 2017 ). In Leishmania , the inhibition of the MetRS or TyrRS leads to reduced protein synthesis and condensed parasite infectivity, supporting their role as key components of the translational machinery (Nasim & Qureshi, 2023 ). 7. L. amazonensis A0A640KNR1 LtaPh_2521300 Caspase family p20 domain-containing protein. Although classical caspases are absent in protozoa, Leishmania possesses metacaspases, which contain a p20 catalytic domain and function as cysteine proteases. The identified proteins are involved in the programmed cell death-like pathways, stress response, and the progression of the cell cycle (Vandana et al., 2019). For the survival of the parasite under oxidative stress, the role of the metacaspase with the p20 domain is predominant and is also essential for the differentiation between the promastigote and amastigote phases (Aghaei et al., 2025 ). The metacaspase gene disruption can cause defects in autophagy, decreased infectivity and impaired parasite proliferation, which underscore the key role in cellular homeostasis (Aghaei et al., 2025 ). 8. E9ARK7 LMXM_18_0670 Citrate synthase. This is an important mitochondrial enzyme that catalyses the primary step of the tricarboxylic acid (TCA) cycle by shrinking oxaloacetate to form citrate. Citrate synthase is important for carbon metabolism, ATP generation, and biosynthetic pathways essential for parasite growth (Ranjan et al., 2024 ). Previous research on kinetoplastid parasites indicates that citrate synthase is important for continuing mitochondrial function and metabolic flexibility throughout the transition between promastigote and amastigote stages (Marchese et al., 2018 ). Reduction of the citrate synthase expression leads to impaired respiration and reduced parasite viability, underscoring its role for survival in nutrient-limited environments within the host (Marchese et al., 2018 ). 9. L. tarentolae A0A640K9H5 LtaPh_0808151 Cathepsin L-like protease. Cathepsin L-like proteases, also known as cysteine proteases (CPA/CPB), are major virulence factors in L. infantum . These enzymes facilitate parasite survival by degrading host proteins, modulating macrophage signalling, and aiding immune evasion. This protein helps in the entry of the parasite, nutrient acquisition and the reduction of the antigen expression, which supports the intracellular replication. However, these proteins are also required for the parasite surface molecules processing and were explored as potential vaccine and drug targets due to their roles in pathogenicity (Silva-Almeida et al., 2012 ). 10. A0A640KBV6 LtaPh_1009541 Leishmanolysin. Leishmanolysin, commonly known as GP63, is a zinc-dependent metalloprotease mostly expressed on the surface of L. infantum promastigotes. GP63 is an important virulence factor that protects the parasite from complement-mediated lysis and controls host immune responses by slicing signalling molecules. Additionally, it destroys the extracellular matrix leads to parasite dissemination and helping the survival of the parasite in the macrophages by impairing the phagolysosomal maturation. Due to its high immunogenicity and wide functional roles, GP63 is broadly explored as a potential drug target in visceral leishmaniasis (Guay-Vincent et al., 2022 ; Isnard et al., 2012 ). 11. L. tropica A0A3S7WRN3 LdCL_110015200 K Homology domain-containing protein. K Homology (KH) domain-containing proteins function primarily as RNA-binding regulators that regulate mRNA stability, translation, and stage-specific gene expression. Transcriptional control is not available in Leishmania ; the KH-domain proteins play an important role in post-transcriptional regulation and serve the parasite to survive between the different stages of the life cycle. These proteins help to bind the U-rich elements, and they relate to RNA-processing complexes that affect the stress response, surface antigens, as well as virulence linked transcript. This domain also helps in regulating the amastigote survival pathways, making them potential candidates for understanding parasite differentiation and potential drug targets (Ferreira et al., 2020 ; Haskell & Zinovyeva, 2021 ). 12. A4I7Z8 SODB2 LINJ_32_1920 Superoxide dismutase. In Leishmania , the superoxide dismutase plays an important role as an antioxidant enzyme, which converts the superoxide radicals into hydrogen peroxide, and helps in protecting the parasite from oxidative stress. Leishmania relies heavily on Fe-SODs, producing these enzymes structurally and functionally distinct from host SODs. SODs are essential for survival within the macrophages, contribute to virulence, and support resistance to reactive oxygen species (ROS). Inhibition of SODs has been reported to reduce parasite viability, underscoring them as promising drug targets (Ghosh et al., 2003 ; Roy et al., 2025 ). Discussion The exploratory study from this comparative secretome analysis provides a more comprehensive understanding of protein secretion across pathogenic and non-pathogenic Leishmania species than what had been reported previously by Pissarra et al. (Pissarra et al., 2022 ). While previous work was involved in defining a conserved core secretome, our findings indicate that Leishmania secretomes show far wider functional diversity and species specificity. The detection of additional catalytic, lipid-associated, nucleic-acid-binding, and proteolytic proteins suggests a more detailed interplay between parasite biology, environmental niche, and tissue tropism than previously recognised. The proteins that were not identified in previous datasets likely reflect differences in annotation pipelines, search algorithms, or mass-spectrometry depth; however, their identification here highlights the importance of continuously updating proteomic databases and analytical context to better reflect biological complexity. Furthermore, one methodological limitation in the study by Pissarra et al. ( 2022 ) concerns their use of an NNscore > 0.5 in SecretomeP to predict non-classical secretion. This low threshold is known to produce a high false-positive rate in kinetoplastids, leading to less accurate identification of proteins within the secretome (Pissarra et al., 2022 ). Consequently, their reported “core secretome” may include cytosolic proteins incorrectly classified as secreted, masking species-specific differences that became clearer in our more stringent re-analysis. The enrichment of ATP-binding proteins, helicases, and oxidoreductases in L. donovani and L. infantum is in line with the metabolic reprogramming and stress adaptation required for visceral infection. These findings align with previous studies demonstrating that RNA-binding proteins, redox regulators, and ATP-dependent chaperones are essential for parasite survival in macrophages and contribute to virulence (Fonseca Pires et al., 2014 ; Leclercq et al., 2013 ). Similarly, the common protease activity observed in L. braziliensis has support in the literature, as serine proteases and metalloproteases are known to drive tissue degradation, extracellular matrix breakdown, and severe mucocutaneous pathology (M Guedes et al., 2007 ; Souza-Melo et al., 2023 ; Zabala-Peñafiel et al., 2021 ). These proteases, which were understated in earlier comparative analyses, appear to be more diverse and abundant than previously thought. The lipid-centric metabolic profile of L. amazonensis identified here repeats findings that this species deploys host lipids and modulates membrane structure to facilitate intracellular replication (De Cicco et al., 2012 ; Gdovinova & Descoteaux, 2025 ). The present study enhances this understanding by demonstrating that lipid-binding and lipid-metabolising enzymes are major secreted components, emphasising their role in disease progression. Likewise, the stress-response dominated secretome of L. tropica supports its known adaptability to diverse cutaneous environments and fluctuating immune pressures (Pissarra et al., 2022 ). These species-specific functional enrichments help clarify how differences in biological processes shape distinct disease indicators. The finding that L. tarentolae exhibits significant overlap with pathogenic species in several molecular and biological functions challenges the conventional view that non-pathogenic species possess fundamentally different secretome architectures. Instead, the presence of structural, biosynthetic, and redox-related proteins supports the hypothesis that non-pathogenic species retain ancestral pathways essential for survival but lack specific virulence determinants. Earlier genomic studies had proposed that L. tarentolae lost only a subset of virulence genes rather than experiencing widespread functional degeneration (Andrade et al., 2024 ; Novo et al., 2015 ). The present secretome analysis supports this model, demonstrating that many secretory features associated with pathogenicity are conserved across the genus. The expanded diversity of proteins uncovered in this study has significant implications for developing species-specific diagnostic tools, biomarkers, and vaccine targets. Highly expressed heat-shock proteins, redox enzymes, lipid-metabolic proteins, and proteases identified here have been previously implicated as immunomodulators or potential vaccine candidates (Chowdhury et al., 2025 ; Rostami & Khamesipour, 2021 ). The newly identified species-specific proteins, particularly in L. amazonensis and L. braziliensis , may serve as more accurate biomarkers for differential diagnosis or as immunogenic components in species-targeted vaccine strategies. Since secreted proteins are often the first point of contact with the host immune system, a more comprehensive understanding of species-specific secretomes can directly inform translational research aimed at improving disease management. Conclusion This study provides a comprehensive and refined comparative analysis of the promastigote secretomes of six Leishmania species, L. braziliensis, L. donovani, L. infantum, L. amazonensis, L. tropica , and L. tarentolae , revealing a broader and more functionally diverse landscape of secreted proteins than previously documented, particularly when compared with the core secretome described by Pissarra et al. ( 2022 ). Through re-evaluation of the datasets, the present work identified numerous additional proteins and functional categories that had not been previously recognised, highlighting substantial species-specific variation. Visceralising species ( L. donovani and L. infantum ) displayed strong enrichment in ATP-binding and nucleotide-binding proteins, oxidoreductases, and stress-regulatory pathways, reflecting the metabolic flexibility and intracellular survival strategies required for their dissemination into visceral organs. Cutaneous species such as L. braziliensis were characterised by a protease-rich profile, including serine-type endopeptidases and extracellular matrix–modifying enzymes directly related to tissue destruction and mucocutaneous pathology. L. amazonensis exhibited a pronounced enrichment in lipid-binding and lipid-metabolising enzymes, supporting its reliance on lipid remodelling mechanisms that drive its diffuse cutaneous disease phenotype. Meanwhile, L. tropica showed elevated expression of stress-response and signalling-related proteins, consistent with its capacity to persist in fluctuating cutaneous environments. Unexpectedly, the non-pathogenic L. tarentolae shared several functional features with pathogenic species, including structural, biosynthetic, and redox-related proteins, indicating that evolutionarily conserved secretory pathways remain intact even in species lacking human pathogenicity. Overall, this study's findings demonstrate that Leishmania secretomes are highly species-specific and significantly more complex than previously reported. By identifying additional proteins across diverse functional categories, ranging from binding and catalytic activities to proteolytic, lipid-metabolic, and redox-regulatory functions, this work emphasises that a core-proteome approach alone cannot fully capture the biological diversity present within the genus. These species-specific signatures not only enhance our understanding of the molecular basis of distinct clinical manifestations but also provide a valuable resource for identifying new diagnostic markers, therapeutic targets, and vaccine candidates. The expanded secretome profiles obtained here underscore the importance of species-level proteomic analysis for understanding host-parasite interactions and highlight the need for continued refinement of proteomic pipelines and annotation methods. Collectively, this combined summary and conclusion strengthen the increasing recognition that Leishmania biology is shaped by both conserved evolutionary mechanisms and finely tuned species-specific adaptations, offering new avenues for translational research aimed at improving leishmaniasis diagnosis, treatment, and control. Declarations Conflict of interest No conflict of interest. Acknowledgement We would like to acknowledge the Institute of Bioinformatics, Bangalore, India, for undertaking this study and analysis. Availability of data and materials All data supporting the findings of this study are included in the article and its supplementary information files. Funding source The authors declare that this study was conducted without any financial support. Ethics approval The Institutional Ethics Committee has confirmed that no ethical approval is required for this study. References Abhishek, K., Sardar, A. H., Das, S., Kumar, A., Ghosh, A. K., Singh, R., Saini, S., Mandal, A., Verma, S., Kumar, A., Purkait, B., Dikhit, M. R., & Das, P. (2017). Phosphorylation of Translation Initiation Factor 2-Alpha in Leishmania donovani under Stress Is Necessary for Parasite Survival. Molecular and Cellular Biology , 37 (1). https://doi.org/10.1128/mcb.00344-16 Aghaei, M., Aghaei, S., Shahmoradi, Z., & Hejazi, S. H. (2025). 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Genetics and molecular biology , 35 , 1-17. https://doi.org/10.1590/S1415-47572012005000008 Tonui, W. K., Mejia, J. S., Hochberg, L., Mbow, M. L., Ryan, J. R., Chan, A. S. T., Martin, S. K., & Titus, R. G. (2004). Immunisation with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major . Infection and Immunity , 72 (10), 5654–5661. https://doi.org/10.1128/IAI.72.10.5654-5661.2004 Tsigankov, P., Gherardini, P. F., Helmer-Citterich, M., & Zilberstein, D. (2012). What has proteomics taught us about Leishmania development? Parasitology , 139 (9), 1146–1157. https://doi.org/10.1017/S0031182012000157 Vandana, Dixit, R., Tiwari, R., Katyal, A., & Pandey, K. C. (2019). Metacaspases: Potential Drug Target Against Protozoan Parasites. Frontiers in Pharmacology , 10 . https://doi.org/10.3389/fphar.2019.00790 Walker, J., Vasquez, J. J., Gomez, M. A., Drummelsmith, J., Burchmore, R., Girard, I., & Ouellette, M. (2006). Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes. Molecular and Biochemical Parasitology , 147 (1), 64–73. https://doi.org/10.1016/j.molbiopara.2006.01.008 WHO . (2023). Zabala-Peñafiel, A., Dias-Lopes, G., Cysne-Finkelstein, L., Conceição-Silva, F., Miranda, L. de F. C., Fagundes, A., Schubach, A. de O., Fernandes Pimentel, M. I., Souza-Silva, F., Machado, L. de A., & Alves, C. R. (2021). Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony. Scientific Reports , 11 (1). https://doi.org/10.1038/s41598-021-93665-z Zijlstra, E. E. (2016). The immunology of post-kala-azar dermal leishmaniasis (PKDL). In Parasites and Vectors (Vol. 9, Issue 1). BioMed Central Ltd. https://doi.org/10.1186/s13071-016-1721-0 Supplementary Files SupplementaryFigure1.pdf Graphicalabstact.pdf SupplementaryFigure2.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor invited by journal 28 Jan, 2026 Editor assigned by journal 01 Jan, 2026 First submitted to journal 29 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8472209","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584067330,"identity":"803ab772-5392-4c24-9f3d-ff3240ae60d3","order_by":0,"name":"Soumi Chowdhury","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Soumi","middleName":"","lastName":"Chowdhury","suffix":""},{"id":584067331,"identity":"8b275d99-c617-408d-b21b-d84eeb2f4b01","order_by":1,"name":"Karthick Vasudevan","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Karthick","middleName":"","lastName":"Vasudevan","suffix":""},{"id":584067332,"identity":"68358898-faa6-4bef-84d7-569426a3b05c","order_by":2,"name":"Shubhankar Pawar","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Shubhankar","middleName":"","lastName":"Pawar","suffix":""},{"id":584067333,"identity":"3b791ba7-7dc2-456b-a193-8650ef942281","order_by":3,"name":"Molieswar Jaikumar","email":"","orcid":"","institution":"CCMB: Centre for Cellular and Molecular Biology CSIR","correspondingAuthor":false,"prefix":"","firstName":"Molieswar","middleName":"","lastName":"Jaikumar","suffix":""},{"id":584067334,"identity":"57836511-d793-4b32-a9bf-e2384e34543a","order_by":4,"name":"Piyush Mohaptra","email":"","orcid":"","institution":"CCMB: Centre for Cellular and Molecular Biology CSIR","correspondingAuthor":false,"prefix":"","firstName":"Piyush","middleName":"","lastName":"Mohaptra","suffix":""},{"id":584067335,"identity":"10d5daa9-05e0-4a59-bb59-7ea2aa9a6142","order_by":5,"name":"Nitin Tupperwar","email":"","orcid":"","institution":"CCMB: Centre for Cellular and Molecular Biology CSIR","correspondingAuthor":false,"prefix":"","firstName":"Nitin","middleName":"","lastName":"Tupperwar","suffix":""},{"id":584067336,"identity":"5734d724-7f7c-429e-87fb-ea611c5c16e7","order_by":6,"name":"Harsh Pawar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYBACAyBmZqiQSGBjYGxgqACLARmEtZyBajkDEmIjRgtjG0MCmAfRQsBh5uxnH38unGeRx8fe3PbgQMW9aP75zY0ffzDY5Ms7YNdi2ZNuJj1zm0QxG8/BdoMDZ4pzZxxjbJbmYUiz3HgAh8MOpLEx826TSGwDIumPbQm5DccY25gZGA4bGOLwksH5Z8yfeedAtEgcBGqZD9TC+AOflhtpDNK8DUhaNgC1MPAAtcjj8L7BjWds0jzHwH5pkzhwJiF347FEoF8M0gwMcGk5n8b8maemLk++vf2ZxIGKhNx5h48//PijwsZAHofDcAEDUMiQpgUISLVlFIyCUTAKhi0AAAIjW1cCKdigAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":true,"prefix":"","firstName":"Harsh","middleName":"","lastName":"Pawar","suffix":""}],"badges":[],"createdAt":"2025-12-29 10:39:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8472209/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8472209/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101790690,"identity":"9296c9f1-4c7f-4099-9022-96ae0fcd8a57","added_by":"auto","created_at":"2026-02-03 16:06:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow for identifying hidden secreted proteins across seven \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species using proteogenomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the proteogenomic workflow used to analyse the secretomes of seven \u003cem\u003eLeishmania\u003c/em\u003especies (\u003cem\u003eL. amazonensis, L. braziliensis, L. donovani, L. infantum, L. major, L. tarentolae,\u003c/em\u003e and \u003cem\u003eL. tropica\u003c/em\u003e). Raw tandem mass spectrometry (MS/MS) data were searched against species-specific six-frame translated genomes to identify unique peptides, genome search–specific peptides (GSSPs), and novel open reading frames (ORFs). Peptides mapping exclusively to the six-frame translation database and not to the annotated protein database enabled the discovery of previously unannotated, “hidden” secreted proteins conserved or species-specific across the seven \u003cem\u003eLeishmania\u003c/em\u003e secretomes.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/0b47f28b144cdb4e2ca82691.jpeg"},{"id":101880562,"identity":"f4932d91-c170-4a0c-9797-d3978a8443ea","added_by":"auto","created_at":"2026-02-04 15:03:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverlap of secreted proteins identified across seven \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis Venn diagram illustrates the shared and species-specific secretome components identified in \u003cem\u003eL. amazonensis\u003c/em\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e, \u003cem\u003eL. donovani\u003c/em\u003e, \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. major\u003c/em\u003e, \u003cem\u003eL. tarentolae\u003c/em\u003e, and \u003cem\u003eL. tropica\u003c/em\u003e. Each coloured region represents the set of secreted proteins detected in a given species, while overlapping regions indicate proteins conserved among multiple species. The central intersection denotes proteins shared across all seven \u003cem\u003eLeishmania\u003c/em\u003especies, representing the conserved core secretome. Unique and non-overlapping regions highlight species-specific secreted proteins that may contribute to lineage-specific adaptations and virulence traits.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/9256d29004990d6f46706676.jpeg"},{"id":101790688,"identity":"6c7c4f2c-22a9-4ed1-ada9-71cc4962430c","added_by":"auto","created_at":"2026-02-03 16:06:51","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of Predicted Secretomes Between Pissarra et al. (2022) and Chowdhury et al. (2025) Across Six \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenn diagrams illustrating the overlap in predicted secreted proteins between Pissarra et al. (2022) and Chowdhury et al. (2025) for six \u003cem\u003eLeishmania\u003c/em\u003e species:\u003cbr\u003e\n(A) \u003cem\u003eL. amazonensis\u003c/em\u003e, (B) \u003cem\u003eL. braziliensis\u003c/em\u003e, (C) \u003cem\u003eL. donovani\u003c/em\u003e, (D) \u003cem\u003eL. infantum\u003c/em\u003e, (E) \u003cem\u003eL. tarentolae\u003c/em\u003e, and (F) \u003cem\u003eL. tropica\u003c/em\u003e.\u003cbr\u003e\nThe yellow circle illustrates the secreted proteins for each \u003cem\u003eLeishmania\u003c/em\u003especies in Pissarra et al. (2022), and the purple circle shows the predicted secreted proteins in Chowdhury et al. (2025). The central overlapping regions represent the identified proteins in both studies. However, no shared proteins have been identified (intersection = 0) in all species, though species-specific datasets of secreted protiens was indentified in both studies with distinguished variation in the number of unique proteins in each dataset. These findings underscore the methodological and threshold-based differences that produce distinct secretomes across studies.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/55821a953e0f52bc4140d10e.jpeg"},{"id":102398814,"identity":"e74f2877-34fb-4124-8fe0-3ed78289884c","added_by":"auto","created_at":"2026-02-11 10:29:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2060233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/64e9d855-c1b5-457c-acdc-635004f6c409.pdf"},{"id":101790691,"identity":"64802891-71b1-4477-87d4-ef2b868791f3","added_by":"auto","created_at":"2026-02-03 16:06:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":442256,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/54ae872b3ae6a6a607399eaf.pdf"},{"id":102397148,"identity":"8d914693-9d84-46a8-ba9d-e394ade59ad1","added_by":"auto","created_at":"2026-02-11 10:04:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1846034,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstact.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/c8cd13d2c68c7ab557137751.pdf"},{"id":101790694,"identity":"9e4b3430-d9c3-492a-9030-90a00a66d2ac","added_by":"auto","created_at":"2026-02-03 16:06:52","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":539086,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8472209/v1/2a52f1839665527dc2e88c77.pdf"}],"financialInterests":"","formattedTitle":"Mapping the Hidden Secretome in Leishmania Parasites Using a Proteogenomics Approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eLeishmania\u003c/em\u003e, which is a kinetoplastid protozoan parasite, is the causative agent of a wide range of infectious diseases mentioned to as leishmaniasis. This signifies one of the most significant neglected tropical diseases worldwide, infecting annually around 12\u0026nbsp;million people and threatening over 1\u0026nbsp;billion individuals in endemic regions (Alvar et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This disease composite is caused by the obligate intracellular protozoan parasites belonging to the genus \u003cem\u003eLeishmania\u003c/em\u003e, transmitted to mammalian hosts by the bite of the infected female Phlebotomine sand flies (WHO, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The clinical manifestations of leishmaniasis are strangely diverse, ranging from cutaneous lesions to life-threatening visceral disease, depending on the infecting species and complex host-parasite interactions (Burza et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Presently, 20 different \u003cem\u003eLeishmania\u003c/em\u003e species are known as human pathogens, identified into three major subgenera: \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eLeishmania\u003c/em\u003e), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e), and \u003cem\u003eSauroleishmania\u003c/em\u003e, based on the developmental features within the sand fly vector and phylogenetic relationships (Akhoundi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe heterogeneity in clinical demonstrations reflects not only species-specific virulence factors but also differences in tissue tropism, immune evasion mechanisms, and metabolic adaptations (Peacock et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rogers et al., 2011). Cutaneous leishmaniasis (CL), the most common form, is caused by species such as \u003cem\u003eLeishmania (L.) major\u003c/em\u003e, \u003cem\u003eLeishmania (L.) tropica\u003c/em\u003e, \u003cem\u003eLeishmania (L.) mexicana\u003c/em\u003e, \u003cem\u003eLeishmania (L.) amazonensis\u003c/em\u003e, and \u003cem\u003eLeishmania (V.) braziliensis\u003c/em\u003e, resulting in skin lesions that can lead to permanent scarring and social stigmatisation (Akhoundi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Visceral leishmaniasis (VL) is caused by \u003cem\u003eLeishmania (L.) donovani\u003c/em\u003e and \u003cem\u003eLeishmania (L.) infantum\u003c/em\u003e, representing the most severe form, with parasites disseminating to the liver, spleen, and bone marrow, proving fatal if left untreated (Lypaczewski et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zijlstra, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Mucocutaneous leishmaniasis (MCL), mostly associated with \u003cem\u003eL. (V.) braziliensis\u003c/em\u003e infection, affects the mucous membranes of the nose, mouth, and throat, causing severe tissue destruction and functional damage (Burza et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe first \u003cem\u003eLeishmania\u003c/em\u003e genome sequence was published in 2005 (Ivens et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Comparative genomic analyses have represented an unexpectedly high level of genetic conservation among species, with over 7,000 genes shared across the genus (Peacock et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Despite this distinguished genomic similarity, important variation exists in chromosome ploidy, gene copy number, and chromosomal rearrangements, which contribute to phenotypic diversity and clinical outcomes (Rogers et al., 2011; Teixeira et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Interestingly, the inadequate variation observed at the transcriptional level between different life cycle stages, promastigotes in the insect vector and amastigotes in mammalian hosts, proposes that post-transcriptional and post-translational regulatory mechanisms play predominant roles in \u003cem\u003eLeishmania\u003c/em\u003e differentiation, virulence, and pathogenesis. This unusual gene regulation model, distinctive of trypanosomatids, highlights the key importance of proteomic approaches to understand the molecular basis of \u003cem\u003eLeishmania\u003c/em\u003e biology and disease manifestation (Alcolea et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cohen-Freue et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Leifso et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rochette et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rosenzweig et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProteomics has developed as an essential tool for explaining the functional biology of \u003cem\u003eLeishmania\u003c/em\u003e parasites, providing insights into protein expression profiles that cannot be inferred from genomic or transcriptomic data alone (de Jesus et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tsigankov et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Previously, the relative proteomic research between promastigote and amastigote forms represented the stage-specific protein expression profiles in several species, including \u003cem\u003eL. (L.) infantum\u003c/em\u003e (El Fakhry et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), L. \u003cem\u003e(L.) donovani\u003c/em\u003e (Bentel et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), L. \u003cem\u003e(L.) mexicana\u003c/em\u003e (Nugent et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)d \u003cem\u003e(V.) panamensis\u003c/em\u003e (Walker et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Moreover, the comparison between cutaneous and visceral clinical isolates at the proteomic level has identified protein signatures linked with tissue tropism and disease severity, with visceral strains showing enhanced expression of proteins involved in translation, biosynthetic processes, antioxidant defence, and cell signalling (McCall et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLeishmania\u003c/em\u003e secretome is the full set of proteins released into the extracellular environment, which is essential to parasite survival and pathogenesis. These excreted-secreted proteins help begin infection, control host immunity, and support survival within macrophage phagolysosomes (Corrales et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lambertz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Markikou-Ouni et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rosa et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; J. M. Silverman et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; J. M. Silverman et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This secreted \u0026ldquo;molecular arsenal\u0026rdquo; includes virulence factors that suppress host signalling, inhibit macrophage activation, and initiate an anti-inflammatory response (Lemesre et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tonui et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Proteomic profiling of the secretome from various species, including \u003cem\u003eL. (L.) donovani\u003c/em\u003e (Maxwell et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), L. \u003cem\u003e(L.) mexicana\u003c/em\u003e (Hassani et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), L. \u003cem\u003e(V.) braziliensis\u003c/em\u003e (Cuervo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e)d \u003cem\u003e(L.) major\u003c/em\u003e (Atayde et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), has verified that the primary secretion mechanism involves extracellular vesicles, particularly exosomes and microvesicles, which are important for parasite virulence and host-pathogen communication (Atayde et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; J. M. Silverman et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The small fraction of secreted proteins expressed classical N-terminal signal peptides, with the common being released through unusual, non-classical secretory pathways (J. M. Silverman et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Proteomic analysis of promastigote secretomes from seven \u003cem\u003eLeishmania\u003c/em\u003e species shows that nearly one-third of secreted proteins form a conserved core set, reflecting shared mechanisms of host adaptation across diverse lineages (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The analysis of the gene ontology designates that most secreted proteins have catalytic functions crucial for host interaction, and bioinformatic predictions represent that over 42% are exported via non-classical secretion pathways in all species (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, despite advances in \u003cem\u003eLeishmania\u003c/em\u003e proteomics, a large percentage of the predicted proteome remains experimentally unvalidated and is still annotated as hypothetical (Ivens et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This \u0026ldquo;hidden proteome\u0026rdquo; may represent novel virulence factors, stage-specific regulators, metabolic enzymes, immunomodulators, and proteins arising from non-canonical translation (alternative ORFs, uORFs, smORFs) that escape detection due to low abundance and unusual properties (Castellana et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Nesvizhskii, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The exploration of these uncharacterised proteins in the \u003cem\u003eLeishmania\u003c/em\u003e secretome, including several limited to human-pathogenic species, highlights their potential roles in virulence (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProteogenomic approaches in protozoan parasites have generated insights into genome annotation refinement and the discovery of novel protein-coding genes. In \u003cem\u003eL. donovani\u003c/em\u003e, proteogenomics has confirmed predicted genes, identified new ORFs, and refined gene models (Nirujogi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similar analyses in \u003cem\u003eL. major\u003c/em\u003e and \u003cem\u003eL. braziliensis\u003c/em\u003e have uncovered previously unannotated coding sequences, corrected incorrect predictions, and identified novel proteins important for parasite adaptation and biology (Pawar et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shenoy and Chowdhury et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These findings underscore the appropriateness of proteogenomics for trypanosomatids, whose compressed genomes, polycistronic transcription, and extensive post-transcriptional regulation complicate conventional gene prediction.\u003c/p\u003e \u003cp\u003eIn this study, we used a comprehensive proteogenomic approach to identify and characterise the hidden proteome of \u003cem\u003eLeishmania\u003c/em\u003e parasites systematically. To accomplish this, we utilised publicly available mass spectrometry data and searched it against a custom-generated six-frame translated genome database of seven \u003cem\u003eLeishmania\u003c/em\u003e species reference strains. Our current study identified 693 novel secreted proteins that were missed in the previous large-scale secretome study of seven \u003cem\u003eLeishmania\u003c/em\u003e species. Our findings significantly expand the experimentally validated \u003cem\u003eLeishmania\u003c/em\u003e proteome, reveal novel potential virulence factors and drug targets, and provide insights into species-specific adaptations underlying clinical diversity. Overall, these results emphasise the value of GSSP-informed proteogenomics in uncovering novel hidden secreted proteins in \u003cem\u003eLeishmania\u003c/em\u003e parasites.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Generation of protein database and mass spectrometry data analysis\u003c/h2\u003e \u003cp\u003eThe whole genome sequence fasta files (Version 68) for the following seven \u003cem\u003eLeishmania\u003c/em\u003e species (\u003cem\u003eL. amazonensis\u003c/em\u003e MHOM/BR/71973/M2269, \u003cem\u003eL. braziliensis\u003c/em\u003e MHOM/BR/75/M2904, \u003cem\u003eLeishmania donovani\u003c/em\u003e CL-SL, \u003cem\u003eL. infantum\u003c/em\u003e JPCM5, \u003cem\u003eL. major\u003c/em\u003e strain Friedlin, \u003cem\u003eLeishmania tarentolae\u003c/em\u003e Parrot-TarII, and \u003cem\u003eL. tropica\u003c/em\u003e L590) were downloaded from TriTrypDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tritrypdb.org/tritrypdb/app/\u003c/span\u003e\u003cspan address=\"https://tritrypdb.org/tritrypdb/app/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Using in-house Python scripts, a custom-built six-frame translated genome database was generated, with a minimum length of each translated protein entry being 10 amino acids or above. An unbiased approach was used to generate the six-frame translated protein database as previously described (Nirujogi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The publicly available mass spectrometry data (PXD023228) that was previously published by another group was downloaded from the PRIDE proteomics server (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eProteogenomic analysis\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLeishmania\u003c/em\u003e species secretome mass spectrometry data were searched against the custom six-frame translated \u003cem\u003eseven Leishmania species\u003c/em\u003e genome databases. The database-dependent searches were analysed using the MaxQuant environment and Andromeda. The following search parameters were applied: trypsin digestion specified as the proteolytic enzymes, allowing up to one missed cleavage. Peptide mass tolerance was set to 20 ppm, and fragment mass tolerance was set to 0.1 Da. Carbamidomethylation of cysteine was designated as a fixed modification, while oxidation of methionine and acetylation of protein N-termini were included as variable modifications. Peptide data from MaxQuant searches were filtered using a 1% false discovery rate (FDR) threshold. The resulting unique peptides identified through both search algorithms, following database-dependent searches, were subsequently used for downstream analysis (Shenoy and Chowdhury et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe total unique peptides identified from each \u003cem\u003eLeishmania\u003c/em\u003e species secretome searches were initially mapped to the seven \u003cem\u003eLeishmania\u003c/em\u003e species-specific protein database (version 68) downloaded from TriTrypDB. Those peptides that mapped to the respective \u003cem\u003eLeishmania\u003c/em\u003e species protein database were discarded. While the peptides that did not map to the respective \u003cem\u003eLeishmania\u003c/em\u003e species protein database but mapped to the six-frame translated genome database were referred to as genome search-specific peptides (GSSPs) and were further used for analysis. We defined genome search-specific peptides (GSSPs) through mass spectrometry (MS) data analysis that maps specifically to a translated genome database but is not present in the currently annotated protein databases. Hence, these peptides represent experimental evidence of novel or unannotated protein-coding regions within a genome, or indicate inaccuracies in existing genome annotations. These GSSPs were subsequently mapped to the genome to identify novel coding regions and to refine existing gene models, based on their genomic coordinates relative to current annotations (Shenoy and Chowdhury et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bioinformatics analysis:\u003c/h2\u003e \u003cp\u003eThe GSSPs that mapped to the six-frame translated genome database of respective \u003cem\u003eLeishmania species\u003c/em\u003e were further analysed by undertaking BLAST analysis (protein) using UniProt and TriTrypDB databases to identify the genomic region to which these GSSPs map and the conserved orthologous proteins in other related \u003cem\u003eLeishmania\u003c/em\u003e in which these GSSPs map. Using this approach, we could categorise our proteogenomic identifications into novel secreted proteins previously not identified in the seven \u003cem\u003eLeishmania\u003c/em\u003e species. An overview of the proteogenomic workflow used in this study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Overall Composition of the Secretome Across \u003cem\u003eLeishmania\u003c/em\u003e Species\u003c/h2\u003e \u003cp\u003eThe analysis of the seven \u003cem\u003eLeishmania\u003c/em\u003e species secretome MS/MS datasets yielded 7,146 unique peptides following searches against the species-specific six-frame translated genome databases. Peptides mapping to the annotated protein databases were excluded, and the remaining unmapped peptides were classified as genome search-specific peptides (GSSPs), resulting in 693 unique GSSPs. Subsequent genomic mapping of these GSSPs enabled the identification of 469 novel open reading frames (ORFs) across the seven species. These ORFs represent previously unannotated, hidden secreted proteins that were not captured in existing genome annotations, thereby expanding the catalogue of secreted proteins in \u003cem\u003eLeishmania\u003c/em\u003e. An overview of the proteogenomic workflow used in this study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eComparative analysis of the promastigote secretomes of \u003cem\u003eL. amazonensis, L. braziliensis, L. donovani, L. infantum, L. major, L. tarentolae\u003c/em\u003e, and \u003cem\u003eL. tropica\u003c/em\u003e revealed substantial qualitative and quantitative variations across species, extending well beyond what was previously reported in Pissarra et al. (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although Pissarra and colleagues identified a conserved core secretome comprising 306 proteins, our reanalysis of the dataset demonstrated that each species possesses a wider range and more functionally diverse secretome than earlier recognised. Several additional secreted proteins detected in the present proteogenomics study were not missed in Pissarra\u0026rsquo;s reported dataset, suggesting that prior analyses underestimated species-specific and niche-dependent secreted proteins. This inconsistency is likely due to differences in peptide identification thresholds, protein database versions, annotation pipelines, and species-specific proteomic depth. The extended number of secreted proteins identified in this study highlights the complication of secretome biology across pathogenic and non-pathogenic \u003cem\u003eLeishmania\u003c/em\u003e species. The overlap and species-specific indicates to the secretome reveals a wide range of diversity, highlighting the heterogeneity of protein secretion across the genus and pathways that may underlie species-specific virulence and clinical manifestations, as represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Further comparison with previously reported secretomes (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Molecular Function Variability Among Species\u003c/h2\u003e \u003cp\u003eThe catalytic and binding features of the most predominant molecular functional categories have been\u003c/p\u003e \u003cp\u003eidentified across all species, which are in line with the previous studies; though this dataset allows a more granular understanding of functional specialisation. In \u003cem\u003eL. donovani\u003c/em\u003e, the distinct abundance of the ATP and nucleotide-binding and helicase-related proteins was identified. These functions align with its visceral disease phenotype and support enhanced intracellular survival strategies involving nucleic-acid modulation, metabolic flexibility, and host immune evasion. Although Pissarra et al. had noted nucleic-acid binding enrichment in visceralising species, the recent findings extended this range by highlighting additional transport-associated enzymes and regulatory proteins involved in RNA processing. In \u003cem\u003eL. infantum\u003c/em\u003e, ion-binding activities, particularly Zn-binding oxidoreductases and structural molecule categories, were more abundant than previously described. These findings emphasise the importance of redox regulation, ion homeostasis, and metabolic control mechanisms in visceral infection biology. In contrast, \u003cem\u003eL. braziliensis\u003c/em\u003e revealed a uniquely protease-rich secretome dominated by serine-type endopeptidase and extracellular matrix-modifying enzymes, reflecting its capacity to drive tissue destruction and mucosal pathology. These proteolytic signatures extend beyond what Pissarra et al. reported and reveal additional peptidases previously unrecognised. The species-wise distributions of molecular functions and biological processes are shown in \u003cb\u003eSupplementary Figs.\u0026nbsp;1 and 2\u003c/b\u003e, where bar plots represent the variation in functional categories across the six \u003cem\u003eLeishmania\u003c/em\u003e secretomes.\u003c/p\u003e \u003cp\u003eThe distinguishing enzymatic profiles were also detected in \u003cem\u003eL. amazonensis\u003c/em\u003e, which showed increased levels of lipid-binding proteins, lyases, and isomerases. This pattern supports its dependence on lipid-based metabolic pathways and membrane remodelling mechanisms, characteristic of diffuse cutaneous leishmaniasis. \u003cem\u003eL. tropica\u003c/em\u003e presented molecular functional diversity but included several enzymes connected with redox regulation, signal transduction, and protein refolding, which may reflect adaptive responses to environmental stress encountered within the cutaneous niche. Surprisingly, \u003cem\u003eL. tarentolae\u003c/em\u003e shared several functional categories traditionally associated with pathogenic species, including structural molecule activities, biosynthetic enzymes, and redox regulators. This overlap suggests conservation of key physiological functions even in non-pathogenic species and implies that some secreted pathways may serve essential biological roles beyond virulence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Species-Specific Differences in Biological Processes\u003c/h2\u003e \u003cp\u003eBiological process analysis discovered both shared and species-specific features across the \u003cem\u003eLeishmania\u003c/em\u003e secretomes. Core biological processes, including metabolic processes, cellular regulation, and responses to environmental stimuli, were conserved across all species, consistent with their shared need to resist oxidative stress, adapt to nutrient fluctuations, and maintain homeostasis in both vector and host environments. However, species-level determination highlighted several previously unreported patterns.\u003c/p\u003e \u003cp\u003eIn the visceralising species (\u003cem\u003eL. donovani\u003c/em\u003e and \u003cem\u003eL. infantum\u003c/em\u003e), proteins involved in metabolism, RNA processing, stress adaptation, and macromolecule biosynthesis were more dominant than in cutaneous species. These processes likely support the metabolic plasticity required for survival in visceral organs, particularly within macrophages, where parasites encounter high oxidative pressure and nutrient limitation. In \u003cem\u003eL. braziliensis\u003c/em\u003e, biological processes associated with proteolysis and extracellular matrix organisation were dominant, reflecting its pathogenic role in mucocutaneous tissue destruction. \u003cem\u003eL. amazonensis\u003c/em\u003e exhibited enrichment in lipid metabolic processes, fatty acid turnover, and vesicle-mediated transport, consistent with its unique pathology characterised by parasitophorous vacuoles enriched in host lipids. \u003cem\u003eL. tropica\u003c/em\u003e was characterised by enhanced stress-response pathways, including protein refolding and cellular response mechanisms, indicating heightened sensitivity to environmental fluctuations. Meanwhile, \u003cem\u003eL. tarentolae\u003c/em\u003e showed surprisingly high levels of biological processes associated with cellular homeostasis and biosynthesis, suggesting evolutionary conservation of core survival mechanisms regardless of pathogenicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Highly Expressed Functional Groups Relevant to \u003cem\u003eLeishmania\u003c/em\u003e Biology\u003c/h2\u003e \u003cp\u003eAcross all species, the secretome was dominated by several highly expressed protein groups central to \u003cem\u003eLeishmania\u003c/em\u003e survival and virulence. These included heat-shock proteins such as HSP70, HSP83, and HSP60, which are involved in thermotolerance, immune modulation, and the transition between vector and mammalian environments. Protein disulfide isomerases, peroxiredoxins, and trypanothione-dependent redox enzymes were also abundant, consistent with their established roles in antioxidant defence and parasite resilience in oxidative environments. Tubulins, actins, and associated motor proteins were frequently detected, supporting their role in vesicle-mediated secretion and host-parasite interactions. Moreover, numerous metabolic enzymes known to exert moonlighting functions, such as glycolytic enzymes and dehydrogenases at high abundance, underscoring their dual roles in both metabolism and immunomodulation. Compared with prior reports, these proteins were detected with greater species-specific resolution, revealing new insights into their differential contributions to parasite pathogenicity. A summary of representative functionally characterised proteins is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eKey functionally characterised proteins selected across six\u003c/b\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003especies and their predicted biological roles (full list is presented in the supplementary file).\u003c/b\u003e This table represents the key proteins from each \u003cem\u003eLeishmania\u003c/em\u003e species analysed in this study (\u003cem\u003eL. braziliensis\u003c/em\u003e, \u003cem\u003eL. donovani\u003c/em\u003e, \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. amazonensis\u003c/em\u003e, \u003cem\u003eL. tarentolae\u003c/em\u003e, and \u003cem\u003eL. tropica\u003c/em\u003e). For each protein, the UniProt accession number, corresponding gene identifier, functional description, and experimentally supported or literature-based biological role are provided. The listed proteins represent diverse molecular functions, including proteases, metabolic enzymes, mitochondrial translocases, ubiquitination machinery, antioxidant defence proteins, and RNA-binding regulators, highlighting essential processes such as parasite survival, metabolism, stress response, differentiation, host\u0026ndash;pathogen interactions, and virulence. The complete list of identified proteins is provided in the supplementary data.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSl No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBiological role\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. braziliensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A3S7WZY5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLdCL_260021200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMetallopeptidase belonging to the thimet oligopeptidase (TOP) family, a zinc-dependent endopeptidase.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTOP enzymes are involved in peptide processing, protein turnover and regulation of intracellular signalling peptides (Besteiro et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn trypanosomatids, TOP-like enzymes contribute to parasite survival, proteolysis, stress response, and may modulate host\u0026ndash;parasite interactions (Sundar \u0026amp; Singh, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A640KD91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLtaPh_1412600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCore component of the TIM23 mitochondrial translocase complex, which imports preproteins into the inner mitochondrial membrane.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTIM50 is essential for mitochondrial protein import, organelle biogenesis, and parasite viability (Roberts et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn kinetoplastids, TIM complex proteins are critical due to their single, specialised mitochondrion (kinetoplast) (Mani et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. donovani\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A3S7X395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLdCL_300014700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdenosine kinase (AK).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCatalyses the phosphorylation of adenosine to AMP, part of the purine-nucleoside salvage pathway critical for parasite survival. Has been biochemically purified and characterised in \u003cem\u003eL. donovani\u003c/em\u003e, showing unique kinetic and regulatory properties (Boitz et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A3S7WT53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLdCL_140017800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKinesin K39, putative.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMember of the kinesin-motor protein family; the \u0026ldquo;K39\u0026rdquo; repeat region is antigenic and widely used in serodiagnosis of visceral leishmaniasis. It likely plays a role in microtubule-based motility or cytoskeletal organisation in the parasite (Gerald et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA4I1S9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLINJ_25_2400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRING-type E3 ubiquitin transferase.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRING-type E3 ubiquitin ligases are key regulators of the ubiquitin\u0026ndash;proteasome system (UPS) in \u003cem\u003eLeishmania\u003c/em\u003e, directing the ubiquitination and turnover of specific proteins. They contribute to promastigote\u0026ndash;amastigote differentiation, stress adaptation, and intracellular survival within macrophages. UPS components, including RING E3 ligases, are upregulated during oxidative and heat stress and help maintain protein quality. Studies in kinetoplastids also show that RING-type E3 ligases are required for proper cell-cycle progression and DNA replication. E3 ligase consider as potential drug target as the UPS-associated proteins effect the parasite virulence and inhibit the pathway lethal to \u003cem\u003eLeishmania\u003c/em\u003e (Bulatov et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Burge et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA4HX10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLINJ_16_1170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTyrosyl or methionyl-tRNA synthetase-like protein.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTyrosol- and methionyl- tRNA synthetases in aminoscyl-tRNA synthetases (aaRS), are useful enzymes in \u003cem\u003eLeishmania\u003c/em\u003e that help to catalyse the amino acid attachment to their corresponding tRNAs throughout protein synthesis. Mostly, these enzymes in kinetoplastids indicate the unique structural adaptations that differ from mammalian homologues, making them crucial for the parasite's survival and potential drug target (Burge et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several aaRS, including TyrRS and MetRS in \u003cem\u003eL. donovani\u003c/em\u003e, are essential for mitochondrial and cytosolic translation, parasite growth, and survival under stress (Abhishek et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eLeishmania\u003c/em\u003e, the inhibition of the MetRS or TyrRS leads to reduced protein synthesis and condensed parasite infectivity, supporting their role as key components of the translational machinery (Nasim \u0026amp; Qureshi, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. amazonensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A640KNR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLtaPh_2521300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCaspase family p20 domain-containing protein.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAlthough classical caspases are absent in protozoa, \u003cem\u003eLeishmania\u003c/em\u003e possesses metacaspases, which contain a p20 catalytic domain and function as cysteine proteases. The identified proteins are involved in the programmed cell death-like pathways, stress response, and the progression of the cell cycle (Vandana et al., 2019). For the survival of the parasite under oxidative stress, the role of the metacaspase with the p20 domain is predominant and is also essential for the differentiation between the promastigote and amastigote phases (Aghaei et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The metacaspase gene disruption can cause defects in autophagy, decreased infectivity and impaired parasite proliferation, which underscore the key role in cellular homeostasis (Aghaei et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE9ARK7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLMXM_18_0670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCitrate synthase.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThis is an important mitochondrial enzyme that catalyses the primary step of the tricarboxylic acid (TCA) cycle by shrinking oxaloacetate to form citrate. Citrate synthase is important for carbon metabolism, ATP generation, and biosynthetic pathways essential for parasite growth (Ranjan et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Previous research on kinetoplastid parasites indicates that citrate synthase is important for continuing mitochondrial function and metabolic flexibility throughout the transition between promastigote and amastigote stages (Marchese et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Reduction of the citrate synthase expression leads to impaired respiration and reduced parasite viability, underscoring its role for survival in nutrient-limited environments within the host (Marchese et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. tarentolae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A640K9H5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLtaPh_0808151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCathepsin L-like protease.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCathepsin L-like proteases, also known as cysteine proteases (CPA/CPB), are major virulence factors in \u003cem\u003eL. infantum\u003c/em\u003e. These enzymes facilitate parasite survival by degrading host proteins, modulating macrophage signalling, and aiding immune evasion. This protein helps in the entry of the parasite, nutrient acquisition and the reduction of the antigen expression, which supports the intracellular replication. However, these proteins are also required for the parasite surface molecules processing and were explored as potential vaccine and drug targets due to their roles in pathogenicity (Silva-Almeida et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A640KBV6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLtaPh_1009541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLeishmanolysin.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeishmanolysin, commonly known as GP63, is a zinc-dependent metalloprotease mostly expressed on the surface of \u003cem\u003eL. infantum\u003c/em\u003e promastigotes. GP63 is an important virulence factor that protects the parasite from complement-mediated lysis and controls host immune responses by slicing signalling molecules. Additionally, it destroys the extracellular matrix leads to parasite dissemination and helping the survival of the parasite in the macrophages by impairing the phagolysosomal maturation. Due to its high immunogenicity and wide functional roles, GP63 is broadly explored as a potential drug target in visceral leishmaniasis (Guay-Vincent et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Isnard et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. tropica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A3S7WRN3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLdCL_110015200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK Homology domain-containing protein.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK Homology (KH) domain-containing proteins function primarily as RNA-binding regulators that regulate mRNA stability, translation, and stage-specific gene expression. Transcriptional control is not available in \u003cem\u003eLeishmania\u003c/em\u003e; the KH-domain proteins play an important role in post-transcriptional regulation and serve the parasite to survive between the different stages of the life cycle. These proteins help to bind the U-rich elements, and they relate to RNA-processing complexes that affect the stress response, surface antigens, as well as virulence linked transcript. This domain also helps in regulating the amastigote survival pathways, making them potential candidates for understanding parasite differentiation and potential drug targets (Ferreira et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Haskell \u0026amp; Zinovyeva, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA4I7Z8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSODB2 LINJ_32_1920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSuperoxide dismutase.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIn \u003cem\u003eLeishmania\u003c/em\u003e, the superoxide dismutase plays an important role as an antioxidant enzyme, which converts the superoxide radicals into hydrogen peroxide, and helps in protecting the parasite from oxidative stress. \u003cem\u003eLeishmania\u003c/em\u003e relies heavily on Fe-SODs, producing these enzymes structurally and functionally distinct from host SODs. SODs are essential for survival within the macrophages, contribute to virulence, and support resistance to reactive oxygen species (ROS). Inhibition of SODs has been reported to reduce parasite viability, underscoring them as promising drug targets (Ghosh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Roy et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe exploratory study from this comparative secretome analysis provides a more comprehensive understanding of protein secretion across pathogenic and non-pathogenic \u003cem\u003eLeishmania\u003c/em\u003e species than what had been reported previously by Pissarra et al. (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While previous work was involved in defining a conserved core secretome, our findings indicate that \u003cem\u003eLeishmania\u003c/em\u003e secretomes show far wider functional diversity and species specificity. The detection of additional catalytic, lipid-associated, nucleic-acid-binding, and proteolytic proteins suggests a more detailed interplay between parasite biology, environmental niche, and tissue tropism than previously recognised. The proteins that were not identified in previous datasets likely reflect differences in annotation pipelines, search algorithms, or mass-spectrometry depth; however, their identification here highlights the importance of continuously updating proteomic databases and analytical context to better reflect biological complexity. Furthermore, one methodological limitation in the study by Pissarra et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) concerns their use of an NNscore\u0026thinsp;\u0026gt;\u0026thinsp;0.5 in SecretomeP to predict non-classical secretion. This low threshold is known to produce a high false-positive rate in kinetoplastids, leading to less accurate identification of proteins within the secretome (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, their reported \u0026ldquo;core secretome\u0026rdquo; may include cytosolic proteins incorrectly classified as secreted, masking species-specific differences that became clearer in our more stringent re-analysis.\u003c/p\u003e \u003cp\u003eThe enrichment of ATP-binding proteins, helicases, and oxidoreductases in \u003cem\u003eL. donovani\u003c/em\u003e and \u003cem\u003eL. infantum\u003c/em\u003e is in line with the metabolic reprogramming and stress adaptation required for visceral infection. These findings align with previous studies demonstrating that RNA-binding proteins, redox regulators, and ATP-dependent chaperones are essential for parasite survival in macrophages and contribute to virulence (Fonseca Pires et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Leclercq et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, the common protease activity observed in \u003cem\u003eL. braziliensis\u003c/em\u003e has support in the literature, as serine proteases and metalloproteases are known to drive tissue degradation, extracellular matrix breakdown, and severe mucocutaneous pathology (M Guedes et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Souza-Melo et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zabala-Pe\u0026ntilde;afiel et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These proteases, which were understated in earlier comparative analyses, appear to be more diverse and abundant than previously thought.\u003c/p\u003e \u003cp\u003eThe lipid-centric metabolic profile of \u003cem\u003eL. amazonensis\u003c/em\u003e identified here repeats findings that this species deploys host lipids and modulates membrane structure to facilitate intracellular replication (De Cicco et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gdovinova \u0026amp; Descoteaux, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The present study enhances this understanding by demonstrating that lipid-binding and lipid-metabolising enzymes are major secreted components, emphasising their role in disease progression. Likewise, the stress-response dominated secretome of \u003cem\u003eL. tropica\u003c/em\u003e supports its known adaptability to diverse cutaneous environments and fluctuating immune pressures (Pissarra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These species-specific functional enrichments help clarify how differences in biological processes shape distinct disease indicators.\u003c/p\u003e \u003cp\u003eThe finding that \u003cem\u003eL. tarentolae\u003c/em\u003e exhibits significant overlap with pathogenic species in several molecular and biological functions challenges the conventional view that non-pathogenic species possess fundamentally different secretome architectures. Instead, the presence of structural, biosynthetic, and redox-related proteins supports the hypothesis that non-pathogenic species retain ancestral pathways essential for survival but lack specific virulence determinants. Earlier genomic studies had proposed that \u003cem\u003eL. tarentolae\u003c/em\u003e lost only a subset of virulence genes rather than experiencing widespread functional degeneration (Andrade et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Novo et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The present secretome analysis supports this model, demonstrating that many secretory features associated with pathogenicity are conserved across the genus.\u003c/p\u003e \u003cp\u003eThe expanded diversity of proteins uncovered in this study has significant implications for developing species-specific diagnostic tools, biomarkers, and vaccine targets. Highly expressed heat-shock proteins, redox enzymes, lipid-metabolic proteins, and proteases identified here have been previously implicated as immunomodulators or potential vaccine candidates (Chowdhury et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Rostami \u0026amp; Khamesipour, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The newly identified species-specific proteins, particularly in \u003cem\u003eL. amazonensis\u003c/em\u003e and \u003cem\u003eL. braziliensis\u003c/em\u003e, may serve as more accurate biomarkers for differential diagnosis or as immunogenic components in species-targeted vaccine strategies. Since secreted proteins are often the first point of contact with the host immune system, a more comprehensive understanding of species-specific secretomes can directly inform translational research aimed at improving disease management.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a comprehensive and refined comparative analysis of the promastigote secretomes of six \u003cem\u003eLeishmania\u003c/em\u003e species, \u003cem\u003eL. braziliensis, L. donovani, L. infantum, L. amazonensis, L. tropica\u003c/em\u003e, and \u003cem\u003eL. tarentolae\u003c/em\u003e, revealing a broader and more functionally diverse landscape of secreted proteins than previously documented, particularly when compared with the core secretome described by Pissarra et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Through re-evaluation of the datasets, the present work identified numerous additional proteins and functional categories that had not been previously recognised, highlighting substantial species-specific variation. Visceralising species (\u003cem\u003eL. donovani\u003c/em\u003e and \u003cem\u003eL. infantum\u003c/em\u003e) displayed strong enrichment in ATP-binding and nucleotide-binding proteins, oxidoreductases, and stress-regulatory pathways, reflecting the metabolic flexibility and intracellular survival strategies required for their dissemination into visceral organs. Cutaneous species such as \u003cem\u003eL. braziliensis\u003c/em\u003e were characterised by a protease-rich profile, including serine-type endopeptidases and extracellular matrix\u0026ndash;modifying enzymes directly related to tissue destruction and mucocutaneous pathology. \u003cem\u003eL. amazonensis\u003c/em\u003e exhibited a pronounced enrichment in lipid-binding and lipid-metabolising enzymes, supporting its reliance on lipid remodelling mechanisms that drive its diffuse cutaneous disease phenotype. Meanwhile, \u003cem\u003eL. tropica\u003c/em\u003e showed elevated expression of stress-response and signalling-related proteins, consistent with its capacity to persist in fluctuating cutaneous environments. Unexpectedly, the non-pathogenic \u003cem\u003eL. tarentolae\u003c/em\u003e shared several functional features with pathogenic species, including structural, biosynthetic, and redox-related proteins, indicating that evolutionarily conserved secretory pathways remain intact even in species lacking human pathogenicity.\u003c/p\u003e \u003cp\u003eOverall, this study's findings demonstrate that \u003cem\u003eLeishmania\u003c/em\u003e secretomes are highly species-specific and significantly more complex than previously reported. By identifying additional proteins across diverse functional categories, ranging from binding and catalytic activities to proteolytic, lipid-metabolic, and redox-regulatory functions, this work emphasises that a core-proteome approach alone cannot fully capture the biological diversity present within the genus. These species-specific signatures not only enhance our understanding of the molecular basis of distinct clinical manifestations but also provide a valuable resource for identifying new diagnostic markers, therapeutic targets, and vaccine candidates. The expanded secretome profiles obtained here underscore the importance of species-level proteomic analysis for understanding host-parasite interactions and highlight the need for continued refinement of proteomic pipelines and annotation methods. Collectively, this combined summary and conclusion strengthen the increasing recognition that \u003cem\u003eLeishmania\u003c/em\u003e biology is shaped by both conserved evolutionary mechanisms and finely tuned species-specific adaptations, offering new avenues for translational research aimed at improving leishmaniasis diagnosis, treatment, and control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the Institute of Bioinformatics, Bangalore, India, for undertaking this study and analysis.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are included in the article and its supplementary information files.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this study was conducted without any financial support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institutional Ethics Committee has confirmed that no ethical approval is required for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbhishek, K., Sardar, A. 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Trypanosomatid comparative genomics: contributions to the study of parasite biology and different parasitic diseases. \u003cem\u003eGenetics and molecular biology\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e, 1-17. https://doi.org/10.1590/S1415-47572012005000008 \u003c/li\u003e\n\u003cli\u003eTonui, W. K., Mejia, J. S., Hochberg, L., Mbow, M. L., Ryan, J. R., Chan, A. S. T., Martin, S. K., \u0026amp; Titus, R. G. (2004). Immunisation with \u003cem\u003eLeishmania major\u003c/em\u003e exogenous antigens protects susceptible BALB/c mice against challenge infection with \u003cem\u003eL. major\u003c/em\u003e. \u003cem\u003eInfection and Immunity\u003c/em\u003e, \u003cem\u003e72\u003c/em\u003e(10), 5654\u0026ndash;5661. https://doi.org/10.1128/IAI.72.10.5654-5661.2004\u003c/li\u003e\n\u003cli\u003eTsigankov, P., Gherardini, P. F., Helmer-Citterich, M., \u0026amp; Zilberstein, D. (2012). What has proteomics taught us about \u003cem\u003eLeishmania\u003c/em\u003e development? \u003cem\u003eParasitology\u003c/em\u003e, \u003cem\u003e139\u003c/em\u003e(9), 1146\u0026ndash;1157. https://doi.org/10.1017/S0031182012000157\u003c/li\u003e\n\u003cli\u003eVandana, Dixit, R., Tiwari, R., Katyal, A., \u0026amp; Pandey, K. C. (2019). Metacaspases: Potential Drug Target Against Protozoan Parasites. \u003cem\u003eFrontiers in Pharmacology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e. https://doi.org/10.3389/fphar.2019.00790\u003c/li\u003e\n\u003cli\u003eWalker, J., Vasquez, J. J., Gomez, M. A., Drummelsmith, J., Burchmore, R., Girard, I., \u0026amp; Ouellette, M. (2006). Identification of developmentally-regulated proteins in \u003cem\u003eLeishmania panamensis\u003c/em\u003e by proteome profiling of promastigotes and axenic amastigotes. \u003cem\u003eMolecular and Biochemical Parasitology\u003c/em\u003e, \u003cem\u003e147\u003c/em\u003e(1), 64\u0026ndash;73. https://doi.org/10.1016/j.molbiopara.2006.01.008\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eWHO\u003c/em\u003e. (2023).\u003c/li\u003e\n\u003cli\u003eZabala-Pe\u0026ntilde;afiel, A., Dias-Lopes, G., Cysne-Finkelstein, L., Concei\u0026ccedil;\u0026atilde;o-Silva, F., Miranda, L. de F. C., Fagundes, A., Schubach, A. de O., Fernandes Pimentel, M. I., Souza-Silva, F., Machado, L. de A., \u0026amp; Alves, C. R. (2021). Serine proteases profiles of \u003cem\u003eLeishmania (Viannia) braziliensis\u003c/em\u003e clinical isolates with distinct susceptibilities to antimony. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(1). https://doi.org/10.1038/s41598-021-93665-z\u003c/li\u003e\n\u003cli\u003eZijlstra, E. E. (2016). The immunology of post-kala-azar dermal leishmaniasis (PKDL). In \u003cem\u003eParasites and Vectors\u003c/em\u003e (Vol. 9, Issue 1). BioMed Central Ltd. https://doi.org/10.1186/s13071-016-1721-0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Leishmania, Secretome, Proteogenomic, Mass spectrometry, Novel protein-coding regions, Comparative proteomics","lastPublishedDoi":"10.21203/rs.3.rs-8472209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8472209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeishmaniasis affects millions of people worldwide and is caused by obligate intracellular protozoan parasites transmitted through infected phlebotomine sand flies. Clinical outcomes range from self-healing cutaneous lesions to fatal visceral disease across twenty recognised human-pathogenic species. Although \u003cem\u003eLeishmania\u003c/em\u003egenomes are remarkably conserved, substantial phenotypic diversity arises through post-transcriptional and post-translational regulatory mechanisms, highlighting the essential role of proteomic investigations. The \u003cem\u003eLeishmania\u003c/em\u003esecretome contains virulence factors crucial for establishing infection, modulating host immunity, and promoting parasite survival within macrophages. Previous proteomic studies have characterised secreted proteins across multiple species, identifying a conserved core secretome predominantly released via extracellular vesicles through non-classical pathways. However, large portions of the predicted proteome remain experimentally unvalidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo uncover overlooked proteins, we conducted a comprehensive proteogenomic analysis using publicly available mass spectrometry datasets. Custom six-frame translated genome databases were generated for seven reference \u003cem\u003eLeishmania\u003c/em\u003e species, enabling the identification of genome search-specific peptides.\u003c/p\u003e\n\u003cp\u003eThis approach helps to detect the novel secreted proteins that have been missed in previous secretome researches together with low-abundance features, hypothetical proteins and the proteins that are from the canonical translation procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis proteogenomic study identified 469 secreted proteins in the seven \u003cem\u003eLeishmania\u003c/em\u003especies that were not reported previously. The proteins were significantly explored and validated in the \u003cem\u003eLeishmania\u003c/em\u003e proteome and disclose a higher level of functional diversity. The visceralising species were abundant in ATP-binding proteins and oxidoreductases, while the cutaneous species were represented with proteasome-rich secretome profiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe identification of these further secreted proteins highlights the boundaries of previous studies and emphasises the higher resolution provided by the proteogenomic approaches. The species-specific categorical and functional differences explored in this study can contribute to diverse tissue tropisms, host-pathogen interactions and the clinical indicators of leishmaniasis. The newly identified proteins in this study likely include previously unidentified virulence factors with potential significance for parasite variations, pathogenesis, and therapeutic interference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study reveals that the proteogenomic analysis is a prevailing approach for exploring the hidden secretome of \u003cem\u003eLeishmania\u003c/em\u003e species. These findings advance the understanding of parasite evolution and biology by identifying 469 novel secreted proteins and detecting the significant species-specific functional diversity; also highlight potential indicators for future functional research and therapeutic interventions.\u003c/p\u003e","manuscriptTitle":"Mapping the Hidden Secretome in Leishmania Parasites Using a Proteogenomics Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 16:06:46","doi":"10.21203/rs.3.rs-8472209/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-02T13:52:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T06:34:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Parasitic Diseases","date":"2026-01-28T12:53:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-02T02:54:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Parasitic Diseases","date":"2025-12-30T02:50:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"636a21ea-51a7-4187-8acc-61c6e4680f9a","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T16:06:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 16:06:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8472209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8472209","identity":"rs-8472209","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}