Percorrer por autor "Martins, Diogo"
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- An endoribonuclease of the YicC-like family delays sporulation via sRNA degradation in Clostridioides difficilePublication . Martins, Diogo; Salgueiro, Bruno; Sobral, Daniel; Gragera, Marcos; Hensel, Zach; Henriques, Adriano O.; Romão, Célia V.; Serrano, MónicaClostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.
- A single amino acid substitution in CspA increases germination sensitivity and broadens bile salt germinant specificity in clostridioides difficile sporesPublication . Roseiro, Isabel; Nunes, Alexandra; Martins, Diogo; Alves, Frederico; Persson, Søren; Henriques, Adriano O.; Oleastro, Mónica; Serrano, MónicaOnce regarded primarily as a healthcare-associated pathogen, Clostridioides difficile has increasingly been reported as a cause of community-acquired infection, raising questions about the contribution of animal and environmental reservoirs to persistence and transmission. Ribotype 033 (RT033) is predominantly associated with animal and environmental reservoirs, yet key traits underlying its ecology are poorly defined. Here we show that RT033 strains produce spores with enhanced germination sensitivity and expanded germinant specificity, enabling germination at low concentrations of bile-salts and in response to bile-salts that are typically inhibitory, including those more typical of animal hosts. Genetic analysis identified a single amino acid substitution (R1036I) in CspA, a component of the CspBAC germination apparatus, as the determinant of this phenotype. Expression of the RT033 cspBAC operon in a laboratory strain was sufficient to confer increased sensitivity and expanded bile-salt responsiveness. The R1036I substitution disrupts a conserved salt bridge at the CspA:CspC interface while not causing major destabilisation of the complex in the absence or presence of germinants and co-germinants. Together, our findings reveal a naturally occurring rewiring of bile-salt sensing in spores of RT033 strains that likely reflects adaptation to animal-associated bile-acid environments and may influence persistence, transmission, and zoonotic potential of this lineage.