REGULATION OF CLOSTRIDIODES DIFFICILE CRISPR-CAS SYSTEM BY BIOFILM-ASSOCIATED FACTORS AND GLUCOSE



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Abstract

Clostridioides difficile is a spore-forming enteropathogenic anaerobic bacterium and one of the most common opportunistic human pathogens. Its pathogenicity relies on the production of toxins, sporulation, biofilm formation, and the ability to withstand numerous stresses encountered in the host environment. In addition to these well-known mechanisms, C. difficile possesses a remarkably complex CRISPR-Cas system characterized by two cas operons and multiple CRISPR arrays. While CRISPR-Cas systems are primarily studied as adaptive immune mechanisms against bacteriophages and mobile genetic elements, accumulating evidence suggests they may also be integrated into broader regulatory networks that contribute to bacterial physiology, adaptation, and virulence. However, the regulation and functional dynamics of this system in C. difficile remain largely unexplored.

In this study, we investigated the regulation of the C. difficile CRISPR-Cas system under biofilm-inducing factors. Quantitative PCR analysis revealed the induction of several CRISPR arrays and the partial cas operon under high intracellular levels of the secondary messenger cyclic di-guanosine monophosphate, a key regulator of bacterial phenotypic shifts. These results were partially confirmed by interference efficiency assays. A secondary bile salt, sodium deoxycholate, known to trigger biofilm formation, also increased both cas operons and one CRISPR array expression, suggesting its role for CRISPR-Cas system regulation during host-associated stress Moreover we identified glucose as a regulatory factor for C. difficile CRISPR-Cas system. Elevated glucose concentration in the medium induced the expression of the partial cas operon and CRISPR3-4 arrays. However, at the same time it functionally suppressed the interference efficiency of the system.

Together, our findings demonstrate that the C. difficile CRISPR-Cas system is responsive to biofilm-inducing signals and nutrient availability, linking its regulation to key aspects of bacterial physiology and adaptation to the host. This work also highlights the potential for non-canonical regulatory roles of CRISPR-Cas in C. difficile survival and pathogenesis.

About the authors

Anna S. Maikova

St. Petersburg Pasteur Institute, St. Petersburg, Russian Federation;
Peter the Great St. Petersburg polytechnic university, St. Petersburg, Russian Federation

Author for correspondence.
Email: ann-maikova@yandex.ru
ORCID iD: 0000-0002-0222-9939
SPIN-code: 3834-0677
Scopus Author ID: 57189218939
ResearcherId: AAF-9383-2019

Ph.D. in microbiology, researcher of the Laboratory for molecular genetics of pathogens

Россия, 197101, Russian Federation, St. Petersburg, Mira str., 14

Dmitry Evgenievich Polev

St. Petersburg Pasteur Institute, St. Petersburg, Russian Federation

Email: polev@pasteurorg.ru

PhD in Biology, Senior Researcher, Head of the Metagenomic Research Group

Россия

Mikhail A. Khodorkovskii

Peter the Great St. Petersburg polytechnic university, St. Petersburg, Russian Federation

Email: khodorkovskii@mail.ru

Ph.D. in Physical and Mathematical Sciences, Head of the research complex «Nanobiotechnologies»,

Россия, 195251, Russian Federation, St. Petersburg, Polytechnicheskaya str., 29B

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