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Proteomic analysis of Plasma-derived Extracellular Vesicles reveals different biomarkers for monitoring visceral leishmaniasis progression

ABSTRACT: Visceral leishmaniasis (VL), the most severe form of leishmaniasis, lacks standardized and validated early predictors for cure and relapse, which is especially important for individuals at high-risk. Here, we searched for protein biomarkers in plasma extracellular vesicles (EVs). EVs from patients with active VL (n=12) and patients at post-treatment intervals (1, 3, and 6 months; n=12 per time interval) were analysed by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) to characterize their proteomic profiles. Six candidate biomarkers were further tested by ELISA using total plasma. The comparison of the protein profiles of plasma EVs identified 132 human proteins differentially abundant in active VL compared to successfully cured patients, with 64 upregulated and 68 downregulated proteins in VL. Additionally, the analysis highlighted pathogenic mechanisms associated with VL and pathways associated to effective cure. Levels of SAA, and ITGB1 proteins showed a differential expression when analyzed in total plasma, emerging as promising easy-to-measure biomarkers for patients management. Remarkably, proteins from Leishmania spp. were identified in the EV samples, highlighting a novel source of parasite-derived biomarkers in human samples. Plasma EVs contain protein biomarkers to monitor VL treatment success, with some of them detectable using total plasma at 1 month after treatment. This study also provides a proteomic landscape of plasma derived EVs involved in VL, offering insights into disease pathogenesis.

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ORGANISM(S): Homo Sapiens (human) Leishmania Infantum

TISSUE(S): Blood Plasma

DISEASE(S): Leishmaniasis

SUBMITTER: Ana Montero Calle

LAB HEAD: Rodrigo Barderas

PROVIDER: PXD060604 | Pride | 2025-08-27

REPOSITORIES: Pride

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Project description:Leishmania is an intracellular protozoan parasite and the etiological agent of a vector-borne disease known as leishmaniasis. This neglected tropical disease exhibits high morbidity and mortality. It is endemic in 97 countries and 700.000–1.000.000 new cases are estimated to occur each year. Leishmania-HIV co-infection has been an emergent public health problem in the last twenty years and has been reported in 35 endemic countries. HIV-infected people are especially vulnerable to visceral leishmaniasis (VL), and VL accelerates HIV replication and advancement to AIDS. In the context of HIV co-infection, the challenges associated with VL disease management are very significant. Disease presentation is often atypical and therapeutical responses are limited with recurrent relapses. A better understanding of the disease and also new biomarkers for infection detection, treatment prognosis and relapse are needed. Extracellular Vesicles (EVs), are considered a resource with great potential for generating a higher understanding of complex biological processes and also as a potential source of biomarkers. To evaluate the potential of plasma EVs in the context of VL and to find new possible molecules of interest for disease management in the context of HIV/VL coinfection, EVs were recovered from the plasma of an HIV+VL+ patient over a two-year period, and compared to age and sex-matched HIV and HIV-VL- controls. The approach selected for plasma EVs recovery was Size-Exclusion Chromatography followed by fraction selection using common EVs markers and proteomic analysis of fractions of interest by MS. The proteomic analysis performed confirmed the identification of common EVs biomarkers. The capacity to detect Leishmania proteins was limited with no identifications with more than one UP existed. Moreover no Leishmania protein identification was common between time points. Concernng human proteins, several proteins were detected with distinct abundance in the patient compared to both control groups. Using GO approach, the proteins were associated with specific biological processes, like MHC-I that provide a complementary overview of the patient immunological condition. Among the proteins detected, we highlighted the Macrophage receptor with collagenous structure (MARCO). This protein was detected in the patient in 4 out of 5 time points. Significantly, MARCO was also detected in plasma EVs from 5 other VL patients that were analysed. The presence of MARCO in the plasma EVs preparations was also confirmed by Western Blot. In conclusion, it was demonstrated that EVs analysis in the context of VL can contribute to understand the ongoing pathological process. Moreover, these EV are a source of possible biomarkers for VL monitoring. MARCO was a protein of interest being only identified in the VL patients. Ultimately, further studies need to be performed to generate a critical mass of data on EVs in the context of this and other infections to generate improvements in the clinical management of these vulnerable populations.

Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

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	TMT_Exos_Leish_F1_12.raw	Raw
	TMT_Exos_Leish_F2.raw	Raw
	TMT_Exos_Leish_F3.raw	Raw

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Proteomic analysis of Plasma-derived Extracellular Vesicles reveals different biomarkers for monitoring visceral leishmaniasis progression

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