Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

International audiencePathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria

Production and evaluation of a new set of recombinant antigens for the serological diagnosis of human cysticercosis

International audienceHuman cysticercosis caused by Taenia soliun (T. soliun) is endemic in certain areas of Latin America, Asia and Sub-Saharan Africa. Neurocysticercosis (NCC) is mainly diagnosed by neuroimaging, which, in most cases, is unavailable in endemic areas. Due to their high sensitivity and specificity, serological tests such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) based on the glycosylated fraction of the cyst CS50 are widely used for the detection of the anti-cysticercus IgG antibodies despite their significant cost and the need of cysticercus material. Given their cost-effectivess and simplicity, immunoassays based on recombinant proteins could provide new alternatives for human cysticercosis diagnosis: such tests would be aimed at screening those people living in remote areas who need further examination. To date, however, no test using recombinant antigens is commercially available.Herein, five recombinant proteins (R14, R18, R93.1, R914.1, and R915.2) were produced, three of which (R93.1, R914.1, and R915.2) were newly identified from the cyst fluid. Evaluation of the diagnostic performance of these recombinant antigens by ELISA was done using sera from 200 epileptic and non-epileptic individuals in comparison with the WB-CS50 as the reference serological method.Recombinant proteins-based ELISA showed a level of diagnostic performance that is inferior than the reference serological method, but similar to that of the native antigen ELISA for human cysticercosis (commonly used for screening). Further optimization of expression conditions is still needed in order to improve proteins solubility and enhance diagnostic performance for human cysticercosis detection. However, this preliminary evaluation of the recombinant antigens has shown their potential valuable use for screening cysticercosis in patients with epilepsy attending dispensaries in remote areas. Future studies should be conducted to evaluate our recombinant antigens in a large group of patients with different stages of NCC, and in correlation with imaging findings

Binding interface between the Salmonella σ(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

International audienceIn many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ S accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ S binding to the Crl protein increases σ S activity by favouring its association to E. Taking advantage of evolution of the σ S sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ S to the S. Typhimurium σ S-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ S and to generate in silico models of the σ S-Crl complex constrained by mutational analysis. The σ S-Crl models suggest that the identified arginine in σ S interacts with an aspartate of Crl that is required for σ S binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ S-Crl complex. In bacteria, a primary housekeeping sigma factor and one or more alternative σ factors associate with the catalytically active RNA polymerase (RNAP) core enzyme (α 2β β 'ω , E), to form the holoenzyme Eσ , and direct transcription initiation of specific subsets of genes 1,2. In many Gram-negative bacteria, σ S /RpoS is produced during late exponential phase, or in response to stress, to modify global gene transcription and to allow stationary phase survival and general stress resistance 3–5. In the wide host-range pathogen S. Typhimurium, σ S is not only required for general stress resistance, but also for virulence, biofilm formation and development of the red dry and rough (rdar) morphotype, a colony morphology caused by the production of amyloid fibers (curli) and cellulose 6–8. The efficiency of formation of the housekeeping and alternative Eσ can be modulated by regulatory factors that bind E and/or σ 5,9. So far, Crl is the only known σ S-dedicated regulatory factor that enhances σ S activity through a direct interaction, favouring Eσ S formation 7,10–15. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level tha

Seroprevalence of Cysticercosis among Epileptic Patients Attending Neurological Units in the Urban Area of Abidjan

International audienceCysticercosis is one of the main causes of secondary epilepsy in sub-Saharan Africa. To estimate the seroprevalence of cysticercosis among epileptic patients, we conducted a cross-sectional study of patients attending neurology consultation in Abidjan, Côte d’Ivoire. Methods: Patients’ socio-demographic and lifestyle data were collected as well as blood samples for serological testing using ELISA and Western blot based on IgG antibodies detection. For qualitative variables comparison, Chi2 or Fisher tests were used; a Student’s t-test was used to compare quantitative variables. A multivariate logistic regression model was fit to identify risks factors. Results: Among 403 epileptic patients included in the study, 55.3% were male; the median age was 16.9 years; 77% lived in Abidjan; 26.5% were workers. Most patients included in the study had tonic-clonic seizures (80%), and 11.2% had focal deficit signs. The seroprevalence of cysticercosis was 6.0%. The risk was higher in patients over 30 years old (aOR = 5.1 (1.3–20.0)) than in patients under 16. The risk was also considerably high in patients who reported epileptics in the family (aOR = 5 (1.7–14.6)). The risk was three-fold less in females than in males. Conclusions: This study highlighted the exposure of epileptic patients to Taenia solium larvae in an urban area. The risk of positive serology was increased with age, male gender, and family history of epilepsy

Seroprevalence of porcine cysticercosis in traditional farms in South-Eastern Côte d'Ivoire

Background: Porcine cysticercosis is an endemic parasitic zoonosis in many developing countries. The objective of this study was to estimate the seroprevalence of porcine cysticercosis in traditional pig farms in the departments of Dabou, Aboisso and Agboville. Methods: Blood samples were taken from pigs and analyzed by ELISA (IgG) and western blot. Data on farming practices and pig characteristics were collected. Multivariate logistic regression models were constructed to identify risk factors. Results: A total of 668 pigs were sampled from 116 farms and 639 samples were analyzed. The seroprevalence of cysticercosis was estimated at 13.2%. Overweight [OR = 2.6; 95%CI (1.3–4.9)] and fat pigs [OR = 2.3; 95%CI (1.0–4.8)] were twice as likely to be seropositive for cysticercosis. This risk was increased in farms using well water for drinking [OR = 2.5; 95%CI (1.0–6.3)] as well as those reporting veterinary care of the animals (OR = 2.9; 95%CI (1.2–7.3)). Conclusions: This study demonstrated the circulation of Taenia solium in pig farms in southern Côte d'Ivoire

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

International audienceThe multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

International audienceThe current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs

A comparison of four serological assays for detecting anti–SARS-CoV-2 antibodies in human serum samples from different populations

International audienceIt is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region