Inheritance and maintenance of small RNA-mediated epigenetic effects.

Heritable traits are predominantly encoded within genomic DNA, but it is now appreciated that epigenetic information is also inherited through DNA methylation, histone modifications, and small RNAs. Several examples of transgenerational epigenetic inheritance of traits have been documented in plants and animals. These include even the inheritance of traits acquired through the soma during the life of an organism, implicating the transfer of epigenetic information via the germline to the next generation. Small RNAs appear to play a significant role in carrying epigenetic information across generations. This review focuses on how epigenetic information in the form of small RNAs is transmitted from the germline to the embryos through the gametes. We also consider how inherited epigenetic information is maintained across generations in a small RNA-dependent and independent manner. Finally, we discuss how epigenetic traits acquired from the soma can be inherited through small RNAs.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development.

Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.

Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis.

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.

Functional Characterization of the m6A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite.

Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease.

Germline inherited small RNAs facilitate the clearance of untranslated maternal mRNAs in C. elegans embryos.

Inheritance and clearance of maternal mRNAs are two of the most critical events required for animal early embryonic development. However, the mechanisms regulating this process are still largely unknown. Here, we show that together with maternal mRNAs, C. elegans embryos inherit a complementary pool of small non-coding RNAs that facilitate the cleavage and removal of hundreds of maternal mRNAs. These antisense small RNAs are loaded into the maternal catalytically-active Argonaute CSR-1 and cleave complementary mRNAs no longer engaged in translation in somatic blastomeres. Induced depletion of CSR-1 specifically during embryonic development leads to embryonic lethality in a slicer-dependent manner and impairs the degradation of CSR-1 embryonic mRNA targets. Given the conservation of Argonaute catalytic activity, we propose that a similar mechanism operates to clear maternal mRNAs during the maternal-to-zygotic transition across species.

Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants.

PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.

A secreted Heat shock protein 90 of Trichomonas vaginalis.

Trichomonas vaginalis is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since Trichomonas Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.

Antigenic characterization of 52-55kDa protein isolated from Trypanosoma evansi and its application in detection of equine trypanosomosis.

Trypanosoma evansi is a haemo-protozoan parasite responsible for the disease surra, an economically important disease of wide range of domestic and wild animals. The present diagnostic methods using soluble antigens have inherent problems like lack of standardized and reproducible antigens, as well as ethical issues. This entails further efforts for search of defined antigenic molecules with satisfying sensitivity and specificity for sero-epidemiology of trypanosomosis. In present investigation, we have identified and purified 52-55kDa immuno-dominant protein cluster in molecular mass ranges by preparatory SDS-PAGE methods from T. evansi proteome. The purified protein was further characterized by hyper immune serum raised in rabbits and also further evaluated for its immunodiagnostic potential using experimentally infected horse serum samples by different immunological tests. The immunoblot, ELISA and dot blot assay using purified cluster in infected pooled serum samples showed detection of infection early as 10th days post infection till termination of experiment. The observations revealed that purified cluster is expressed not only at early stage but also persisted and detected throughout course of infection. Further, whole cell lysate antigen separated out and detected 141 spots by 2-D gel electrophoresis. The isoelectric focussing (PI) of 52-55kDa was determined in pH range between 6.9 and 7.5 along with two other cluster of proteins recognised by immune sera of ponies infected with T. evansi. MS/MS analysis of the purified protein cluster identified five proteins i.e. pyruvate kinase 1, beta tubulin, paraflagellar rod protein, alanine aminotransferase and variable surface glycoprotein showing homology to protein present in Trypanosome database. These identified proteins may be useful for development of vaccines and diagnostic targets against animal trypanosomosis.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata-infected counterpart was utilized to test the effects of geldanamycin and the derivative 17-AAG. T. annulata-infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17-AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

The Potential of Lactobacillus casei and Entercoccus faecium Combination as a Preventive Probiotic Against Entamoeba.

Travellers' diarrhoea caused by enteric protozoa like Entamoeba histolytica is among the most common protozoan diseases in developing countries. In developing countries, amoebiasis is the second most prevalent protozoan disease. This protozoan parasite is often known to coexist as a part of the normal gut microbiota. It is estimated that around 50-60 % of population in developing countries might be harbouring Entamoeba in an asymptomatic manner. Due to physiological perturbation or upon immuno-compromise, it can become virulent and then cause diarrhoea, bloody stools and may invade other organs if left untreated. Nitroimidazole drugs, namely metronidazole and tinidazole, are widely used to treat protozoan infections. These drugs often show dose-dependent side effects. With emerging antibiotic resistance, novel therapeutics to prevent parasitic infections is required. This study aims to study effect of probiotics on prevention of Amoebiasis. In this study, we have investigated the effect of selected probiotics on the growth of Entamoeba. From the list of probiotics being currently used, five bacterial strains were selected for testing. These probiotic strains were co-cultured with Entamoeba, and their effect on Entamoeba proliferation was checked. Of the five probiotics chosen, individual treatments of Lactobacillus casei and Enterococcus faecium showed a significant reduction of up to 71 % in parasite survival only at higher CFUs. When the two probiotics were used in combination, the percentage of survival reduced gradually further to 80 % at a total CFU of 109 cells/ml of bacteria. The study lays the foundation for providing cost-effective prophylactic treatment for amoebiasis without the overuse of antibiotics.

First Structural View of a Peptide Interacting with the Nucleotide Binding Domain of Heat Shock Protein 90.

The involvement of Hsp90 in progression of diseases like cancer, neurological disorders and several pathogen related conditions is well established. Hsp90, therefore, has emerged as an attractive drug target for many of these diseases. Several small molecule inhibitors of Hsp90, such as geldanamycin derivatives, that display antitumor activity, have been developed and are under clinical trials. However, none of these tested inhibitors or drugs are peptide-based compounds. Here we report the first crystal structure of a peptide bound at the ATP binding site of the N-terminal domain of Hsp90. The peptide makes several specific interactions with the binding site residues, which are comparable to those made by the nucleotide and geldanamycin. A modified peptide was designed based on these interactions. Inhibition of ATPase activity of Hsp90 was observed in the presence of the modified peptide. This study provides an alternative approach and a lead peptide molecule for the rational design of effective inhibitors of Hsp90 function.

Heat Shock Protein 90 regulates encystation in Entamoeba.

Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely - trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

Heat shock protein 90 as a potential drug target against surra.

Trypanosomiasis is caused by Trypanosoma species which affect both human and animal populations and pose a major threat to developing countries. The incidence of animal trypanosomiasis is on the rise. Surra is a type of animal trypanosomiasis, caused by Trypanosoma evansi, and has been included in priority list B of significant diseases by the World Organization of Animal Health (OIE). Control of surra has been a challenge due to the lack of effective drugs and vaccines and emergence of resistance towards existing drugs. Our laboratory has previously implicated Heat shock protein 90 (Hsp90) from protozoan parasites as a potential drug target and successfully demonstrated efficacy of an Hsp90 inhibitor in cell culture as well as a pre-clinical mouse model of trypanosomiasis. This article explores the role of Hsp90 in the Trypanosoma life cycle and its potential as a drug target. It appears plausible that the repertoire of Hsp90 inhibitors available in academia and industry may have value for treatment of surra and other animal trypanosomiasis.

Identification of heat shock factor binding protein in Plasmodium falciparum.

BACKGROUND: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. METHODS: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. RESULTS: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. CONCLUSIONS: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 µM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) µM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

Heat shock protein 90 inhibitors as broad spectrum anti-infectives.

Combating stress is one of the prime requirements for any organism. For parasitic microbes, stress levels are highest during the growth inside the host. Their survival depends on their ability to acclimatize and adapt to new environmental conditions. Robust cellular machinery for stress response is, therefore, both critical and essential especially for pathogenic microorganisms. Microbes have cleverly exploited stress proteins as virulence factors for pathogenesis in their hosts. Owing to its ability to sense and respond to the stress conditions, Heat shock protein 90 (Hsp90) is one of the key stress proteins utilized by parasitic microbes. There are growing evidences for the critical role played by Hsp90 in the growth of pathogenic organisms like Candida, Giardia, Plasmodium, Trypanosoma, and others. This review, therefore, explores potential of exploiting Hsp90 as a target for the treatment of infectious diseases. This molecular chaperone has already gained attention as an effective anti-cancer drug target. As a result, a lot of research has been done at laboratory, preclinical and clinical levels for several Hsp90 inhibitors as potential anti-cancer drugs. In addition, lot of data pertaining to toxicity studies, pharmacokinetics and pharmacodynamics studies, dosage regime, drug related toxicities, dose limiting toxicities as well as adverse drug reactions are available for Hsp90 inhibitors. Therefore, repurposing/ repositioning strategies are also being explored for these compounds which have gone through advanced stage clinical trials. This review presents a comprehensive summary of current status of development of Hsp90 as a drug target and its inhibitors as candidate anti-infectives. A particular emphasis is laid on the possibility of repositioning strategies coupled with pharmaceutical solutions required for fulfilling needs for ever growing pharmaceutical infectious disease market.