A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes.

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1-G2 tether and S-M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement-loss of orthogonal orientation and ability to separate and move apart-are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

Novel insertions in the mitochondrial maxicircle of Trypanosoma musculi, a mouse trypanosome.

Trypanosoma musculi is a, globally distributed, mouse-specific haemoflagellate, of the family Trypanosomatidae, which shares similar characteristics in morphology with Trypanosoma lewisi. The kinetoplast (mitochondrial) DNA of Trypanosomatidae flagellates is comprised of catenated maxicircles and minicircles. However, genetic information on the T. musculi kinetoplast remains largely unknown. In this study, the T. musculi maxicircle genome was completely assembled, with PacBio and Illumina sequencing, and the size was confirmed at 34 606 bp. It consisted of 2 distinct parts: the coding region and the divergent regions (DRs, DRI and II). In comparison with other trypanosome maxicircles (Trypanosoma brucei, Trypanosoma cruzi and T. lewisi), the T. musculi maxicircle has a syntenic distribution of genes and shares 73.9, 78.0 and 92.7% sequence identity, respectively, over the whole coding region. Moreover, novel insertions in MURF2 (630 bp) and in ND5 (1278 bp) were found, respectively, which are homologous to minicircles. These findings support an evolutionary scenario similar to the one proposed for insertions in Trypanosoma cruzi, the pathogen of American trypanosomiasis. These novel insertions, together with a deletion (281 bp) in ND4, question the role of Complex I in T. musculi. A detailed analysis of DRII indicated that it contains numerous repeat motifs and palindromes, the latter of which are highly conservative and contain A5C elements. The comprehensively annotated kinetoplast maxicircle of T. musculi reveals a high degree of similarity between this parasite and the maxicircle of T. lewisi and suggests that the DRII could be a valuable marker for distinguishing these evolutionarily related species.

Nile tilapia (Oreochromis niloticus) can be experimentally infected with both marine and freshwater fish trypanosomes.

Trypanosomes are haemoflagellates found in vertebrate species and many of them can cause death in infected hosts including fish and humans. With the development of high-density farming in marine and freshwater fish aquaculture systems, severe disease or death, caused by trypanosomiasis, has been frequently reported. However, due to the lack of a model system, particularly for marine fish trypanosomes, and a paucity in the understanding of the biology and pathogenesis of these parasites, effective treatment for fish trypanosomiasis is significantly hampered. The goldfish is the common model system for freshwater fish trypanosomes, mainly of the species Trypanosoma carassii, while a similar model for marine fish trypanosomes has not yet been established. To address this issue, we found that Nile tilapia (Oreochromis niloticus) could be easily infected with a marine fish trypanosome, Trypanosoma epinepheli isolated from Lates calcarifer. Obvious clinical symptoms, associated with a high parasitemia (>108/ml), were found in the infected tilapias and more than 70% mortality was recorded in individuals within 20 days of infection. Interestingly, we also found that the Nile tilapia could also be infected with a freshwater fish trypanosome isolated from the largemouth bass (Micropterus salmoides) and caused significant death (more than 13%) in infected fish. This system not only provides an economical and effective laboratory model to study the biology and pathogenesis of marine and freshwater fish trypanosomes, but also provides a useful platform to develop vaccines and screen compounds for the protection and treatment of fish trypanosomiasis.

Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi.

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

Infection with Trypanosoma lewisi or Trypanosoma musculi may promote the spread of Toxoplasma gondii.

Toxoplasma gondii can infect almost all warm-blooded vertebrates with pathogensis being largely influenced by the host immune status. As important epidemiological hosts, rodents are globally distributed and are also commonly found infected with haemoflagellates, such as those in the genus Trypanosoma. We here address whether and how co-infection with trypanosomes can influence T. gondii infection in laboratory models. Rats of five strains, co-infected with T. lewisi and mice of four strains, co-infected with T. musculi, were found to be more or less susceptible to T. gondii infection, respectively, with corresponding increased or decreased brain cyst burdens. Downregulation of iNOS expression and decreased NO production or reverse were observed in the peritoneal macrophages of rats or mice, infected with trypanosomes, respectively. Trypanosoma lewisi and T. musculi can modulate host immune responses, either by enhancement or suppression and influence the outcome of Toxoplasma infection.

The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia.

Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.

Encephalitis is mediated by ROP18 of Toxoplasma gondii, a severe pathogen in AIDS patients.

The neurotropic parasite Toxoplasma gondii is a globally distributed parasitic protozoan among mammalian hosts, including humans. During the course of infection, the CNS is the most commonly damaged organ among invaded tissues. The polymorphic rhoptry protein 18 (ROP18) is a key serine (Ser)/threonine (Thr) kinase that phosphorylates host proteins to modulate acute virulence. However, the basis of neurotropism and the specific substrates through which ROP18 exerts neuropathogenesis remain unknown. Using mass spectrometry, we performed proteomic analysis of proteins that selectively bind to active ROP18 and identified RTN1-C, an endoplasmic reticulum (ER) protein that is preferentially expressed in the CNS. We demonstrated that ROP18 is associated with the N-terminal portion of RTN1-C and specifically phosphorylates RTN1-C at Ser7/134 and Thr4/8/118. ROP18 phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells. Remarkably, ROP18 phosphorylation of RTN1-C enhances glucose-regulated protein 78 (GRP78) acetylation by attenuating the activity of histone deacetylase (HDAC), and this event is associated with an increase of neural apoptosis. These results clearly demonstrate that both RTN1-C and HDACs are involved in T. gondii ROP18-mediated pathogenesis of encephalitis during Toxoplasma infection.

Non-equivalence in old- and new-flagellum daughter cells of a proliferative division in Trypanosoma brucei.

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.

Human African trypanosomiasis: the current situation in endemic regions and the risks for non-endemic regions from imported cases.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense and caused devastating epidemics during the 20th century. Due to effective control programs implemented in the last two decades, the number of reported cases has fallen to a historically low level. Although fewer than 977 cases were reported in 2018 in endemic countries, HAT is still a public health problem in endemic regions until it is completely eliminated. In addition, almost 150 confirmed HAT cases were reported in non-endemic countries in the last three decades. The majority of non-endemic HAT cases were reported in Europe, USA and South Africa, due to historical alliances, economic links or geographic proximity to disease-endemic countries. Furthermore, with the implementation of the 'Belt and Road' project, sporadic imported HAT cases have been reported in China as a warning sign of tropical diseases prevention. In this paper, we explore and interpret the data on HAT incidence and find no positive correlation between the number of HAT cases from endemic and non-endemic countries. This data will provide useful information for better understanding the imported cases of HAT globally in the post-elimination phase.

Temperature is a key factor influencing the invasion and proliferation of Toxoplasma gondii in fish cells.

Toxoplasma gondii has long been considered a ubiquitous parasite possessing the capacity of infecting virtually all warm-blooded animals globally. Occasionally, this parasite can also infect cold-blooded animals such as fish if their body temperature reaches 37 °C. However, we are currently lacking an understanding of key details such as the minimum temperature required for T. gondii invasion and proliferation in these cold-blooded animals and their cells. Here, we performed in vitro T. gondii infection experiments with rat embryo fibroblasts (REF cells), grouper (Epinephelus coioides) splenocytes (GS cells) and zebra fish (Danio rerio) hepatocytes (ZFL cells), at 27 °C, 30 °C, 32 °C, 35 °C and 37 °C, respectively. We found that T. gondii tachyzoites could penetrate REF, GS nd ZFL cells at 27 °C but clear inhibition of multiplication was observed. Intriguingly, the intracellular tachyzoites retained the ability to infect mice after 12 days of incubation in GS cells cultured at 27 °C as demonstrated by bioassay. At 30 °C, 32 °C and 35 °C, we observed that the mammalian cells (REF cells) and fish cells (GS and ZFL cells) could support T. gondii invasion and replication, which showed a temperature-dependent relationship in infection and proliferation rates. Our data demonstrated that the minimum temperature for T. gondii invasion and replication was 27 °C and 30 °C respectively, which indicated that temperature should be a key factor for T. gondii invasion and proliferation in host cells. This suggests that temperature-dependent infection determines the differences in the capability of T. gondii to infect cold- and warm-blooded vertebrates.

Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii.

Mesenchymal stromal cells (MSCs) have recently been shown to play important roles in mammalian host defenses against intracellular pathogens, but the molecular mechanism still needs to be clarified. We confirmed that human MSCs (hMSCs) prestimulated with IFN-γ showed a significant and dose-dependent ability to inhibit the growth of two types of Toxoplasma gondii [type I RH strain with green fluorescent proteins (RH/GFP) or type II PLK strain with red fluorescent proteins (PLK/RED)]. However, in contrast to previous reports, the anti-T. gondii activity of hMSCs was not mediated by indoleamine 2,3-dioxygenase (IDO). Genome-wide RNA sequencing (RNA-seq) analysis revealed that IFN-γ increased the expression of the p65 family of human guanylate-binding proteins (hGBPs) in hMSCs, especially hGBP1. To analyze the functional role of hGBPs, stable knockdowns of hGBP1, -2, and -5 in hMSCs were established using a lentiviral transfection system. hGBP1 knockdown in hMSCs resulted in a significant loss of the anti-T. gondii host defense property, compared with hMSCs infected with nontargeted control sequences. hGBP2 and -5 knockdowns had no effect. Moreover, the hGBP1 accumulation on the parasitophorous vacuole (PV) membranes of IFN-γ-stimulated hMSCs might protect against T. gondii infection. Taken together, our results suggest that hGBP1 plays a pivotal role in anti-T. gondii protection of hMSCs and may shed new light on clarifying the mechanism of host defense properties of hMSCs.

Nitric oxide blocks the development of the human parasite Schistosoma japonicum.

Human schistosomiasis, caused by Schistosoma species, is a major public health problem affecting more than 700 million people in 78 countries, with over 40 mammalian host reservoir species complicating the transmission ecosystem. The primary cause of morbidity is considered to be granulomas induced by fertilized eggs of schistosomes in the liver and intestines. Some host species, like rats (Rattus norvegicus), are naturally intolerant to Schistosoma japonicum infection, and do not produce granulomas or pose a threat to transmission, while others, like mice and hamsters, are highly susceptible. The reasons behind these differences are still a mystery. Using inducible nitric oxide synthase knockout (iNOS-/-) Sprague-Dawley rats, we found that inherent high expression levels of iNOS in wild-type (WT) rats play an important role in blocking growth, reproductive organ formation, and egg development in S. japonicum, resulting in production of nonfertilized eggs. Granuloma formation, induced by fertilized eggs in the liver, was considerably exacerbated in the iNOS-/- rats compared with the WT rats. This inhibition by nitric oxide acts by affecting mitochondrial respiration and energy production in the parasite. Our work not only elucidates the innate mechanism that blocks the development and production of fertilized eggs in S. japonicum but also offers insights into a better understanding of host-parasite interactions and drug development strategies against schistosomiasis.

The eIF3 complex of Trypanosoma brucei: composition conservation does not imply the conservation of structural assembly and subunits function.

The multisubunit eukaryotic initiation factor 3 (eIF3) plays multiple roles in translation but is poorly understood in trypanosomes. The putative subunits eIF3a and eIF3f of Trypanosoma brucei (TbIF3a and TbIF3f) were overexpressed and purified, and 11 subunits were identified, TbIF3a through l minus j, which form a tight complex. Both TbIF3a and TbIF3f are essential for the viability of T. brucei RNAi knockdown of either of them severely reduced total translation and the ratio of the polysome/80S peak area. TbIF3f and TbIF3a RNAi cell lines were modified to express tagged-TbIF3a and -TbIF3f, respectively. RNAi in combination with affinity purification assays indicated that both subunits are variably required for TbIF3 stability and integrity. The relative abundance of other subunits in the TbIF3f-tag complex changed little upon TbIF3a depletion; while only subunits TbIF3b, i, and e copurified comparably with TbIF3a-tag upon TbIF3f depletion. A genome-wide UV-crosslinking assay showed that several TbIF3 subunits have direct RNA-binding activity, with TbIF3c showing the strongest signal. In addition, CrPV IRES, but neither EMCV IRES nor HCV IRES, was found to mediate translation in T. brucei These results together imply that the structure of TbIF3 and the subunits function have trypanosome-specific features, although the composition is evolutionarily conserved.

The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo.

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients.

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii.

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.

Divergent mitochondrial respiratory chains in phototrophic relatives of apicomplexan parasites.

Four respiratory complexes and ATP-synthase represent central functional units in mitochondria. In some mitochondria and derived anaerobic organelles, a few or all of these respiratory complexes have been lost during evolution. We show that the respiratory chain of Chromera velia, a phototrophic relative of parasitic apicomplexans, lacks complexes I and III, making it a uniquely reduced aerobic mitochondrion. In Chromera, putative lactate:cytochrome c oxidoreductases are predicted to transfer electrons from lactate to cytochrome c, rendering complex III unnecessary. The mitochondrial genome of Chromera has the smallest known protein-coding capacity of all mitochondria, encoding just cox1 and cox3 on heterogeneous linear molecules. In contrast, another photosynthetic relative of apicomplexans, Vitrella brassicaformis, retains the same set of genes as apicomplexans and dinoflagellates (cox1, cox3, and cob).

[Pathogen Identification in an Imported Case of Cutaneous Leishmaniasis].

In this study, immune and molecular biological methods were used to identify the pathogen in a blood sample from a patient with dermatosis. Venous blood was collected and tested with Leish rK39 dipsticks. The lesion sample was collected and fixed in 75% ethanol, and DNA was extracted. The internal transcribed spacer 1 of rDNA and N-acetylglucosamine-1-phosphate transferase of Leishmania were amplified with PCR using primers LITSR-L5.8S and NAGTL1s-NAGTL4, respectively. The amplified products were sequenced and analyzed by BLAST. Weakly positive results were obtained for the gold-labeled Leish rK39 dipstick serological test. PCR resulted in products of 404 bp and 1 405 bp with primers LITSR-L5.8S and NAGTL1-NAGTL4, respectively. Both were 99.7% homologous to the corresponding sequence of Leishmania major. The accession number of the two sequences were KU975160 and KX150476. The case of dermatosis is diagnosed as imported cutaneous leishmaniasis and the pathogen is L. major.

Solanesyl diphosphate synthase, an enzyme of the ubiquinone synthetic pathway, is required throughout the life cycle of Trypanosoma brucei.

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.

Investigation of infectivity of neonates and adults from different rat strains to Toxoplasma gondii Prugniaud shows both variation which correlates with iNOS and Arginase-1 activity and increased susceptibility of neonates to infection.

Mouse models differ considerably from humans with regard to clinical symptoms of toxoplasmosis caused by Toxoplasma gondii and, by comparison, the rat model is more representative of this disease in humans. In the present study, we found that different strains of adult and newborn rats (Lewis, Wistar, Sprague Dawley, Brown Norway and Fischer 344) exhibited remarkable variation in the number of brain cysts following inoculation with the T.gondii Prugniaud strain. In adult rats, large numbers of cysts (1231 ± 165.6) were observed in Fischer 344, but none in the other four. This situation was different in newborn rats aged from 5 to 20 days old. All Fischer 344 and Brown Norway newborns were cyst-positive while cyst-positive infection in Sprague Dawley neonates ranged from 54.5% to 60% depending on their age at infection. In Wistar and Lewis rat neonates, however, cyst-positivity rates of 0-42.9% and 0-25% were found respectively. To investigate whether rat strain differences in infectivity could be related to inherent strain and genetic differences in the host immune response, we correlated our data with previously reported strain differences in iNOS/Arginase ratio in adult rats and found them to be linked. These results show that interactions between host genetic background and age of rat influence T.gondii infection.

Split photosystem protein, linear-mapping topology, and growth of structural complexity in the plastid genome of Chromera velia.

The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here, we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulsed-field gel electrophoresis and polymerase chain reaction, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions, and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function.