Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5′ end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration

Virulence test using nematodes to prescreen Nocardia species capable of inducing neurodegeneration and behavioral disorders

Background. Parkinson's disease (PD) is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain-some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. No cardia are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system(CNS) invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are N. farcinica, N. nova, N. brasiliensis and N. cyriacigeorgica. Some studies have highlighted the ability of N. cyriacigeorgica to induce Parkinson's disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. Methods. Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. Results. The nematode Caenorhabditis elegans allowed us to demonstrate that Nocardia strains belonging to N. cyriacigeorgica and N. farcinica species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of Nocardia strains to induce neurodegeneration. These behavioral disorders were induced by N. cyriacigeorgica species (N. cyriacigeorgica GUH-2 and N. cyriacigeorgica 44484) and N. farcinica 10152. Discussion. We conclude that C. elegans is a good model for detecting Nocardia strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to characterize the induced behavioral disorders and bacterial dissemination

Bacteriome genetic structures of urban deposits are indicative of their origin and impacted by chemical pollutants

Abstract Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16 S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits

Incidence of hydrological, chemical, and physical constraints on bacterial pathogens, Nocardia cells, and fecal indicator bacteria trapped in an urban stormwater detention basin in Chassieu, France

International audienc

Spatio-temporal variations in chemical pollutants found among urban deposits match changes in thiopurine S-methyltransferase-harboring bacteria tracked by the tpm metabarcoding approach

Authors thank the OTHU network for technical assistance and financial supports, the School of Integrated Watershed Sciences H2O'LYON (ANR 17-EURE-0018), and the Urban School of Lyon (ANR-17-CONV-0004) for their advices and supports in the elaboration of this multi-disciplinary research initiative.International audienceThe bTPMT (bacterial thiopurine S-methyltransferase), encoded by the tpm gene, can detoxify metalloid-containing oxyanions and xenobiotics. The hypothesis of significant relationships between tpm distribution patterns and chemical pollutants found in urban deposits was investigated. The tpm gene was found conserved among eight bacterial phyla with no sign of horizontal gene transfers but a predominance among gamma proteobacteria. A DNA metabarcoding approach was designed for tracking tpm-harboring bacteria among polluted urban deposits and sediments recovered for more than six years in a detention basin (DB). This DB recovers runoff waters and sediments from a zone of high commercial activities. The PCR products from DB samples led to more than 540,000 tpm reads after DADA2 or MOTHUR bio-informatic manipulations that were allocated to more than 88 and less than 634 sequence variants per sample. The tpm community patterns were significantly different between the recent urban deposits and those that had accumulated for more than 2 years in the DB, and between those of the DB surface and the DB settling pit. These groups of samples had distinct mixture of priority pollutants. Significant relationships between tpm ordination patterns, sediment accumulation time periods and location, and concentrations in PAH, chlorpyrifos, and 4-nonylphenols (NP) were observed. These correlations matched the higher occurrences of, among others, Aeromonas, Pseudomonas, and Xanthomonas tpm-harboring bacteria in recent urban DB deposits more contaminated with chrysene and alkylphenol ethoxylates. Highly significant drops in tpm reads allocated to Aeromonas species were recorded in the oldest DB sediments accumulating naphthalene and metallic pollutants. Degraders of urban pollutants such as P. aeruginosa and P. putida showed conserved distribution patterns over time but P. syringae phytopathogens were more abundant in the oldest sediments. TPMT-harboring bacteria can be used to assess the incidence of high risk priority pollutants on environmental systems

Caractérisation des sédiments d’un bassin de retenue-décantation des eaux pluviales et éléments pour la gestion

National audienceStormwater runoffs are loaded with metals, PAHs, pesticides and pathogenic bacteria, conveyed for many in particulate form. One of the techniques used to manage this contaminated stormwater is to use retention basins in order to allow settling of the suspended solids. The deposits thus set up represent zones of contamination and a particular waste. The deposits thus constituted represent waste that must be managed. The objectives of this paper are: i) to characterize the sediments of a stormwater retention basin, ii) to identify the variability and heterogeneity of its microbiological, physical, chemical and ecotoxicological characteristics and to highlight the key factors that could explain this variability, iii) establish correlations between characteristics and identify trends in the change of sediment quality. The deriving results show that the median diameter of the sediments is between 50 and 150 μm. They are mostly contaminated with PAH, metals and 4-n-nonylphenol. Concerning the microbiological characterization, the bacteria “cores” observed in the sediments are composed of the bacterial genus which may contain pathogenic forms: Mycobacteria, Pseudomonas, Aeromonas, Nocardia. Nocardia counts are higher in sediments composed of fine and light particles (grain size between 1.1 and 3.3 μm). The ecotoxicity analysis of these sediments using the “ostracodes” test revealed that “fresh” sediments collected during a storm event were more ecotoxic than old ones. Sediment characteristics trended to become homogenous after 5 years of monitoring. Regarding investigations on the identification of the sources of microbiological contaminants observed in the retention basin, socio-urban observations revealed a direct relationship between the microbiological contamination of runoff and the presence of items and marks leave by socio-economic activities undertaken on the watershed. These results are useful for the managers in order to guide the choice or to stimulate the development of sectors or strategies of treatment and valorization of these sediments