Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Parasitic egg recognition using convolution and attention network.

Intestinal parasitic infections (IPIs) caused by protozoan and helminth parasites are among the most common infections in humans in low-and-middle-income countries. IPIs affect not only the health status of a country, but also the economic sector. Over the last decade, pattern recognition and image processing techniques have been developed to automatically identify parasitic eggs in microscopic images. Existing identification techniques are still suffering from diagnosis errors and low sensitivity. Therefore, more accurate and faster solution is still required to recognize parasitic eggs and classify them into several categories. A novel Chula-ParasiteEgg dataset including 11,000 microscopic images proposed in ICIP2022 was utilized to train various methods such as convolutional neural network (CNN) based models and convolution and attention (CoAtNet) based models. The experiments conducted show high recognition performance of the proposed CoAtNet that was tuned with microscopic images of parasitic eggs. The CoAtNet produced an average accuracy of 93%, and an average F1 score of 93%. The finding opens door to integrate the proposed solution in automated parasitological diagnosis.

VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles.

Trichomonas vaginalis is a common sexually transmitted extracellular parasite that adheres to epithelial cells in the human urogenital tract. Extracellular vesicles (EVs) have been described as important players in the pathogenesis of this parasite as they deliver proteins and RNA into host cells and modulate parasite adherence. EVs are heterogeneous membrane vesicles released from virtually all cell types that collectively represent a new dimension of intercellular communication. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery contributes to several key mechanisms in which it reshapes membranes. Based on this, some components of the ESCRT have been implicated in EVs biogenesis in other cells. Here, we demonstrated that VPS32, a member of ESCRTIII complex, contribute to the biogenesis and cargo sorting of extracellular vesicles in the parasite T. vaginalis. Moreover, we observe that parasites overexpressing VPS32 have a striking increase in adherence to host cells compared to control parasites; demonstrating a key role for this protein in mediating host: parasite interactions. These results provide valuable information on the molecular mechanisms involved in extracellular vesicles biogenesis, cargo-sorting, and parasite pathogenesis.

Trogocytosis in Unicellular Eukaryotes.

Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a whole cell or a particle. Trogocytosis has been demonstrated in a broad range of cell types in multicellular organisms and is also known to be involved in a plethora of functions. In immune cells, trogocytosis is involved in the "cross-dressing" between antigen presenting cells and T cells, and is thus considered to mediate intercellular communication. On the other hand, trogocytosis has also been reported in a variety of unicellular organisms including the protistan (protozoan) parasite Entamoeba histolytica. E. histolytica ingests human T cell line by trogocytosis and acquires complement resistance and cross-dresses major histocompatibility complex (MHC) class I on the cell surface. Furthermore, trogocytosis and trogocytosis-like phenomena (nibbling of a live cell, not previously described as trogocytosis) have also been reported in other parasitic protists such as Trichomonas, Plasmodium, Toxoplasma, and free-living amoebae. Thus, trogocytosis is conserved in diverse eukaryotic supergroups as a means of intercellular communication. It is depicting the universality of trogocytosis among eukaryotes. In this review, we summarize our current understanding of trogocytosis in unicellular organisms, including the history of its discovery, taxonomical distribution, roles, and molecular mechanisms.

Epidemiology of Intestinal Parasites in a University Hospital in Northern Cyprus: A 4-year Retrospective Experience

Objective: Intestinal parasitic infections (IPI) are considered as one of the most important public health problems that cause morbidity and mortality. For this reason, to determine their prevalence it is critical for prevention. This study aimed to determine the prevalence of intestinal parasites. Methods: In our study, a total of 4.957 patients registered to our hospital with gastrointestinal symptoms between January 2016 and December 2019 were retrospectively analysed. Their stool samples were examined macroscopically and microscopically. In the microscopy, native-lugol and formol ethyl acetate concentration methods were used. Crypto-Giardia-Entamoeba antigen test was applied. All cases were evaluated in terms of age, gender, year and season. Results: In our study group, 239 (4.8%) patients were detected as positive for intestinal parasites. Among these patients, 129 (54%) were male and 110 (46%) were female. No statistically significant difference was found between IPI and gender (p=0.228). Blastocystis hominis (76.2%) and Giardia intestinalis (12.1%) were the most common parasites. According to age groups, most intestinal parasites are found in 16-45 years old and least in 0-15-years-old (p=0.0001). A significant increase was found in positive intestinal parasite cases especially after 2018 (p=0.0001). Our study determined that intestinal parasites were observed most frequently in autumn (p=0.033). Conclusion: The prevalence of IPI in our country is low. However, due to the increasing trend of IPI since 2018, necessary measures must be implemented to prevent further increase in the number of cases. In addition, reasons behind the rising cases of intestinal parasites during the autumn months in which rainfall begins require further investigation.

Parallel functional reduction in the mitochondria of apicomplexan parasites.

Gregarines are an early-diverging lineage of apicomplexan parasites that hold many clues into the origin and evolution of the group, a remarkable transition from free-living phototrophic algae into obligate parasites of animals.1 Using single-cell transcriptomics targeting understudied lineages to complement available sequencing data, we characterized the mitochondrial metabolic repertoire across the tree of apicomplexans. In contrast to the large suite of proteins involved in aerobic respiration in well-studied parasites like Toxoplasma or Plasmodium,2 we find that gregarine trophozoites have significantly reduced energy metabolism: most lack respiratory complexes III and IV, and some lack the electron transport chains (ETCs) and tricarboxylic acid (TCA) cycle entirely. Phylogenomic analyses show that these reductions took place several times in parallel, resulting in a functional range from fully aerobic organelles to extremely reduced "mitosomes" restricted to Fe-S cluster biosynthesis. The mitochondrial genome has also been lost repeatedly: in species with severe functional reduction simply by gene loss but in one species with a complete ETC by relocating cox1 to the nuclear genome. Severe functional reduction of mitochondria is generally associated with structural reduction, resulting in small, nondescript mitochondrial-related organelles (MROs).3 By contrast, gregarines retain distinctive mitochondria with tubular cristae, even the most functionally reduced cases that also lack genes associated with cristae formation. Overall, the parallel, severe reduction of gregarine mitochondria expands the diversity of organisms that contain MROs and further emphasizes the role of parallel transitions in apicomplexan evolution.

New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa.

A survey for species of the genus Trichoderma occurring as endophytes of Coffea, and as mycoparasites of coffee rusts (Hemileia), was undertaken in Africa; concentrating on Cameroon and Ethiopia. Ninety-four isolates of Trichoderma were obtained during this study: 76 as endophytes of healthy leaves, stems and berries and, 18 directly from colonized rust pustules. A phylogenetic analysis of all isolates used a combination of three genes: translation elongation factor-1α (tef1), rpb2 and cal for selected isolates. GCPSR criteria were used for the recognition of species; supported by morphological and cultural characters. The results reveal a previously unrecorded diversity of Trichoderma species endophytic in both wild and cultivated Coffea, and mycoparasitic on Hemileia rusts. Sixteen species were delimited, including four novel taxa which are described herein: T. botryosum, T. caeruloviride, T. lentissimum and T. pseudopyramidale. Two of these new species, T. botryosum and T. pseudopyramidale, constituted over 60% of the total isolations, predominantly from wild C. arabica in Ethiopian cloud forest. In sharp contrast, not a single isolate of Trichoderma was obtained using the same isolation protocol during a survey of coffee in four Brazilian states, suggesting the existence of a 'Trichoderma void' in the endophyte mycobiota of coffee outside of Africa. The potential use of these African Trichoderma isolates in classical biological control, either as endophytic bodyguards-to protect coffee plants from Hemileia vastatrix, the fungus causing coffee leaf rust (CLR)-or to reduce its impact through mycoparasitism, is discussed, with reference to the on-going CLR crisis in Central America.

Promises and Pitfalls of Parasite Patch-clamp.

Protozoan parasites acquire essential ions, nutrients, and other solutes from their insect and vertebrate hosts by transmembrane uptake. For intracellular stages, these solutes must cross additional membranous barriers. At each step, ion channels and transporters mediate not only this uptake but also the removal of waste products. These transport proteins are best isolated and studied with patch-clamp, but these methods remain accessible to only a few parasitologists due to specialized instrumentation and the required training in both theory and practice. Here, we provide an overview of patch-clamp, describing the advantages and limitations of the technology and highlighting issues that may lead to incorrect conclusions. We aim to help non-experts understand and critically assess patch-clamp data in basic research studies.

Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

New Lineage of Microbial Predators Adds Complexity to Reconstructing the Evolutionary Origin of Animals.

The origin of animals is one of the most intensely studied evolutionary events, and our understanding of this transition was greatly advanced by analyses of unicellular relatives of animals, which have shown many "animal-specific" genes actually arose in protistan ancestors long before the emergence of animals [1-3]. These genes have complex distributions, and the protists have diverse lifestyles, so understanding their evolutionary significance requires both a robust phylogeny of animal relatives and a detailed understanding of their biology [4, 5]. But discoveries of new animal-related lineages are rare and historically biased to bacteriovores and parasites. Here, we characterize the morphology and transcriptome content of a new animal-related lineage, predatory flagellate Tunicaraptor unikontum. Tunicaraptor is an extremely small (3-5 µm) and morphologically simple cell superficially resembling some fungal zoospores, but it survives by preying on other eukaryotes, possibly using a dedicated but transient "mouth," which is unique for unicellular opisthokonts. The Tunicaraptor transcriptome encodes a full complement of flagellar genes and the flagella-associated calcium channel, which is only common to predatory animal relatives and missing in microbial parasites and grazers. Tunicaraptor also encodes several major classes of animal cell adhesion molecules, as well as transcription factors and homologs of proteins involved in neurodevelopment that have not been found in other animal-related lineages. Phylogenomics, including Tunicaraptor, challenges the existing framework used to reconstruct the evolution of animal-specific genes and emphasizes that the diversity of animal-related lineages may be better understood only once the smaller, more inconspicuous animal-related lineages are better studied. VIDEO ABSTRACT.

Spermatological characteristics of Sclerodistomoides pacificus (Digenea, Sclerodistomoididae) a parasite of the flying fish Cheilopogon pinnatibarbatus (Teleostei, Exocoetidae).

Sclerodistomoides pacificus is the only species described now in Sclerodistomoididae. We present in this paper the first ultrastructural data of the mature spermatozoon of a species from the genus Sclerodistomoides. Adult specimens of S. pacificus (Digenea: Hemiuroidea: Sclerodistomoididae), were parasites of the gall-bladder of the teleost fish Cheilopogon pinnatibarbatus captured in the Atlantic Ocean, near Dakar (Senegal). The male gamete is a filiform cell which exhibits a similar ultrastructural organization to that reported in most species belonging to the Hemiuroidea with two axonemes of the 9 + '1' pattern of trepaxonematans, a nucleus, a mitochondrion, external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior region of the spermatozoon, and parallel cortical microtubules disposed in one side of the spermatozoon. However, the present study allowed describing for the first time a moniliform mitochondrion in the Hemiuroidea. The presence of a moniliform mitochondrion and the absence of filamentous external ornamentation described in other Hemiuridae: Lecithochirium microstomum, L. musculus and Hemiurus appendiculatus are a good tool for phylogenetic purposes in the Hemiuroidea. Moreover, spermatological organisation and model are discussed in context with those of previous studies in the Hemiuroidea.

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Isolation and Functions of Extracellular Vesicles Derived from Parasites: The Promise of a New Era in Immunotherapy, Vaccination, and Diagnosis.

Experimental and epidemiological evidence shows that parasites, particularly helminths, play a central role in balancing the host immunity. It was demonstrated that parasites can modulate immune responses via their excretory/secretory (ES) and some specific proteins. Extracellular vesicles (EVs) are nano-scale particles that are released from eukaryotic and prokaryotic cells. EVs in parasitological studies have been mostly employed for immunotherapy of autoimmune diseases, vaccination, and diagnosis. EVs can carry virulence factors and play a central role in the development of parasites in host cells. These molecules can manipulate the immune responses through transcriptional changes. Moreover, EVs derived from helminths modulate the immune system via provoking anti-inflammatory cytokines. On the other hand, EVs from parasite protozoa can induce efficient immunity, that makes them useful for probable next-generation vaccines. In addition, it seems that EVs from parasites may provide new diagnostic approaches for parasitic infections. In the current study, we reviewed isolation methods, functions, and applications of parasite's EVs in immunotherapy, vaccination, and diagnosis.

New species of Laboulbenia (Laboulbeniales, Ascomycota) on Gerridae (Hemiptera, Insecta), a new host family.

Four new species of Laboulbenia (Laboulbeniales, Ascomycota) occurring on Gerridae (Hemiptera, Insecta), a new host family, are described from six Central and South American countries: Bolivia, Brazil, Ecuador, Panama, Peru, and Venezuela. The new species are Laboulbenia brachymetrae, L. cylindrostethi, L. neogerris, and L. tachygerris.

Taxonomic notes on eight species of obligate mycoparasites in the genus Syncephalis isolated from soil and dung.

Species of Syncephalis (Zoopagomycotina, Piptocephalidaceae) are obligate mycoparasites that grow on common saprobic species of Mortierellomycotina and Mucoromycotina in soil and dung. Despite their ubiquitous occurrence across the globe, fungi in the genus Syncephalis are understudied, and there are few modern taxonomic treatments of these fungi. In order to clarify species concepts in the genus, we provide morphological data and discuss seven classical Syncephalis species: S. basibulbosa, S. cordata, S. depressa, S. hypogena, S. intermedia, S. nodosa, and S. sphaerica. Three of these species are only known as herbarium specimens (S. basibulbosa, S. cordata, S. intermedia). We have isolated co-cultures of the remaining parasites (S. depressa, S. nodosa, and S. sphaerica) on their host fungi both from nature and from culture collections. The remaining taxon (S. hypogena) was revived from a lyophilized culture. We provide photos and updated descriptions for all of these species as well as new geographic data and references to documented herbarium specimens for each taxon. In addition, we also describe the new species S. latigena.

Comparison of Light Microscopy and Polymerase Chain Reaction for the Detection of Haemoparasites in Cattle in Nigeria.

PURPOSE: Haemoparasitic diseases are among the important factors that threaten cattle health and productivity especially in the sub-Saharan region. In Nigeria, their detection using sensitive molecular techniques is scanty. This study was designed to investigate and to reevaluate the repertoire of haemoparasites of cattle in Ibadan, Nigeria with a comparative evaluation of light microscopy (LM) and polymerase chain reaction (PCR) methods. METHODS: Blood samples from 100 cattle slaughtered at Ibadan abattoirs were examined using LM and PCR techniques for haemoparasite detection. The PCR reactions using three primer sets targeting the 16S rRNA genes for Hemoplasma spp. and Anaplasma/Ehrlichia spp. and 18S rRNA genes of Babesia/Theleiria spp. were done. A few randomly selected amplicons from each set were sequenced and analysed. RESULTS: A total infection rate of 34% by LM including Hemoplasma spp. (17%), Anaplasma spp. (16%), microfilaria (5%) and Trypanosoma spp. (12%) was recorded. While, 86% positivity was recorded with PCR amplification as follows: Hemoplasma spp. (64%), Babesia/Theleiria spp. (46%) and Anaplasma/Ehrlichia spp. (5%). Comparison of LM and PCR findings showed that no LM Anaplasma spp.-positive samples and 7 out of the 17 LM hemoplasma-positive cattle were confirmed by PCR. In addition, LM led to misdiagnosis of 46 Babesia/Theleiria spp.-positive samples. Amplicon sequencing and phylogenetic analysis of Babesia/Theileria spp.-positive samples revealed Theileria velifera and Theileria annulata. In the Anaplasma/Ehrlichia spp.-positive samples, only Anaplasma marginale was characterized. Mycoplasma wenyonii, "Candidatus Mycoplasma haemobos" and Pseudomonas fluorescens like were characterized among the hemoplasma-infected cattle. CONCLUSIONS: The first report of "Candidatus Mycoplasma haemobos" and Pseudomonas fluorescens like in Nigerian cattle is herewith documented. The alarming LM misdiagnosis of haemoparasites during this study confirms its limitations as it fails to identify many parasites and emphasizes the need for inclusion of molecular techniques to improve their detection. The study also shows for the first time the high prevalence of haemotropic mycoplasma in Nigerian cattle via molecular diagnostic methods, thus indicating a strong need for the investigation of their zoonotic implications.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

[Advances in researches of exosomes and other extracellular vesicles in parasites and parasitic diseases].

Exosomes are tiny vesicles secreted by most endogenous cells, and the extracellular vesicles (EVs) are specifically secreted by cells. Recently, it was found that exosomes contain a large quantity of important substances such as proteins, nucleic acids, and lipids, which play important roles in material exchange and information transmission in cell-cell communication, and in modulating the immune response, metabolism, and expansion, metastasis, and drug resistance of tumors. This paper summarizes the recent researches on exosomes in parasites and parasitic diseases and hopes to be helpful for improving the researches of parasites and parasitic diseases.

Application of Autofluorescence for Confocal Microscopy to Aid in Archaeoparasitological Analyses.

Confocal laser scanning microscopy (CLSM) was used to examine archaeoparasitological specimens from coprolites associated with La Cueva de los Muertos Chiquitos (CMC) located near present-day Durango, Mexico. The eggs for 4 different types of parasites recovered from CMC coprolites were imaged using CLSM to assist with identification efforts. While some of the parasite eggs recovered from CMC coprolites were readily identified using standard light microscopy (LM), CLSM provided useful data for more challenging identifications by highlighting subtle morphological features and enhancing visualization of parasite egg anatomy. While other advanced microscopy techniques, such as scanning electron microscopy (SEM), may also detect cryptic identifying characters, CLSM is less destructive to the specimens. Utilizing CLSM allows for subsequent examinations, such as molecular analyses, that cannot be performed following SEM sample preparation and imaging. Furthermore, CLSM detects intrinsic autofluorescence molecules, making improved identification independent of resource and time-intensive protocols. These aspects of CLSM make it an excellent method for assisting in taxonomic identification and for acquiring more detailed images of archaeoparasitological specimens.