A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei

Trypanosoma brucei is a unicellular parasite that causes devastating diseases like sleeping sickness in humans and the “nagana” disease in cattle in Africa. Fundamental to the establishment and prolongation of a trypanosome infection is the parasite's ability to escape the mammalian host's immune system by antigenic variation, which relies on periodic changes of a protein surface coat.

From the origin and molecular diversity of the amastins, to the origin and diversity of intracellular parasitism from human Trypanosomatids

The large families of amastins from Leishmania donovani, L. infantum, L. major, L. braziliensis and Trypanosoma cruzi are strongly associated with the evolution of intracellular parasitism of rich cells in human MHC.1 molecules such as the macrophages, dendritic cells, and Langerhans cells by these parasites, recognize the MHC-1 molecules as host receptor. The internalization and transport of the paraste in the cytoplas of infected cell is facilitated by the MHC-1 recycle and endosome formation drag and transport the parasite in the cytoplasm of infected cell. The microbody amastins participate as coreceptor potency the infection, the tropism of L. major and L. braziliensis by the cells from the skin is facilitated by two molecular interactions, the first molecular interaction is faclitated by the amastins interact the human MHC-1 molecules, and the second molecular interaction is facilitated by the numerous microbody amastins; which also participate in the biogenesis of the small prasitophorous vcuole from L. major, and large parasitophorous vacuole from L. braziliensis. All amastins from these parasites developed deactivation domains, in different grade L. donovani develop an amastin surface coat specialized in deactivation of infected macrophages heavily glycosylated developed 38 amastins with 38 glycosylation Asp. N-Glycosylation sites and 45 N-glucosamina glycosylation sites, whereas L. infantum, L. major and L. braziliensis developed one half of glycosylated amastins in asparagine N-glycosylation sites, and T. cruzi did not developed none glycosylated amastin. The amastins surface coat from L. donovani is rich in phosphorylation sites, developed 45 amastins with 45 casein kinase II phosphorylations sites, and 48 amastins with 48 protein kinase phosphorylation sites. L. infantum, L. braziliensis, and T. cruzi developed 32, 42, and 8 amastins, with 94, 114, 21 casein kinase II phosphorylation sites; in similar way developed 35, 38, 11 amastins with 89, 78, and 22 protein kinase phosphorylation sites. The family of amastins from L. donovani develop 137 phosphoserines. and 128 phosphothreonine, L. major developed 14 phosphoserine and 4 phosphothreonine; L. infantum 1 phophoserine and 7 phosphothreonine; L. braziliensis did not developed phosphoserine and phosphothreonine and T. cruzi 4 phosphoserine and 4 phosphothreonine. The results show that amastin surface coat is equiped with numerous phosphorylations sites atractive for phosphohrylases from the infected host contribute with the dephosphorylation and deactivation of infectetd host cells. The amastins from L. major develop a membrane amastin with laminin G domain, which can interact with the collagen and heparin sulfate proteoglycan sites from the extracellular matrix of the skin tissue. Furthermore develop 14 amastins with tyrosine sulfation site, evade the activation of receptor of chemokines and the activation of the immune response by chemokines. There is an alternative mechanism of polarization of the immune response from protective TH1 to non protective TH2. The parasite nutrition is mediated by amastins that dissimilate the MHC-1 molecules and other subsets of proteins, the dissimilation products can be translocated through of the parasite cell membrane and employed as nutrient source.

Fungal Cell Wall Proteins and Signaling Pathways Form a Cytoprotective Network to Combat Stresses

Candida species are part of the normal flora of humans, but once the immune system of the host is impaired and they escape from commensal niches, they shift from commensal to pathogen causing candidiasis. Candida albicans remains the primary cause of candidiasis, accounting for about 60% of the global candidiasis burden. The cell wall of C. albicans and related fungal pathogens forms the interface with the host, gives fungal cells their shape, and also provides protection against stresses. The cell wall is a dynamic organelle with great adaptive flexibility that allows remodeling, morphogenesis, and changes in its components in response to the environment. It is mainly composed of the inner polysaccharide rich layer (chitin, and β-glucan) and the outer protein coat (mannoproteins). The highly glycosylated protein coat mediates interactions between C. albicans cells and their environment, including reprograming of wall architecture in response to several conditions, such as carbon source, pH, high temperature, and morphogenesis. The mannoproteins are also associated with C. albicans adherence, drug resistance, and virulence. Vitally, the mannoproteins contribute to cell wall construction and especially cell wall remodeling when cells encounter physical and chemical stresses. This review describes the interconnected cell wall integrity (CWI) and stress-activated pathways (e.g., Hog1, Cek1, and Mkc1 mediated pathways) that regulates cell wall remodeling and the expression of some of the mannoproteins in C. albicans and other species. The mannoproteins of the surface coat is of great importance to pathogen survival, growth, and virulence, thus understanding their structure and function as well as regulatory mechanisms can pave the way for better management of candidiasis.

A Reinterpretation of Evidence for the Endothelial Glycocalyx Filtration Structure

The endothelial glycocalyx (eGlx) is thought to be the primary macromolecular filter for fluid flux out of the vasculature. This filter maintains the higher protein concentration within the vessel lumen relative to the tissue. Whilst the arguments for the eGlx being the size filter are convincing the structural evidence has been limited to specialized stains of perfusion fixed tissue, which are further processed for resin embedding for transmission electron microscopy. The staining and processing of the delicate pore structure has left many researchers struggling to interpret the observed surface coat. Previous work has alluded to a 19.5 nm spacing between fibers; however, whilst repeatable it does not give an eGlx pore size consistent with known glycosaminoglycan (GAG) molecular structure due to the required fiber thickness of >10 nm. Here a new interpretation is proposed based on the likelihood that the electron micrographs of are often of collapsed eGlx. The 19.5 nm spacing measured may therefore be the core protein of the proteoglycans (PGs) with the GAGs wrapped up around them rather than in an expanded in vivo state. The concept is explored to determine that this is indeed consistent with experimental measurements of permeability if the syndecans are predominately dimerized. Further an alteration of core protein lattice from hexagonal packing to square packing dramatically changes the permeability which could be facilitated via known mechanisms such as transient actin binding.

Keith Vickerman. 21 March 1933—28 June 2016

Keith Vickerman was a parasitologist and protozoologist who made major contributions to our understanding of the biology of African trypanosomes, the causative agents of human sleeping sickness and nagana in cattle. His first academic post was at University College London, where he quickly mastered the techniques of electron microscopy (EM) and produced some of the best electron micrographs of parasitic protozoa at that time. He was a great believer in observation and deduction, and what began as an exercise in EM led him to investigate two of the then outstanding problems of trypanosome biology: how the parasites manage the transition from the tsetse fly vector to its mammalian host, and how they evade the host's immune response. Morphological changes, he found, were correlated with changes in the single mitochondrion and ensuring biochemical changes during the transition from a glucose-rich environment in mammalian blood to the glucose-poor tsetse gut. It was while comparing bloodstream and tsetse forms that he observed that the trypanosomes possessed a thick surface coat in the blood, which he subsequently identified as the variable antigen that was repeatedly formed and reformed and that this was the basis of antigenic variation—findings that stimulated a vast amount of interest among immunologists, biochemists and geneticists. In his later career a new problem emerged, and he found that a disease devastating stocks of the commercially important Norway lobster, Nephrops norvegicus , thought to be caused by a virus was actually caused by a protozoan, Hematodinium . Keith will always be remembered as one of the founders of modern parasitology.

Surface expressed Plasmodium circumsporozoite protein (CSP) modulates cellular flexibility and motility

Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface protein of sporozoites and a major vaccine candidate. However, neither the structure nor the role of CSP in sporozoite motility is well understood. Our recent in vitro data, from single molecule pulling experiments suggested a mechanically pliable structure for P. falciparum CSP. By engineering vegetative cells of the cellular slime mold Dictyostelium discoideum with regulatable CSP surface expression, we report evidence for direct involvement of CSP towards conferring elastic properties and motility of the cells. With an increase in the surface CSP levels by 5to8 fold, the Youngs moduli of the cells, observed through atomic force microscopy, decreased around 2 fold, with a concomitant increase in motility by about 2 fold. Interestingly, only full length CSP expression conferred maximal flexibility and motility, as opposed to repeat region alone or the flanking domains of CSP. The enhanced motility of the CSP expressing cells was abrogated with anti CSP antibodies as well as phospholipase cleavage of CSP, indicating specific contribution of CSP towards motility. Measurements of the Youngs moduli of Plasmodium berghei midgut (MG) and salivary gland (SG) sporozoites revealed an inverse correlation with CSP levels with a decrease from 1.1 kPa to 0.3 kPa as the CSP concentration doubled from MG to SG sporozoites. We hypothesize that high CSP level lowers the stiffness of sporozoites possibly through its pliable surface-coat, leading to cellular flexibility. These findings may explain a sporozoites developmental ability to enhance its CSP levels during transition from midgut to salivary glands to suit a migratory mode in the host, needed for successful hepatocyte invasion.

Leishmania donovani metacyclic promastigotes impair phagosome properties in inflammatory monocytes

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and to the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Surface ultrastructure of Blastocystis sp. isolated from cattle

Widisuputri NKA, Lastuti NDR, Suprihati E, Hastutiek P, Plumeriastuti H, Mufasirin, Puspitasari H, Suwanti LT. 2021. Surface ultrastructure of Blastocystis sp. isolated from cattle. Biodiversitas 22: 1514-1518. Blastocystis sp is a protozoan parasite commonly detected in the intestinal tract of humans and various animals that causes zoonotic blastocystosis. The pathogenic potential of Blastocystis is still being evaluated, some Blastocystis sp are completely pathogenic, while others can be considered commensal and hypothetical, related to the role of the surface coat of Blastocystis sp. This study aimed to compare the surface ultrastructure of Blastocystis sp. in cattle with diarrhea and non diarrhea by Scanning Electron Microscope (SEM). Four Blastocystis sp. isolates were selected from the faeces of four positives cattle which consisted of two diarrhea and two non-diarrhea cattle. The result showed that Blastocystis sp. in cattle appeared in round shape and reproduced by binary fission. The surface cell of Blastocystis sp. isolates from diarrhea cattle had a rough surface while organism of non diarrhea cattle isolates was very smooth. Bacteria were seen attached to the surface of Blastocystis sp. from diarrhea cattle faeces. In conclusion, the features of the surface structure of Blastocystis sp. correlated with symptomatic appearance. The surface structure of Blastocystis sp. isolates from cattle with diarrhea was rougher than non diarrhea.

Leishmania donovani metacyclic promastigotes impair phagosome properties in inflammatory monocytes

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and to the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes were readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli was efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91 phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidified poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan was responsible for the inhibition of PV acidification, impairement of the NADPH oxidase assembly was independent of lipophosphoglycan and of the metalloprotease GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination

ABSTRACTVaccine discovery and development critically depends on predictive assays, which prioritise protective antigens. Immunogenicity is considered one important criterion for progression of candidate vaccines to further clinical evaluation, including phase I/II trials. Here, we tested this assumption in an infection and vaccination model for malaria pre-erythrocytic stages. We engineered Plasmodium berghei parasites that harbour a well-characterised epitope for stimulation of CD8+ T cells either as an antigen in the circumsporozoite protein (CSP), the surface coat protein of extracellular sporozoites or in the upregulated in sporozoites 4 (UIS4), a major protein associated with the parasitophorous vacuole membrane (PVM) that surrounds the intracellular exo-erythrocytic forms (EEF). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust and superior antigen-specific CD8+ T cell responses, whilst an EEF antigen evokes poor responses. However, despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine-induced, antigen-specific effector CD8+ T cells results in high levels of protection when targeting both antigens. Our study is the first demonstration that poor immunogenicity of EEF antigens does not preclude their susceptibility to antigen-specific CD8+ T cell killing. Our findings that antigen immunogenicity is an inadequate predictor of vaccine susceptibility have wide-ranging implications on antigen prioritisation for the design and testing of next-generation pre-erythrocytic malaria vaccines.