Biology subjects

Sorg, J. A.

Publications and source records attributed to Sorg, J. A..

10 recordsLinked to original sources

bioRxiv2025

Assessment of chemical methods in the extraction of spore surface layers in Clostridioides difficile spores

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. ImportanceThe 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.

microbiology↗

bioRxiv2025

Phenotypic analysis of various Clostridioides difficile ribotypes reveals consistency among core processes

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. ImportanceC. 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.

microbiology↗

bioRxiv2024

The impact of YabG mutations on C. difficile spore germination and processing of spore substrates

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (catalytically inactive), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

microbiology↗

bioRxiv2023

The small acid-soluble proteins of Clostridioides difficile regulate sporulation in a SpoIVB2-dependent manner

Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression. ImportanceC. difficile is easily spread through the production of highly resistant spores. Understanding how spores are formed could yield valuable insight into how the sporulation process can be halted to render spores that are sensitive to cleaning methods. Here, we identify another protein involved in the sporulation process that is seemingly controlled by the small acid-soluble proteins (SASPs). This discovery allows us to better understand how the C. difficile SASPs may bind to specific sites on the genome to regulate gene expression.

microbiology↗

bioRxiv2022

Clostridioides difficile bile salt hydrolase activity has substrate specificity and affects biofilm formation

The Clostridioides difficile pathogen is responsible for nosocomial infections. Germination is an essential step for the establishment of C. difficile infection (CDI) because toxins that are secreted by vegetative cells are responsible for the symptoms of CDI. Germination can be stimulated by the combinatorial actions of certain amino acids and either conjugated or deconjugated cholic acid-derived bile salts. During synthesis in the liver, cholic acid- and chenodeoxycholic acid-class bile salts are conjugated with either taurine or glycine at the C24 carboxyl. During GI transit, these conjugated bile salts are deconjugated by microbes that express bile salt hydrolases (BSHs). Here, we surprisingly find that several C. difficile strains have BSH activity. We observed this activity in both C. difficile vegetative cells and in spores and that the observed BSH activity was specific to taurine-derived bile salts. Additionally, we find that this BSH activity can produce cholate for metabolic conversion to deoxycholate by C. scindens. The C. scindens-produced deoxycholate signals to C. difficile to initiate biofilm formation. Our results show that C. difficile BSH activity has the potential to influence the interactions between microbes and this could extend to the GI setting. ImportanceBoth primary and secondary bile salts are well-established to impact C. difficile spore germination and vegetative growth. Here, we find that C. difficile vegetative cells, and spores, have bile salt hydrolase activity that is specific to taurine-derived bile salts. When grown in co-culture with the secondary bile salt-producing bacterium, C. scindens, we find that C. difficile-mediated deconjugation of taurocholate, feeds C. scindens cholate. C. scindens 7-dehydroxylates cholate to deoxycholate. The C. scindens-produced deoxycholate then stimulates biofilm formation by C. difficile cells. Thus, this suggests that the bile salt hydrolase activity expressed by several C. difficile strains could be responsible for modulating in vivo biofilm formation and maintenance in a host.

microbiology↗

bioRxiv2022

Imaging Clostridioides difficile spore germination and germination proteins

Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and co-germinant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory pro-peptide from the pro-SleC cortex lytic enzyme thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, inner spore membrane), these have been based, mostly, on hypotheses or prior data in C. perfringens. In this study, we visualize the germination process using TEM and SEM, and, using immunogold labeling of the spore proteins, find that these proteins are localized to the spore cortex, consistent with the observed, rapid, changes to the spore structure during germination. ImportanceGermination by C. difficile spores is the first step in the establishment of potentially life-threatening CDI. A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form, or trigger germination prematurely when the spore is either in the outside environment or in a host environment that is non-conducive to the establishment of colonization / disease.

microbiology↗

bioRxiv2021

Clostridioides difficile SpoVAD and SpoVAE interact and are required for DPA packaging into spores

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., dry heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is packaged into the spore core, thereby replacing the majority of water. DPA is synthesized by the mother cell and its packaging into the spore core is regulated by the spoVA operon that has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA packaging into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC / SpoVAD / SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA packaging into the spore core. ImportanceC. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is packaged into the spore core, this study may provide insight in how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.

microbiology↗

bioRxiv2021

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main /{beta} SASPs, SspA and SspB, and two annotated SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated SASPs do not contribute to UV resistance. In addition, none of these SASPs contribute to the resistance of tested chemicals. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate / serve as a checkpoint for spore formation, a previously unreported function of SASPs. ImportanceC. difficile infections remain problematic and elimination of spores within an environment is essential to limit person-to-person spread. A deeper understanding of how spores resist cleaning efforts could lead to better strategies to eradicate the spores in a contaminated environment. The small acid-soluble proteins (SASPs), found in all endospore-forming organisms, are one mechanism that allows for spore resilience. Here, we find that C. difficile SspA and SspB protect against UV light. Unexpectedly, these SASPs also regulate spore formation, a role not described for any SASP to date.

microbiology↗

bioRxiv2021

The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. ImportanceC. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile lifecycle, other non-antibiotic treatment options are in urgent need of development. Towards this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology - Stickland metabolism. Here, we use our CRISPR-Cas9 system to knock in a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology - from vegetative growth to spore formation and outgrowth post-germination.

microbiology↗

bioRxiv2018

The requirement for the amino acid co-germinant during C. difficile spore germination is influenced by mutations in yabG and cspA

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A defined yabG mutant exacerbated the EMS mutant phenotype. Moreover, we found that various mutations in cspA caused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, our study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants during C. difficile spore germination and that two pseudoproteases (CspC and CspA) function as the C. difficile germinant receptors.

microbiology↗