Marathe, S. A. | Search bioRxiv

bioRxiv2023

Clustered Regularly Interspaced Short Palindromic Repeats-Cas system regulates Salmonella virulence

ObjectivesInvestigating the type 1-E CRISPR-Cas-mediated regulation of Salmonella pathogenesis. MethodsWe assessed the pathogenicity of the wild-type and CRISPR-Cas knockout strains using infection models. The mechanisms were elucidated using antimicrobial assays and expression analysis. ResultsCRISPR-Cas knockout strains were defective in invasion and proliferation in intestinal epithelial cells and macrophages. However, proliferation defects were not observed in the Gp91phox-/- macrophages, suggesting the systems role in antioxidant defence. The knockout strains show hampered colonization in in-vivo infection models, possibly due to increased sensitivity against innate immune barriers like antimicrobial peptides, complement proteins and oxidative stress. The expression studies of various virulence regulators: pmr genes, anti-oxidant genes, SPI-1 and SPI-2 encoded master regulators, and effectors showed repressed expression in the knockout strains. Some of these genes could be directly regulated by the CRISPR-spacers owing to partial complementarity between the sequences. ConclusionOverall, our study shows that the CRISPR-Cas system positively regulates Salmonella pathogenesis by regulating the expression of different virulence factors.

microbiology↗

bioRxiv2022

The CRISPR-Cas System differentially regulates surface-attached and pellicle-biofilm in Salmonella enterica serovar Typhimurium

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has been studied for its role in biofilm regulation and expression of outer membrane proteins in Salmonella. We investigated the CRISPR-Cas mediated biofilm regulation in Salmonella enterica serovar Typhimurium by deleting CRISPR-Cas components,{Delta} crisprI,{Delta} crisprII, {Delta}{Delta}crisprI crisprII, and{Delta} cas op. We determined that the system positively regulates surface-biofilm while inhibiting pellicle-biofilm. In knockout strains, flagellar (fliC, flgK) and curli (csgA) genes were repressed, causing reduced surface-biofilm. Conversely, they displayed altered pellicle-biofilm architecture possessing bacterial multilayers and a denser ECM with enhanced cellulose and lesser Curli, ergo weaker pellicle. The intracellular cellulose concentration was less in the knockout strains due to upregulation of bcsC, necessary for cellulose export. We hypothesized that exported cellulose integrates into the pellicle. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD, hence csgA and bcsA. The conflicting upregulation of bcsC, the last gene of bcsABZC operon, could be independently regulated by the CRISPR-Cas system owing to a partial match between the CRISPR-spacers and bcsC gene. The CRP-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of Yddx governing the availability of {sigma}28 factor that further regulates class3 flagellar genes (fliC, flgK). Additionally, the variations in the LPS profile and expression of LPS-related genes (rfaC, rfbG, rfbI) in the knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle-biofilm.

microbiology↗