Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Genomics and morphometrics reveal the adaptive evolution of pikas.

Pikas (Lagomorpha: Ochotonidae) are small mouse-like lagomorphs. To investigate their adaptation to different ecological environments during their dispersal from the Qinghai-Xizang (Tibet) Plateau (QTP), we collected 226 pikas and measured 20 morphological characteristics and recorded habitat information. We also sequenced the genome of 81 specimens, representing 27 putative pika species. The genome-wide tree based on 4 090 coding genes identified five subgenera, i.e., Alienauroa, Conothoa, Lagotona, Ochotona, and Pika, consistent with morphometric data. Morphologically, Alienauroa and Ochotona had similar traits, including smaller size and earlier divergence time compared to other pikas. Consistently, the habitats of Alienauroa and Ochotona differed from those of the remaining subgenera. Phylogenetic signal analysis detected 83 genes significantly related to morphological characteristics, including several visual and hearing-related genes. Analysis of shared amino acid substitutions and positively selected genes (PSGs) in Alienauroa and Ochotona identified two genes, i.e., mitochondrial function-related TSFM (p.Q155E) and low-light visual sensitivity-related PROM1 (p.H419Y). Functional experiments demonstrated that TSFM-155E significantly enhanced mitochondrial function compared to TSFM-155Q in other pikas, and PROM1-419Y decreased the modeling of dynamic intracellular chloride efflux upon calcium uptake. Alienauroa and Ochotona individuals mostly inhabit different environments (e.g., subtropical forests) than other pikas, suggesting that a shift from the larger ancestral type and changes in sensory acuity and energy enhancement may have been required in their new environments. This study increases our understanding of the evolutionary history of pikas.

Perfect diffractive circular metagrating for Bessel beam transformation.

Bessel beams, with their non-diffractive property, have attracted great interest in recent years. Optical needle shaping of Bessel beams is highly desired in many applications, however, this typically requires low numerical aperture (NA) bulky 4f confocal systems incorporated with spatial light modulators or round filters. Here, we employ a circular dielectric metagrating for perfect Bessel beam transformation at a desired wavelength. The dielectric metagrating exhibits a high transmissive diffraction efficiency (up to 75%) for a broadband (460 nm to 560 nm), wide-angle range, and dual-polarization response, which is capable of a high-performance transformation of Bessel beams with arbitrary NAs. Our results show potential for special-beam-required applications such as light storage, imaging, and optical manipulation.

Characterisation of novel functionality within the Blastocystis tryptophanase gene.

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.

Salt-inducible kinase inhibition sensitizes human acute myeloid leukemia cells to all-trans retinoic acid-induced differentiation.

Differentiation therapies with all-trans retinoic acid (ATRA) have been successful in treating acute promyelocytic leukemia, a rare subtype of acute myeloid leukemia (AML). However, their efficacy is limited in the case of other AML subtypes. Here, we show that the combination of ATRA with salt-inducible kinase (SIK) inhibition significantly enhances ATRA-mediated AML differentiation. SIK inhibition augmented the ability of ATRA to induce growth inhibition and G1 cell cycle arrest of AML cells. Moreover, combining ATRA and SIK inhibition synergistically activated the Akt signaling pathway but not the MAPK pathway. Pharmacological blockade of Akt activity suppressed the combination-induced differentiation, indicating an essential role for Akt in the action of the combination treatment. Taken together, our study reveals a novel role for SIK in the regulation of ATRA-mediated AML differentiation, implicating the combination of ATRA and SIK inhibition as a promising approach for future differentiation therapy.

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.

The complete mitochondrial genome of Carpodacus pulcherrimus (Passeriformes: Fringillidae).

The Himalayan Beautiful Rosefinch Carpodacus pulcherrimus, belongs to the family Fringillidae, distributed in central Himalayas from India (Himachal Pradesh) to southwest China and Bhutan. The conservation status of this species is least concern (LC) in IUCN. In this study, the complete mitogenome of C. pulcherrimus was determined. The mitogenome is a circular molecule of 16,797 bp in length, containing 13 protein-coding genes, 2 ribosome RNA genes, 22 transfer RNA genes, and 1 non-coding region. We reconstructed a phylogenetic tree based on Bayesian inference for other 14 Fringillidae species. The new mitogenome data would provide useful information for application in conservation genetics and further clarify phylogenetic evolution of this species.

The complete mitochondrial genome of Phoenicurus frontalis (Passeriformes: Muscicapidae).

The Blue-fronted Redstart Phoenicurus frontalis (Muscicapidae) belongs to the family Muscicapidae, distributed in central China, Qinghai-Tibet plateau and the Himalayas. The conservation status of this species is Least Concern (LC) in IUCN. In this study, the complete mitogenome of P. frontalis was determined. The mitogenome is a circular molecule of 16,776 bp in length, containing 13 protein-coding genes, 2 ribosome RNA genes, 22 transfer RNA genes, and 1 non-coding region. We reconstructed a phylogenetic tree based on Bayesian inference for 15 Passeriformes species. The new mitogenome data would provide useful information for application in conservation genetics and further clarify the phylogenetic evolution of this species.

Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes.

The microbial parasite Blastocystis colonizes the large intestines of numerous animal species and increasing evidence has linked Blastocystis infection to enteric diseases with signs and symptoms including abdominal pain, constipation, diarrhea, nausea, vomiting, and flatulence. It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of Blastocystis cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for Blastocystis subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5' UTR was identified. Using this promoter combined with the legumain 3' UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in Blastocystis. This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for Blastocystis is a significant advance for Blastocystis research both in vitro and in vivo. This technique will spearhead further research to understand the parasite's biology, its role in health and disease, along with novel ways to combat the parasite.

Complete mitochondrial genome of Procapra picticaudata, with phylogenetic implication.

The Tibetan gazelle Procapra picticaudata is endemic to the Tibetan plateau. The species is listed as a Near Threatened (NT) species by the IUCN Red List of Threatened Animals and the Red List of China's Vertebrates. In this study, we sequenced the complete mitochondrial genome of P. picticaudata and examined its phylogenetic position with other nine species in Artiodactyla. The complete mitochondrial genome is 16,620 bp in length and contained 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and 1 control region. Our data would provide reference information for further study of this species and be useful for evolutionary and phylogenetics studies for this NT species.

The complete mitochondrial genome of Semnopithecus schistaceus.

Semnopithecus schistaceus Hodgson, 1840 belongs to subfamily Colobinae, family Cercopithecidae. This species was once mixed with S. entellus. The conservation status of this species is Least Concern (LC) in IUCN. In China, this species has been considered as Critically Endangered (CR) by the Red List of China's vertebrates. In this study, the complete mitogenome of S. schistaceus was determined. The mitogenome is a circular molecule of 16,534 bp in length, containing 13 protein－coding genes, 2 ribosome RNA genes, 1 light strand replication origin (OL), 22 transfer RNA genes, and 1 non－coding region. We reconstructed a phylogenetic tree based on Bayesian inference for 19 primates species. The Cyt b p-distance is 0.029 between S. schistaceu and S. entellus. Thus, the taxonomic status of these two species remains to be further studied.

How many species of Apodemus and Rattus occur in China? A survey based on mitochondrial cyt b and morphological analyses.

Apodemus (mice) and Rattus (rats) are the top rodent reservoirs for zoonoses in China, yet little is known about their diversity. We reexamined the alpha diversity of these two genera based on a new collection of specimens from China and their cyt b sequences in GenBank. We also tested whether species could be identified using external and craniodental measurements exclusively. Measurements from 147 specimens of Apodemus and 236 specimens of Rattus were used for morphological comparisons. We analysed 74 cyt b sequences of Apodemus and 100 cyt b sequences of Rattus to facilitate phylogenetic estimations. Results demonstrated that nine species of Apodemus and seven species of Rattus, plus a new subspecies of Rattus nitidus, are distributed in China. Principal component analysis using external and craniodental measurements revealed that measurements alone could not separate the recognized species. The occurrence of Rattus pyctoris in China remains uncertain.

An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei.

In Trypanosoma brucei, the tetracycline-inducible system enables tightly-regulated, highly-efficient expression of recombinant proteins or double-stranded RNA in both procyclic and bloodstream form cells, providing useful molecular genetic tools to study gene functions. An alternative, vanillic acid-inducible system is recently described for procyclic T. brucei, providing ∼18-fold increase in GFP reporter expression upon induction (Sunter JD. Mol. Biochem. Parasitol. 2016, 207:45-48). Here we describe a cumate-inducible system that allows efficient, tunable gene expression showing >300-fold increase in GFP expression upon induction. The cumate-inducible system can be used alone or together with the tetracycline-inducible system, in both procyclic and bloodstream form T. brucei. Efficient cumate-inducible expression is also achieved in T. brucei-infected mice.

BAPTA-AM decreases cellular pH, inhibits acidocalcisome acidification and autophagy in amino acid-starved T. brucei.

To investigate the role of Ca2+ signaling in starvation-induced autophagy in Trypanosoma brucei, the causative agent of human African trypanosomiasis, we used cell-permeant Ca2+ chelator BAPTA-AM and cell impermeant chelator EGTA, and examined the potential involvement of several intracellular Ca2+ signaling pathways in T. brucei autophagy. The results showed an unexpected effect of BAPTA-AM in decreasing cellular pH and inhibiting acidocalcisome acidification in starved cells. The implication of these results in the role of Ca2+ signaling and cellular/organellar pH in T. brucei autophagy is discussed.

ATP-driven and AMPK-independent autophagy in an early branching eukaryotic parasite.

Autophagy is a catabolic cellular process required to maintain protein synthesis, energy production and other essential activities in starved cells. While the exact nutrient sensor(s) is yet to be identified, deprivation of amino acids, glucose, growth factor and other nutrients can serve as metabolic stimuli to initiate autophagy in higher eukaryotes. In the early-branching unicellular parasite Trypanosoma brucei, which can proliferate as procyclic form (PCF) in the tsetse fly or as bloodstream form (BSF) in animal hosts, autophagy is robustly triggered by amino acid deficiency but not by glucose depletion. Taking advantage of the clearly defined adenosine triphosphate (ATP) production pathways in T. brucei, we have shown that autophagic activity depends on the levels of cellular ATP production, using either glucose or proline as a carbon source. While autophagosome formation positively correlates with cellular ATP levels; perturbation of ATP production by removing carbon sources or genetic silencing of enzymes involved in ATP generation pathways, also inhibited autophagy. This obligate energy dependence and the lack of glucose starvation-induced autophagy in T. brucei may reflect an adaptation to its specialized, parasitic life style.

Acidocalcisome is required for autophagy in Trypanosoma brucei.

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca(2+) and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.

Menhaden oil, but not safflower or soybean oil, aids in restoring the polyunsaturated fatty acid profile in the novel delta-6-desaturase null mouse.

BACKGROUND: Polyunsaturated fatty acids (PUFA) have diverse biological effects, from promoting inflammation to preventing cancer and heart disease. Growing evidence suggests that individual PUFA may have independent effects in health and disease. The individual roles of the two essential PUFA, linoleic acid (LA) and α-linolenic acid (ALA), have been difficult to discern from the actions of their highly unsaturated fatty acid (HUFA) downstream metabolites. This issue has recently been addressed through the development of the Δ-6 desaturase knock out (D6KO) mouse, which lacks the rate limiting Δ-6 desaturase enzyme and therefore cannot metabolize LA or ALA. However, a potential confounder in this model is the production of novel Δ-5 desaturase (D5D) derived fatty acids when D6KO mice are fed diets containing LA and ALA, but void of arachidonic acid. OBJECTIVE: The aim of the present study was to characterize how the D6KO model differentially responds to diets containing the essential n-6 and n-3 PUFA, and whether the direct provision of downstream HUFA can rescue the phenotype and prevent the production of D5D fatty acids. METHODOLOGY: Liver and serum phospholipid (PL) fatty acid composition was examined in D6KO and wild type mice fed i) 10% safflower oil diet (SF, LA rich) ii) 10% soy diet (SO, LA+ALA) or iii) 3% menhaden oil +7% SF diet (MD, HUFA rich) for 28 days (n = 3-7/group). RESULTS: Novel D5D fatty acids were found in liver PL of D6KO fed SF or SO-fed mice, but differed in the type of D5D fatty acid depending on diet. Conversely, MD-fed D6KO mice had a liver PL fatty acid profile similar to wild-type mice. CONCLUSIONS: Through careful consideration of the dietary fatty acid composition, and especially the HUFA content in order to prevent the synthesis of D5D fatty acids, the D6KO model has the potential to elucidate the independent biological and health effects of the parent n-6 and n-3 fatty acids, LA and ALA.

A role of autophagy in Trypanosoma brucei cell death.

The early branching eukaryote Trypanosoma brucei contains functional autophagy machinery that allows regulated degradation of its own cellular components. In this study, we examined the function of two Atg8 genes, TbAtg8.1 and TbAtg8.2, in starvation-induced autophagosome formation and cell death in procyclic T. brucei. Upon starvation, both TbAtg8.1 and TbAtg8.2 localize to punctate structures characteristic of autophagosomes as shown by fluorescence and electron microscopy, and wortmannin and chloroquine treatments. While TbAtg8.1 depletion has no detectable effects on TbAtg8.2 recruitment to autophagosomes, TbAtg8.2 depletion greatly reduced the autophagosome relocation of TbAtg8.1. Depletion of TbAtg8.1 and 8.2, individually or together, promote cell survival under starvation conditions. Taken together, these observations confirm the presence of an autophagy-related cell death pathway in T. brucei, where TbAtg8.1 and TbAtg8.2 play essential but distinct roles in autophagosome formation and cell death.

Semicarbazide-sensitive amine oxidase kills African trypanosomes in vitro.

The African trypanosome Trypanosoma brucei is the cause of sleeping sickness in humans and Nagana in animals. Here we report that semicarbazide-sensitive amine oxidases (SSAOs), enzymes that are abound in T. brucei mammal hosts, eliminate trypanosomes by oxidation of its substrate in vitro. SSAO and its endogenous substrate methylamine are not toxic to T. brucei, but parasites were killed in the presence of both of them. SSAO inhibitors antagonized the SSAO-methylamine induced toxicity on T. brucei. The trypanocidal activity was mainly associated with formaldehyde generated in the SSAO mediated oxidation of methylamine. This finding suggests that SSAO may play some roles in non-specific defense of trypanosome infection in mammals.