Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling

Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved

Organoids as tools to investigate gastrointestinal nematode development and host interactions

Gastrointestinal nematodes are a diverse class of pathogens that colonise a quarter of the world’s human population and nearly all grazing livestock. These macroparasites establish, and some migrate, within host gastrointestinal niches during their life cycles and release molecules that condition the host mucosa to enable chronic infections. Understanding how helminths do this, and defining the molecules and mechanisms involved in host modulation, holds promise for novel strategies of anthelmintics and vaccines, as well as new knowledge of immune regulation and tissue repair. Yet the size and complexity of these multicellular parasites, coupled with the reliance on hosts to maintain their life cycles, present obstacles to interrogate how they interact with the gastric and intestinal epithelium, stroma and immune cells during infection, and also to develop protocols to genetically modify these parasites. Gastrointestinal organoids have transformed research on gastric and gut physiology during homeostasis and disease, including investigations on host-pathogen interactions with viruses, bacteria, protozoa and more recently, parasitic nematodes. Here we outline applications and important considerations for the best use of organoids to study gastrointestinal nematode development and interactions with their hosts. The careful use of different organoid culture configurations in order to achieve a closer replication of the in vivo infection context will lead not only to new knowledge on gastrointestinal nematode infection biology, but also towards the replication of their life cycles in vitro, and the development of valuable experimental tools such as genetically modified parasites

Identification of microsatellite markers linked to the blast resistance gene Pi-1(t) in rice

The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F3 segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F3 generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes

RNA in DEAE-Sepharose Fraction 7 induces apoptosis in human monocytes.

<p><i>A.</i> Apoptosis induced by DEAE-Sepharose Fraction 7 (F7), F7 treated with proteinase K (F7+ProtK), F7 treated with DNase1 (F7+DNase1), F7 treated with RNaseV1 (F7+RNaseV1), CF, CF treated with RNaseV1 (CF+RNaseV1), CF-Man, CF-Man treated with RNaseV1 (CF-Man+RNaseV1). Apoptosis was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive. *** significance p<0.001, ** significance p<0.01 as compared to unstimulated control or between treated samples. <i>B.</i> SDS-PAGE with ethidum bromide staining and <i>C.</i> silver staining of F7 and F7 treated with proteinase K, DNase1, or RNaseV1.</p