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 and MaxQuant software were estimated for 11 species from six 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. Placed 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 within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides.

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.

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 α-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. SIGNIFICANCE: Leishmaniasis disease manifests in two main clinical forms according to the infecting Leishmania species and host immune responses, cutaneous and visceral leishmaniasis. In Brazil, cutaneous leishmaniasis (CL) is associated with L. braziliensis and L. amazonensis, while visceral leishmaniasis, also called kala-azar, is caused by L. infantum. Leishmania parasites remodel their proteomes during growth phase transition and changes in their mileu imposed by the host, including temperature. In this study, we performed a quantitative mass spectrometry-based proteomics to compare the proteome of three New world Leishmania species, L. amazonensis (La), L. braziliensis (Lb) and L. infantum (syn L. chagasi) (Lc) in three conditions: a) exponential phase at 25 °C (Exp); b) stationary phase at 25 °C (Sta25) and; c) stationary phase subjected to temperature stress at 34 °C (Sta34). This study provides an in-depth proteome analysis of three Leishmania spp. with varying pathophysiological outcomes, 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.

Protein glycosylation in Leishmania spp.

Protein glycosylation is a co- and post-translational modification that, in Leishmania parasites, plays key roles in vector-parasite-vertebrate host interaction. In the mammalian host, Leishmania protein glycosylation is involved in virulence, host cell invasion, and immune evasion and modulation. The Leishmania glycocalyx is composed by a dense array of glycoconjugates including lipophosphoglycan, glycoinositolphospholipids, glycoproteins and proteophosphoglycans which varies in composition between Leishmania species and developmental stages. The current knowledge on Leishmania protein glycosylation is quite limited. The development of novel analytical tools to characterize the Leishmania glycoproteome and the expanding toolbox to modulate the parasite glycocode will help in deciphering the processes involved in Leishmania-host interaction. This review will recapitulate the current knowledge of Leishmania protein glycosylation, and glycan structures reported, and the potential application of mass spectrometry-based analysis for system-wide Leishmania glycoproteome and glycome analysis.