BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract.OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts.METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy.FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms.MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O 2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and PLOS NEGLECTED TROPICAL DISEASES

Trichomonas vaginalis is a sexually transmitted anaerobic parasite that infects humans causing trichomoniasis, a common and ubiquitous sexually transmitted disease. The life cycle of this parasite possesses a trophozoite form without a cystic stage. However, the presence of nonproliferative and nonmotile, yet viable and reversible spherical forms with internalized flagella, denominated pseudocysts, has been commonly observed for this parasite. To understand the mechanisms involved in the formation of pseudocysts, we performed a mass spectrometry-based high-throughput quantitative proteomics study using a label-free approach and functional assays by biochemical and flow cytometric methods. We observed that the morphological transformation of trophozoite to pseudocysts is coupled to (i) a metabolic shift toward a less glycolytic phenotype; (ii) alterations in the abundance of hydrogenosomal iron–sulfur cluster (ISC) assembly machinery; (iii) increased abundance of regulatory particles of the ubiquitin–proteasome system; (iv) significant alterations in proteins involved in adhesion and cytoskeleton reorganization; and (v) arrest in G2/M phase associated with alterations in the abundance of regulatory proteins of the cell cycle. These data demonstrate that pseudocysts experience important physiological and structural alterations for survival under unfavorable environmental conditions.

Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.

BackgroundCulex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.MethodsWe used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.ResultsZymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.ConclusionWe demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.