Biology subjects

Mule, S. N.

Publications and source records attributed to Mule, S. N..

4 recordsLinked to original sources

bioRxiv2023

The protein map of the protozoan parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum during growth phase transition and temperature stress

Leishmania parasites cause a spectrum of diseases termed leishmaniasis, which manifests in two main clinical forms, cutaneous and visceral leishmaniasis. Leishmania promastigotes transit from proliferative exponential to quiescent stationary phases inside the insect vector, a relevant step that recapitulates early molecular events of metacyclogenesis. During the insect blood meal of the mammalian hosts, the released parasites interact initially with the skin, an event marked by temperature changes. Deep knowledge on the molecular events activated during Leishmania-host interactions in each step is crucial to develop better therapies and to understand the pathogenesis. In this study, the proteomes of Leishmania (Leishmania) amazonensis (La), Leishmania (Viannia) braziliensis (Lb), and Leishmania (Leishmania) infantum (syn L. L. chagasi) (Lc) were analyzed using quantitative proteomics to uncover the proteome modulation in three different conditions related to growth phases and temperature shifts: 1) exponential phase (Exp); 2) stationary phase (Sta25) and; 3) stationary phase subjected to heat stress (Sta34). Functional validations were performed using orthogonal techniques, focusing on -tubulin, gp63 and heat shock proteins (HSPs). Species-specific and condition-specific modulation highlights the plasticity of the Leishmania proteome, showing that pathways related to metabolism and cytoskeleton are significantly modulated from exponential to stationary growth phases, while protein folding, unfolded protein binding, signaling and microtubule-based movement were differentially altered during temperature shifts. This study provides an in-depth proteome analysis of three Leishmania spp., and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts.

microbiology↗

bioRxiv2023

ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which have important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation. AUTHOR SUMMARYGlycosylation is a co- and/or post-translational modification regulated by the addition and removal of glycans. This process shapes the cellular glycome, which in turn, holds significant implications in various biological processes. Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, a globally concerning neglected tropical disease affecting 6 to 8 million individuals worldwide, exerts a profound influence on host glycoprotein sialylation. Remarkably, T. cruzi is incapable of synthesizing sialic acid (Sia) and relies on acquiring it from host glycoconjugates. In mammals, the formation of polysialic acid (polySia) is mediated by polysialyltransferases, such as ST8Sia2. The functional relevance of polySia has been extensively documented in the nervous system. Nevertheless, its role within the context of infectious processes remains largely unexplored. Herein, we demonstrate that in T. cruzi-infected host cells, the expression of the ST8Sia2 enzyme is downregulated, resulting in diminished levels of polysialylation. Furthermore, a reduction in the levels of NCAM1 and SCN5A was observed, which can be attributed to the decreased host polysialylation. Moreover, enzymatic removal of polySia, along with chemical inhibition and genetic silencing of ST8Sia2, led to a marked increase in the number of intracellular parasites. We posit that ST8Sia2 inhibition favors T. cruzi infection, thereby elucidating novel avenues for understanding the mechanisms associated with Chagas disease pathogenesis, prominently featuring the pivotal role of host polysialylation.

microbiology↗

bioRxiv2023

Leishmaniinae: evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer (PD) and MaxQuant (MQ) softwares were estimated for 11 species from 6 Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia) and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Positioned basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships consistent with genera, subgenera, and species-specific biological characteristics, able to resolve within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unravelled. In additional to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, disease presentations, tissue tropisms, potential to jump from insects to vertebrates including humans, and potential targets for species-specific diagnostic and drug development.

evolutionary biology↗

bioRxiv2021

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion under denaturing conditions, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of the formerly N-glycosylated peptides. LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases.

biochemistry↗