Biology subjects

Cid-Rojas, F.

Publications and source records attributed to Cid-Rojas, F..

5 recordsLinked to original sources

bioRxiv2026

Distinctive spore architecture and developmental biology of Turicibacter sanguinis reveal unexpected diversity among gut spore formers

Sporulation is a widespread but incompletely characterized trait among gut commensals, where it underpins microbial persistence, transmission, and ecological resilience. Most insights into spore biology derive from Bacilli and Clostridia, yet little is known about sporulation in phylogenetically distant gut-associated lineages. Turicibacter sanguinis, a strict anaerobe linked to host serotonin metabolism, lipid homeostasis, and neurodegenerative disease, represents one such understudied taxon. Here, we integrate ultrastructural, physiological, and comparative genomic analyses to define the sporulation and germination program of T. sanguinis. We show that T. sanguinis forms heat-resistant spores with a canonical core-cortex-coat architecture but displays previously undescribed features including a dual-layered outer envelope and bimodal electron-dense coat morphotypes. Developmental stages of sporulation follow canonical stages of Bacillus- and Clostridium-like sporulation while genomic analyses reveal a hybrid regulatory architecture combining Clostridial-type Spo0A initiation with Bacillus-like late-stage sigma factor control. Germination assays and genomic signatures further indicate a nutrient-responsive, Bacillus-like pathway involving Ger-family receptors, SpoVA-mediated Ca-DPA release, and CwlJ- and SleM-type cortex hydrolases. Together, these findings identify T. sanguinis as a distinct spore-forming lineage within the human gut microbiota and expand the known diversity of sporulation strategies across the Firmicutes.

microbiology↗

bioRxiv2025

Characterization of a hypersporulating strain derivative of Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive and obligate anaerobic spore-former pathogen. C. difficile spores are essential for the transmission and recurrence of C. difficile infections (CDI). A major challenge in sporulation studies in C. difficile is the low yield and asynchronous nature of this process. In this work, a hypersporulating strain, derivative of R20291, with an early sporulation onset and enhanced sporulation efficiency was isolated by serendipity. This strain had 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs revealed that spores of both strains have similar hair-like projections, electron-dense outer exosporium layer features. Whole genome sequencing and genomic analyses revealed that the hypersporulating strain had a 2356 bp-deletion spanning three ORF, including a non-essential proC1 involved in proline metabolism, and a missense mutation in rsbV, an anti-anti-SigB factor of RsbW. These observations suggest that this RsbV-variant might contribute to constitutive repression of the SigB-dependent general stress response, and therefore, derepressing sporulation in this hypersporulating strain.

microbiology↗

bioRxiv2025

Identification of Novel Cryptic and Classical Clades in Clostridioides difficile

Clostridioides difficile is a major cause of nosocomial infections and antimicrobial-associated diarrhea, with significant global health implications. While five classical clades (C1 to C5) have traditionally encompassed most pathogenic strains, recent genomic studies have uncovered several highly divergent cryptic clades (C-I to C-V), suggesting greater taxonomic and evolutionary complexity. In this study, we performed a comprehensive whole-genome analysis of 25,144 publicly available C. difficile genomes, supplemented with 21 novel isolates from Costa Rica and Brazil. Using average nucleotide identity (ANI), recombination-corrected core-genome phylogenies, we confirmed the presence of all known classical and cryptic clades and identified two novel classical clades (C6 and C7) and two previously undescribed cryptic clades (C-VI and C-VII). We further detected evidence for bifurcation within cryptic clade C-III, supporting its division into two lineages C-IIIa and C-IIIb. ANI comparisons revealed that cryptic clades share less than 95% identity with classical C. difficile strains but remain more similar to C. difficile than to any other Peptostreptococcaceae species, reinforcing their placement within the species boundary. Core-genome recombination analyses revealed limited gene flow between cryptic and classical clades, except for Clade C7, which exhibited intermediate recombination patterns and may represent a transitional lineage. These findings expand the known diversity of C. difficile, provide a revised genomic framework for clade classification, and underscore the evolutionary depth and ecological breadth of cryptic lineages with potential clinical relevance.

microbiology↗

bioRxiv2025

Phylogenomic analysis of the collagen-like BclA proteins in Clostridioides difficile

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium and a major nosocomial pathogen, notable for its high genetic diversity. C. difficile has been classified into five classical phylogenetic clades (C1 to C5) and five cryptic clades (C-I to C-V), reflecting its extensive genome plasticity. In addition, C. difficile spores are considered essential for the onset, persistence and transmission of the disease, and their exosporium layer has hair-like projections formed by the BclA-family of proteins (BclA1, BclA2 and BclA3). Previous work in C1 and C2 strains has demonstrated that the absence of these proteins affects spore germination, pathogenesis, persistence, and recurrence of CDI. Nevertheless, the conservation of BclAs across different C. difficile clades remains unclear. In this work, genomics analysis in more than 25,000 C. difficile genomes revealed that the prevalence and variability of the BclAs was not conserved across classical and cryptic clades. The most represented clade on the dataset was C1, where roughly 50% of the genomes possessed all three bclA genes. Pseudogenization of bclA1 or all bclAs was observed in C2 and C3, respectively. Additionally, the absence of bclA1 in C4 and of both bclA1 & bclA3 in C5, further demonstrates the divergence of BclAs among C. difficile clades. Subsequent analysis revealed high variability in the central collagen-like region (CLR) of all three BclAs, and a highly conserved bclA1 pseudogenization event in most members of C2. Overall, the extensive variability of bclA, attributed to the CLR, prevalence of a bclA1 pseudogenization and complete absence of bclAs in certain clades, is likely to impact spore morphogenesis and pathogenesis across different C. difficile clades.

microbiology↗

bioRxiv2021

Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores.

Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

microbiology↗