Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei

BACKGROUND Vacuolar H-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. METHODS In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. RESULTS TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. CONCLUSIONS TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum

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 T. b. 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, United States 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

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

Prevalence and co-infection of Toxoplasma gondii and Neospora caninum in Apodemus sylvaticus in an area relatively free of cats

The protozoan parasite Toxoplasma gondii is prevalent worldwide and can infect a remarkably wide range of hosts despite felids being the only definitive host. As cats play a major role in transmission to secondary mammalian hosts, the interaction between cats and these hosts should be a major factor determining final prevalence in the secondary host. This study investigates the prevalence of T. gondii in a natural population of Apodemus sylvaticus collected from an area with low cat density (<2·5 cats/km2). A surprisingly high prevalence of 40·78% (95% CI: 34·07%–47·79%) was observed despite this. A comparable level of prevalence was observed in a previously published study using the same approaches where a prevalence of 59% (95% CI: 50·13%–67·87%) was observed in a natural population of Mus domesticus from an area with high cat density (>500 cats/km2). Detection of infected foetuses frompregnant dams in both populations suggests that congenital transmission may enable persistence of infection in the absence of cats. The prevalences of the related parasite, Neospora caninum were found to be low in both populations (A. sylvaticus: 3·39% (95% CI: 0·12%–6·66%); M. domesticus: 3·08% (95% CI: 0·11%–6·05%)). These results suggest that cat density may have a lower than expected effect on final prevalence in these ecosystems

Quantum Melting and Absence of Bose-Einstein Condensation in Two-Dimensional Vortex Matter

We demonstrate that quantum fluctuations suppress Bose-Einstein condensation of quasi-two-dimensional bosons in a rapidly-rotating trap. Our conclusions rest in part on an effective-Lagrangian description of the triangular vortex lattice, and in part on microscopic Bogoliubov equations in the rapid-rotation limit. We obtain analytic expressions for the collective-excitation dispersion, which, in a rotating system, is quadratic rather than linear. Our estimates for the boson filling factor at which the vortex lattice melts at zero temperature due to quantum fluctuations are consistent with recent exact-diagonalization calculations.Comment: 4 pages, 1 figures, version to appear in Phys. Rev. Let

Investigation into the genetic diversity in toll-like receptors 2 and 4 in the European badger Meles meles

The Toll-like receptor (TLR) genes are a conserved family of genes central to the innate immune response to pathogen infection. They encode receptor proteins, recognise pathogen associated molecular patterns (PAMPs) and trigger initial immune responses. In some host-pathogen systems, it is reported that genetic differences, such as single nucleotide polymorphisms (SNPs), associate with disease resistance or susceptibility. Little is known about TLR gene diversity in the European badger (Meles meles). We collected DNA from UK badgers, carried out PCR amplification of the badger TLR2 gene and exon 3 of TLR4 and determined DNA sequences for individual badgers for TLR2 (n=61) and TLR4 exon 3 (n=59). No polymorphism was observed in TLR4. Three TLR2 amino acid haplotype variants were found. Ninety five percent of badgers were homozygous for one common haplotype (H1), the remaining three badgers had genotypes H1/H3, H1/H2 and H2/H2. By broad comparison with other species, diversity in TLR genes in badgers seems low. This could be due to a relatively localised sampling or inherent low genetic diversity. Further studies are required to assess the generality of the low observed diversity and the relevance to the immunological status of badgers

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 occurrence of malignancy in Trypanosoma brucei brucei by rapid passage in mice

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals

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 is 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. [Abstract copyright: Copyright © 2020. Published by Elsevier Inc.

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. Down-regulation 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