Biology subjects

Pizarro-Guajardo, M.

Publications and source records attributed to Pizarro-Guajardo, M..

11 recordsLinked to original sources

bioRxiv2026

Repurposing nystatin and taurocholate to target intracellular spore persistence during antibiotic treatment to prevent recurrent Clostridioides difficile infection.

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..

pharmacology and toxicology↗

bioRxiv2026

Gut complement C1q and C3 interact with Clostridioides difficile spores via CdeM and contribute to pathogenesis.

Clostridioides difficile infection (CDI) is characterized by toxin-mediated epithelial injury, intestinal inflammation, and a high frequency of disease recurrence. Although complement components are produced locally within the gastrointestinal tract, their contribution to C. difficile pathogenesis remains poorly understood. Here, we investigated the interaction of the complement proteins C1q and C3 with C. difficile spores and their contribution to spore-host interactions and disease outcome. We found that C1q and C3 are accessible within the healthy ileal mucosa and that TcdB intoxication differentially remodels their spatial availability, decreasing accessible C1q while increasing accessible C3 at higher toxin concentrations. C. difficile spores associated with both complement components in vivo and directly interacted with purified human C1q and C3 in vitro, with immunogold electron microscopy localizing these interactions to the outer exosporium. Although the collagen-like BclA proteins modulated complement binding under some conditions, they were not required for C1q or C3 association. Far-Western analysis coupled to mass spectrometry identified the exosporium proteins CotE and CdeM as candidate complement-interacting proteins, and purified CdeM inclusion bodies directly associated with both C1q and C3. Exposure of spores to human serum promoted their interaction with intestinal epithelial cells, whereas depletion of either C1q or C3 markedly reduced spore internalization and reconstitution with the corresponding purified protein partially restored entry. C3-derived species, including C3dg, were selectively associated with spores but not vegetative cells, without reducing spore viability. Finally, C1q deficiency did not alter the initial onset of CDI but accelerated recovery and markedly attenuated recurrent disease following vancomycin treatment. Together, these findings identify intestinal complement as a previously unrecognized component of the C. difficile spore-host interface and reveal C1q and C3 as modulators of spore epithelial entry, persistence, and recurrent disease.

microbiology↗

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

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↗

bioRxiv2025

Clostridioides difficile major toxins remodel the intestinal epithelia, affecting spore adherence/internalization into intestinal tissue and their association with gut vitronectin.

The most common cause of healthcare-associated diarrhea and colitis in the U.S., is Clostridioides difficile, a spore-forming pathogen. Two toxins, TcdA and TcdB, are major virulence factors essential for disease manifestations, while C. difficile spores are essential for disease transmission and recurrence. Both toxins cause major damage to the epithelial barrier, trigger massive inflammation, and reshape the microbiome and metabolic composition, facilitating C. difficile colonization. C. difficile spores, essential for transmission and recurrence of the disease, persist adhered and internalized in the intestinal epithelia. Studies have suggested that toxin-neutralization in combination with antibiotic during CDI treatment in humans significantly reduces disease recurrence, suggesting a link between toxin-mediated damage and spore persistence. Here, we show that TcdA/TcdB-intoxication of intestinal epithelial Caco-2 cells leads to remodeling of accessible levels of fibronectin (Fn) and vitronectin (Vn) and their cognate alpha-integrin subunits. While TcdB-intoxication of intestinal tissue had no impact in accessible levels of Fn and Vn, but significantly increased levels of intracellular Vn. We observed that Fn and Vn released to the supernatant readily bind to C. difficile spores in vitro, while TcdB-intoxication of intestinal tissue led to increased association of C. difficile spores with gut Vn. Toxin-intoxication of the intestinal tissue also contributes to increased adherence and internalization of C. difficile spores. However, TcdB-intoxicated ligated loops infected of mice treated with Bezlotoxumanb (monoclonal anti- TcdB antibodies) did not prevent TcdB-mediated increased spore adherence and internalization into intestinal tissue. This study highlights the importance of studying the impact of C. difficile toxins of host tissues has in C. difficile interaction with host surfaces that may contribute to increased persistence and disease recurrence.

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↗

bioRxiv2021

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease

Inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohns disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBD have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of the IBD and the dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identify a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward toward understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBD.

microbiology↗

bioRxiv2020

Clostridioides difficile spore-entry into intestinal epithelial cells contributes to recurrence of the disease

Clostridioides difficile spores produced during infection are essential for the recurrence of the disease. However, how C. difficile spores persist in the intestinal mucosa to cause recurrent infection remains unknown. Here, we show that C. difficile spores gain entry into the intestinal mucosa via fibronectin-5{beta}1 and vitronectin-v{beta}1 specific-pathways. The spore-surface exosporium BclA3 protein is essential for both spore-entry pathways into intestinal epithelial cells. Furthermore, C. difficile spores of a bclA3 isogenic mutant exhibited reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model of the disease. Inhibition of C. difficile spore-entry led to reduced spore-entry into the intestinal epithelial barrier and recurrence of C. difficile infection in vivo. These findings suggest that C. difficile spore-entry into the intestinal barrier is a novel mechanism of spore-persistence that can contribute to infection recurrence and have implications for the rational design of therapies.

microbiology↗

bioRxiv2020

Nasal immunization with the C-terminal domain of BclA3 induced specific IgG production and attenuated disease symptoms in mice infected with Clostridioides difficile spores

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance a strong immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets of new therapeutic treatments. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a well-established mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some CDI symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization route need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

microbiology↗

bioRxiv2020

Characterization of exosporium layer variability of Clostridioides difficile spores in the epidemically relevant strain R20291.

Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea and spores are the transmission vehicle of the disease. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that the laminations of the spore-coat are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

microbiology↗

bioRxiv2019

cfrB,cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

Cfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

microbiology↗