Biology subjects

Baloh, M.

Publications and source records attributed to Baloh, M..

3 recordsLinked to original sources

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

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↗