About

Daniel Paredes-Sabja is an Associate Professor at Texas A&M University in the Department of Biology, joined the department in 2020. His research focuses on Clostridioides difficile, a Gram-positive, strictly anaerobic, spore-forming bacterium that causes C. difficile infections (CDI), which are a major hospital-acquired pathogen with significant health and economic impacts. His work addresses several key areas including the mechanisms of C. difficile spore-host interactions, exosporium assembly, and therapeutic development against CDI. His research aims to elucidate how C. difficile spores interact with the intestinal mucosa and persist during infection, which is critical for understanding disease recurrence and transmission. He investigates the assembly of the spore's outer exosporium layer, a primary site of interaction with host surfaces, using molecular biology, bacterial genetics, biochemical, structural analysis, omics, and advanced imaging techniques. Additionally, his group works on developing novel therapeutic strategies to prevent spore interaction with the host's intestinal mucosa, their persistence, and transmission, blending expertise from immune-proteomics, immunology, pharmacology, and nanotechnology. His efforts also include adapting platforms for mucosal immunization studies related to SARS-CoV-2. Paredes-Sabja's background includes a Food Engineering degree from Universidad Austral de Chile and a Ph.D. from Oregon State University, with post-doctoral training at the same institution.

Selected publications

• Assessment of chemical methods in the extraction of spore surface layers in <i>Clostridioides difficile</i> spores

  mSphere · 2025-09-15 · 1 citations

  articleOpen accessSenior author

  ABSTRACT Clostridioides difficile spores are essential for initiation, recurrence, and transmission of C. difficile infections (CDI). These outermost layers of the spore, the exosporium and spore coat, are responsible for initial interactions with the host and spore resistance properties, respectively. Several spore coat/exosporium extraction methods have been utilized to study the spore surface, with differing procedures making comparison across studies difficult. Here, we tested how commonly used exosporium and spore coat extraction methods, termed EBB, USD, and Laemmli, remove the spore coat and exosporium layers of C. difficile spores. We assessed the impact of these extraction methods on the spore through transmission electron microscopy, phase contrast microscopy, western blotting, and lysozyme-triggered cortex degradation. Transmission electron microscopy shows that treatment with EBB and USD completely removes the spore coat and exosporium layer while leaving decoated spores intact. Western blots revealed differences in the ability to extract spore surface protein markers (CdeC, CdeM, CotA). In addition, lysozyme was able to degrade the cortex in decoated spores regardless of the treatment employed. Western blot analysis of lysozyme-treated decoated spores reveals that EBB and USD treatment allow for the detection and release of the spore core germination protease, GPR. Our results provide a comparison of commonly used extraction methods in C. difficile spore biology, standardizing their impact on spore coat and exosporium extraction for use in future studies. IMPORTANCE The outermost layers of Clostridioides difficile spores, the exosporium and spore coat, are essential for the spores’ resistance properties and initial interactions with the host. However, there is variability in extraction protocols, making it difficult to compare across studies. This work evaluates the commonly used extraction methods EBB, USD, and Laemmli at removing the exosporium and spore coat and provides a foundation for improved reproducibility. Here, we identified the effectiveness of these different extraction methods, allowing us to better understand these techniques to accurately analyze the spore surface in C. difficile spore research.

  PublisherDOI

• Effect of Pefloxacin on Clostridioides difficile R20291 Persister Cells Formation

  Antibiotics · 2025-06-20

  articleOpen access

  Clostridioides difficile is a Gram-positive bacterium recognized for its ability to produce toxins and form spores. It is mainly accountable for the majority of instances of antibiotic-related diarrhea. Background. Bacterial persister represent a minor fraction of the population that shows temporary tolerance to bactericidal agents, and they pose considerable medical issues because of their link to the rise of antibiotic resistance and challenging chronic or recurrent infections. Our previous research has shown a persister-like phenotype associated with treatments that include pefloxacin. Nonetheless, the mechanism is still mostly unclear, mainly because of the difficulty in isolating this small group of cells. Objectives. To enhance the understanding of C. difficile persister cells, we made an enrichment and characterization of these cells from bacterial cultures during the exponential phase under pefloxacin treatment and lysis treatment. Results. We demonstrate the appearance of cells with lower metabolism and DNA damage. Furthermore, we noted the participation of toxin–antitoxin systems and Clp proteases in the generation of persister cells. Conclusions. This work demonstrates the formation of C. difficile persister cells triggered by a lethal concentration of pefloxacin.

  PublisherOA PDFDOI

• Characterization of a hypersporulating strain derivative of <i>Clostridioides difficile</i> R20291

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-25 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract 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.

  PublisherOA PDFDOI

• Phenotypic analysis of various <i>Clostridioides difficile</i> ribotypes reveals consistency among core processes

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-13 · 2 citations

  preprintOpen access

  Abstract Clostridioides difficile infections (CDI) cause almost 300,000 hospitalizations per year of which ∼15-30% are the result of recurring infections. The prevalence and persistence of CDI in hospital settings has resulted in an extensive collection of C. difficile clinical isolates and their classification, typically by ribotype. While much of the current literature focuses on one or two prominent ribotypes ( e.g ., RT027), recent years have seen several other ribotypes dominate the clinical landscape ( e.g. , RT106 and RT078). Some ribotypes are associated with severe disease and / or increased recurrence rates, but why are certain ribotypes more prominent or harmful than others remains unknown. Because C. difficile has a large, open pan-genome, this observed relationship between ribotype and clinical outcome could be a result of the genetic diversity of C. difficile . Thus, we hypothesize that core biological processes of C. difficile are conserved across ribotypes / clades. We tested this hypothesis by observing the growth kinetics, sporulation, germination, bile acid sensitivity, bile salt hydrolase activity, and surface motility of fifteen strains belonging to various ribotypes spanning each known C. difficile clade. In viewing these phenotypes across each strain, we see that core phenotypes (growth, germination, sporulation, and resistance to bile salt toxicity) are remarkably consistent across clades / ribotypes. This suggests that variations observed in the clinical setting may be due to unidentified factors in the accessory genome or due to unknown host-factors. Importance C. difficile infections impact thousands of individuals every year many of whom experience recurring infections. Clinical studies have reported an unexplained correlation between some clades / ribotypes of C. difficile and disease severity / recurrence. Here, we demonstrate that C. difficile strains across the major clades / ribotypes are consistent in their core phenotypes. This suggests that such phenotypes are not responsible for variations in disease severity / recurrence and are ideal targets for the development of therapeutics meant to treat C. difficile related infections.

  PublisherDOI

• Phylogenomic analysis of the collagen-like BclA proteins in <i>Clostridioides difficile</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-24 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract 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 &amp; 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.

  PublisherOA PDFDOI

• Phenotypic analysis of various <i>Clostridioides difficile</i> ribotypes reveals consistency among core processes

  Applied and Environmental Microbiology · 2025-06-24 · 1 citations

  articleOpen access

  ABSTRACT Clostridioides difficile infections (CDI) cause almost 300,000 hospitalizations per year, of which ~15%–30% are the result of recurring infections. The prevalence and persistence of CDI in hospital settings have resulted in an extensive collection of C. difficile clinical isolates and their classification, typically by ribotype. While much of the current literature focuses on one or two prominent epidemic ribotypes (e.g., RT027), recent years have seen several other ribotypes dominate the clinical landscape (e.g., RT106 and RT078). Some ribotypes are associated with severe disease and/or increased recurrence rates, but why certain ribotypes are more prominent or harmful than others remains unknown. Because C. difficile has a large, open pan-genome, this observed relationship between ribotype and clinical outcome could be a result of the genetic diversity of C. difficile . Thus, we hypothesize that the core biological processes of C. difficile are conserved across ribotypes/clades. We tested this hypothesis by observing the growth kinetics, sporulation, germination, production of toxin A and toxin B, bile acid sensitivity, bile salt hydrolase activity, and surface motility of 15 strains belonging to various ribotypes spanning each known C. difficile clade. In viewing these phenotypes across each strain, we see that core phenotypes (growth, germination, sporulation, and resistance to bile salt toxicity) are remarkably consistent across clades/ribotypes. This suggests that variations observed in the clinical setting may be due to unidentified factors in the accessory genome or due to unknown host factors. IMPORTANCE Clostridioides difficile infections impact thousands of individuals every year, many of whom experience recurring infections. Clinical studies have reported an unexplained correlation between some clades/ribotypes of C. difficile and disease severity/recurrence. Here, we demonstrate that C. difficile strains across major clades/ribotypes are consistent in their core phenotypes. This suggests that such phenotypes are not responsible for variations in disease severity/recurrence and are ideal targets for the development of therapeutics meant to treat C. difficile -related infections.

  PublisherDOI

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

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-29 · 3 citations

  preprintOpen accessSenior authorCorresponding

  Abstract 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.

  PublisherDOI

• Assessment of chemical methods in the extraction of spore surface layers in <i>Clostridioides difficile</i> spores

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-20

  preprintOpen accessSenior authorCorresponding

  Abstract Clostridioides difficile spores are essential for initiation, recurrence, and transmission of C. difficile infections (CDI). These outermost layers of the spore, the exosporium and spore coat, are responsible for initial interactions with the host and spore resistance properties respectively. Several spore coat /exosporium extraction methods have been utilized to study the spore surface with differing procedures making comparison across studies difficult. Here, we tested how commonly used exosporium and spore coat extraction methods, termed EBB, USD, and Laemmli, remove the spore coat and exosporium layers of C. difficile spores. We assessed the impact of these extraction methods on the spore through transmission electron microscopy, phase contrast microscopy, western blotting, and lysozyme triggered cortex degradation. Transmission electron microscopy shows that treatment with EBB and USD, completely remove the spore coat and exosporium layer while leaving decoated spores intact. Western blots revealed differences in the ability to extract spore surface protein markers (CdeC, CdeM, CotA). In addition, lysozyme was able to degrade the cortex in decoated spores regardless of the treatment employed. Western blot analysis of lysozyme treated-decoated spores, reveals that EBB and USD treatment allows for detection and release of the spore core germination protease, GPR. Our results provide a comparison of commonly used extraction methods in C. difficile spore biology, standardizing their impact in spore coat and exosporium extraction for use in future studies. Importance The outermost layers of C. difficile spores, the exosporium and spore coat, are essential for the spores resistance properties and initial interactions with the host. However, there is variability in extraction protocols making it difficult to compare across studies. This work evaluates the commonly used extraction methods EBB, USD, and Laemmli at removing the exosporium and spore coat and provides a foundation for improved reproducibility. Here, we identified the effectiveness of these different extraction methods allowing us to better understand these techniques to accurately analyze spore surface in C. difficile spore research.

  PublisherOA PDFDOI

• Editorial: Pathogenic clostridia

  Anaerobe · 2024-09-14 · 1 citations

  editorial1st author
  PublisherDOI

• Assessment of chemical methods in the removal of the spore coat and exosporium layers of <i>Clostridioides difficile</i> Spores

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-04-08

  preprintOpen accessSenior authorCorresponding

  spores are essential for initiation, recurrence and transmission of the disease. The spore surface layers are composed of an outermost exosporium layer that surrounds another proteinaceous layer, the spore coat. These spore surfaces layers are responsible for initial interactions with the host and spore resistance properties contributing to transmission and recurrence of CDI. During spore-development, assembly of both layers is tightly interconnected thus studying the surface is essential for understanding the assembly of these layers and identification of potential targets for therapeutics. Several spore coat /exosporium extraction methods have utilized different extraction procedures making comparison across studies difficult and their impact on spore surface layer properties remains unclear. Here, we tested how commonly used chemical methods remove the spore coat /exosporium layers, analyzing treated-spores by phase contrast microscopy, transmission electron microscopy, western blotting, and lysozyme-triggered germination to functionally characterize the extraction efficiency of these treatment on these layers. Our results provide a systematic analysis and offer a platform for future spore coat and exosporium-related studies.

  PublisherOA PDFDOI