Phosphorylation and functionality of CdtR in Clostridium difficile

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38–52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin

Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity

Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection

Indexación ScopusClostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5β1 and vitronectin-αvβ1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies. © 2021, The Author(s).https://www-nature-com.recursosbiblioteca.unab.cl/articles/s41467-021-21355-

Functional analysis of the post-transcriptional regulator RsmA reveals a novel RNA-binding site.

The RsmA family of RNA-binding proteins are global post-transcriptional regulators that mediate extensive changes in gene expression in bacteria. They bind to, and affect the translation rate of target mRNAs, a function that is further modulated by one or more, small, untranslated competitive regulatory RNAs. To gain new insights into the nature of this protein/RNA interaction, we used X-ray crystallography to solve the structure of the Yersinia enterocolitica RsmA homologue. RsmA consists of a dimeric beta barrel from which two alpha helices are projected. From structure-based alignments of the RsmA protein family from diverse bacteria, we identified key amino acid residues likely to be involved in RNA-binding. Site-specific mutagenesis revealed that arginine at position 44, located at the N terminus of the alpha helix is essential for biological activity in vivo and RNA-binding in vitro. Mutation of this site affects swarming motility, exoenzyme and secondary metabolite production in the human pathogen Pseudomonas aeruginosa, carbon metabolism in Escherichia coli, and hydrogen cyanide production in the plant beneficial strain Pseudomonas fluorescens CHA0. R44A mutants are also unable to interact with the small untranslated RNA, RsmZ. Thus, although possessing a motif similar to the KH domain of some eukaryotic RNA-binding proteins, RsmA differs substantially and incorporates a novel class of RNA-binding site

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA.We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins

Hepatitis C Virus Infection in San Francisco's HIV-infected Urban Poor: High Prevalence but Low Treatment Rates

OBJECTIVE: To measure Hepatitis C Virus (HCV) prevalence, incidence, and initiation of HCV therapy in a representative HIV-infected cohort of the urban poor. DESIGN: Cohort analysis. SETTING: The Research and Access to Care for the Homeless (REACH) Cohort is a systematic sample of HIV-infected marginally housed individuals identified from single-room occupancy hotels, homeless shelters, and free lunch programs in San Francisco. PARTICIPANTS: Two hundred forty-nine participants with 28.9 months (median) of follow-up were studied. Mean age was 44 (range 24 to 75, standard deviation 8.4) years. Eighty-two percent were male, 43% were African-American, 64% were lifetime injection drug users, and 24% had been on the street or in a shelter in the prior month. INTERVENTIONS: We measured HCV testing and treatment history with structured interviews; additionally, participants were tested for HCV antibodies (EIA-2) with RNA viral load confirmation. MAIN RESULTS: At baseline, 172 (69.1%) were HCV-positive and 182 (73.1%) were HCV-positive at follow-up, including 155 (62.2%) with viremia. HCV-positive status was associated with having injected drugs, elevated serum alanine aminotransferase, homelessness in the last 1 year, and more severe depressive symptoms. The incidence of new HCV infection was 4.63% per person-year (ppy; 95% confidence interval, 2.31 to 8.13) in the entire cohort and 16.77% ppy among injection drug users. The prevalence of HCV antibody-negative HCV-viremia was 13.2% (10/76). Nonwhites were less likely to receive HCV testing and subspecialty referral, controlled for drug use and other confounders. Sixty-eight percent (123/182) were aware treatment was available; however, only 3.8% (7/182) or 1.16% ppy received HCV treatment. CONCLUSIONS: While HCV infection is common, HCV treatment is rare in the HIV-HCV coinfected urban poor. Urban poor, nonwhite individuals are less likely to receive HCV testing and subspecialty referral than their white counterparts. Antibody-negative infection may complicate screening and diagnosis in HIV-infected persons

Organic material decomposition and nutrient dynamics in a mulch system enriched with leguminous trees in the Amazon Decomposição de material orgânico e dinâmica de nutrientes em um sistema de cobertura morta enriquecido com árvores leguminosas na Amazônia

The new techniques proposed for agriculture in the Amazon region include rotational fallow systems enriched with leguminous trees and the replacement of biomass burning by mulching. Decomposition and nutrient release from mulch were studied using fine-mesh litterbags with five different leguminous species and the natural fallow vegetation as control. Samples from each treatment were analyzed for total C, N, P, K, Ca, Mg, lignin, cellulose content and soluble polyphenol at different sampling times over the course of one year. The decomposition rate constant varied with species and time. Weight loss from the decomposed litter bag material after 96 days was 30.1 % for Acacia angustissima, 32.7 % for Sclerolobium paniculatum, 33.9 % for Iinga edulis and the Fallow vegetation, 45.2 % for Acacia mangium and 63.6 % for Clitoria racemosa. Immobilization of N and P was observed in all studied treatments. Nitrogen mineralization was negatively correlated with phenol, C-to-N ratio, lignin + phenol/N ratio, and phenol/phosphorus ratios and with N content in the litterbag material. After 362 days of field incubation, an average (of all treatments), 3.3 % K, 32.2 % Ca and 22.4 % Mg remained in the mulch. Results confirm that low quality and high amount of organic C as mulch application are limiting for the quantity of energy available for microorganisms and increase the nutrient immobilization for biomass decomposition, which results in competition for nutrients with the crop plants.<br>As novas técnicas propostas para a agricultura na Amazônia incluem sistema de rotação de capoeira enriquecido com árvores leguminosas e transformando a queima da biomassa em cobertura morta sobre o solo. A decomposição e a liberação de nutrientes da cobertura morta foram estudadas usando sacos de liteira com malha fina que continham cinco tratamentos com diferentes espécies de leguminosas em comparação a um tratamento-controle com vegetação natural. As amostras para cada tratamento foram analisadas para conteúdos de C total, N, P, K, Ca, Mg, lignina, celulose e polifenóis solúveis em diferentes tempos de amostragem durante um ano. A razão constante de decomposição variou com a espécie e com o tempo. A perda de massa nos sacos de decomposição foi de 30,1 % para Acacia angustissima, de 32,7 % para Sclerolobium paniculatum, de 33,9 % para Inga edulis e para a vegetação secundária, de 45,2 % para Acacia mangium e de 63,6 % para Clitoria racemosa. Foi observada imobilização de N e P em todos os tratamentos, sendo a mineralização do N negativamente correlacionada com o fenol, razão C/N, razão (lignina + fenol)/N, razão fenol/P e o conteúdo de N nos sacos de liteira. Depois de 362 dias de incubação no campo, 3,3 % de K, 32,2 % de Ca e 22,4 % de Mg permaneceram no material em decomposição. Os resultados evidenciaram que a baixa qualidade mineral e a alta quantidade de carbono orgânico e aplicado como cobertura morta podem limitar a quantidade de energia disponível para os microrganismos resultando em uma competição por nutrientes com as plantas agrícolas