Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

Apicomplexa micropore: history, function, and formation.

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.

The Toxoplasma monocarboxylate transporters are involved in the metabolism within the apicoplast and are linked to parasite survival.

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

Towards disentangling the classification of freshwater fish trypanosomes.

Currently, new species of freshwater fish trypanosomes, which are economically important parasites, are being described based on subjectively selected features, i.e., their cell morphology and the host species. We have performed detailed phylogenetic and haplotype diversity analyses of all 18S rRNA genes available for freshwater fish trypanosomes, including the newly obtained sequences of Trypanosoma carassii and Trypanosoma danilewskyi. Based on a sequence similarity of 99.5%, we divide these trypanosomes into 15 operational taxonomic units, and propose three nominal scenarios for distinguishing T. carassii and other aquatic trypanosomes. We find evidences for the existence of a low number of freshwater fish trypanosomes, with T. carassii having the widest geographic and host ranges. Our analyses support the existence of an umbrella complex composed of T. carassii and two sister species. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00191-0.

Functional screening reveals Toxoplasma prenylated proteins required for endocytic trafficking and rhoptry protein sorting.

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

Cryo-Electron Tomography of Toxoplasma gondii Indicates That the Conoid Fiber May Be Derived from Microtubules.

Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis and can infect numerous warm-blooded animals. An improved understanding of the fine structure of this parasite can help elucidate its replication mechanism. Previous studies have resolved the ultrastructure of the cytoskeleton using purified samples, which eliminates their cellular context. Here the application of cryo-electron tomography to visualize T. gondii tachyzoites in their native state is reported. The fine structure and cellular distribution of the cytoskeleton are resolved and analyzed at nanometer resolution. Additionally, the tachyzoite structural characteristics are annotated during its endodyogeny for the first time. By comparing the structural features in mature tachyzoites and their daughter buds, it is proposed that the conoid fiber of the Apicomplexa originates from microtubules. This work represents the detailed molecular anatomy of T. gondii, particularly during the budding replication stage of tachyzoite, and provides a reference for further studies of this fascinating organism.

The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments.

Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.

Does the fish-infecting Trypanosoma micropteri belong to Trypanosoma carassii?

Recently, based on a limited morphological characterisation and partial 18S rRNA gene sequence, Jiang et al. (2019) described Trypanosoma micropteri Jiang, Lu, Du, Wang, Hu, Su et Li, 2019 as a new pathogen of farmed fish. Here we provide evidence based on the expanded sequence dataset, morphology and experimental infections that this trypanosome does not warrant the establishment as a new species, because it is conspecific with the long-term known Trypanosoma carassii Mitrophanow, 1883, a common haemoflagellate parasite of freshwater fish. The former taxon thus becomes a new junior synonym of T. carassii.

Inflammasome Activation Dampens Type I IFN Signaling to Strengthen Anti-Toxoplasma Immunity.

Innate immunity acts as the first line of defense against pathogen invasion. During Toxoplasma gondii infection, multiple innate immune sensors are activated by invading microbes or pathogen-associated molecular patterns (PAMPs). However, how inflammasome is activated and its regulatory mechanisms during T. gondii infection remain elusive. Here, we showed that the infection of PRU, a lethal type II T. gondii strain, activates inflammasome at the early stage of infection. PRU tachyzoites, RNA and soluble tachyzoite antigen (STAg) mainly triggered the NLRP3 inflammasome, while PRU genomic DNA (gDNA) specially activated the AIM2 inflammasome. Furthermore, mice deficient in AIM2, NLRP3, or caspase-1/11 were more susceptible to T. gondii PRU infection, and the ablation of inflammasome signaling impaired antitoxoplasmosis immune responses by enhancing type I interferon (IFN-I) production. Blockage of IFN-I receptor fulfilled inflammasome-deficient mice competent immune responses as WT mice. Moreover, we have identified that the suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator induced by inflammasome-activated IL-1ß signaling and inhibits IFN-I production by targeting interferon regulatory factor 3 (IRF3). In general, our study defines a novel protective role of inflammasome activation during toxoplasmosis and identifies a critical regulatory mechanism of the cross talk between inflammasome and IFN-I signaling for understanding infectious diseases. IMPORTANCE As a key component of innate immunity, inflammasome is critical for host antitoxoplasmosis immunity, but the underlying mechanisms are still elusive. In this study, we found that inflammasome signaling was activated by PAMPs of T. gondii, which generated a protective immunity against T. gondii invasion by suppressing type I interferon (IFN-I) production. Mechanically, inflammasome-coupled IL-1ß signaling triggered the expression of negative regulator SOCS1, which bound to IRF3 to inhibit IFN-I production. The role of IFN-I in anti-T. gondii immunity is little studied and controversial, and here we also found IFN-I is harmful to host antitoxoplasmosis immunity by using knockout mice and recombinant proteins. In general, our study identifies a protective role of inflammasomes to the host during T. gondii infection and a novel mechanism by which inflammasome suppresses IFN-I signaling in antitoxoplasmosis immunity, which will likely provide new insights into therapeutic targets for toxoplasmosis and highlight the cross talk between innate immune signaling in infectious diseases prevention.

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.

iNOS is essential to maintain a protective Th1/Th2 response and the production of cytokines/chemokines against Schistosoma japonicum infection in rats.

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-ß3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis.

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27-37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China.

BACKGROUND: Visceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics. METHODOLOGY/PRINCIPAL FINDINGS: Here, an eleven-year survey (2004-2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship. CONCLUSIONS/SIGNIFICANCE: The above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.

High resistance to Toxoplasma gondii infection in inducible nitric oxide synthase knockout rats.

Nitric oxide (NO) is an important immune molecule that acts against extracellular and intracellular pathogens in most hosts. However, after the knockout of inducible nitric oxide synthase (iNOS -/-) in Sprague Dawley (SD) rats, these iNOS -/- rats were found to be completely resistant to Toxoplasma gondii infection. Once the iNOS -/- rat peritoneal macrophages (PMs) were infected with T. gondii, they produced high levels of reactive oxygen species (ROS) triggered by GRA43 secreted by T. gondii, which damaged the parasitophorous vacuole membrane and PM mitochondrial membranes within a few hours post-infection. Further evidence indicated that the high levels of ROS caused mitochondrial superoxide dismutase 2 depletion and induced PM pyroptosis and cell death. This discovery of complete resistance to T. gondii infection, in the iNOS -/--SD rat, demonstrates a strong link between NO and ROS in immunity to T. gondii infection and showcases a potentially novel and effective backup innate immunity system.

Innate Resistance to Leishmania amazonensis Infection in Rat Is Dependent on NOS2.

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2 -/- rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2 -/- rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

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.

Knockout of NOS2 Promotes Adipogenic Differentiation of Rat MSCs by Enhancing Activation of JAK/STAT3 Signaling.

Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells that possess multilineage differentiation potential and extensive immunomodulatory properties. In mice and rats, MSCs produce nitric oxide (NO), as immunomodulatory effector molecule that exerts an antiproliferative effect on T cells, while the role of NO in differentiation was less clear. Here, we investigated the role of NO synthase 2 (NOS2) on adipogenic and osteogenic differentiation of rat MSCs. MSCs isolated from NOS2-null (NOS2-/-) and wild type (WT) Sprague-Dawley (SD) rats exhibited homogenous fibroblast-like morphology and characteristic phenotypes. However, after induction, adipogenic differentiation was found significantly promoted in NOS2-/- MSCs compared to WT MSCs, but not in osteogenic differentiation. Accordingly, qRT-PCR revealed that the adipogenesis-related genes PPAR-γ, C/EBP-α, LPL and FABP4 were markedly upregulated in NOS2-/- MSCs, but not for osteogenic transcription factors or marker genes. Further investigations revealed that the significant enhancement of adipogenic differentiation in NOS2-/- MSCs was due to overactivation of the STAT3 signaling pathway. Both AG490 and S3I-201, small molecule inhibitors that selectively inhibit STAT3 activation, reversed this adipogenic effect. Furthermore, after high-fat diet (HFD) feeding, knockout of NOS2 in rat MSCs resulted in significant obesity. In summary, NOS2 is involved in the regulation of rat MSC adipogenic differentiation via the STAT3 signaling pathway.

Identification of an orally active carbazole aminoalcohol derivative with broad-spectrum anti-animal trypanosomiasis activity.

Animal trypanosomiasis, caused by the members of subgenus Trypanozoon (Trypanosoma brucei brucei, T. evansi and T. equiperdum), has reduced animal productivity leading to significant negative economic impacts in endemic regions. Due to limited drug discovery and the emergence of drug-resistance over many recent decades, novel and effective compounds against animal trypanosomiasis are urgently required. This study was conducted to evaluate the antitrypanosomal potential of a batch of carbazole aminoalcohol derivatives. Among them, we found that the most effective compound was H1402, which exhibited potent trypanocidal efficacy against the bloodstream-form of T. b. brucei (EC50 = 0.73 ± 0.05 µM) and presented low cytotoxicity against two mammalian cell lines with CC50 > 30 µM. Using a murine model of acute infection, oral administration with H1402 demonstrated a complete clearance of T. b. brucei and all the infected mice were cured when they were treated twice daily for 5 days at a dose of 100 mg/kg. Furthermore, parasites were not detected in mice infected with T. evansi and T. equiperdum (the causative agents of surra and dourine, respectively, in animals) within 30 days following the same regimen with H1402. In addition, H1402 caused severe morphological and ultrastructural destruction to trypanosomes, as well as causing phosphatidylserine externalization, which are suggested to be the most likely cause of cell death. Overall, the present data demonstrated that H1402 could be promising as a rapid, safe and orally active lead compound for the development of new chemotherapeutics for animal trypanosomiasis.

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.