CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems

Abstract

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.

Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3

“…A recent study on the effects of deleting cas 3 in Salmonella enterica showed no differences in liquid growth. In contrast to our results, those researchers found a decrease in intracellular survival in the mutant ( 53 ). The expression profiles of wild-type P. gingivalis and the cas 3 mutant were very similar when the bacteria were placed in human serum-based medium, but when they colonized the human macrophage-like THP-1 cell line, the transcriptome profiles of the wild-type and mutant strains were very different.…”

“…The only activities that were altered under both sets of growing conditions were activities associated with adhesion, probably because fimbrial genes, including fim A, were downregulated in the mutant. In S. enterica , deletion of cas 3 leads to downregulation of saf A, saf B, saf C, and saf D genes in the fimbrial operon ( 53 ). The mechanisms by which cas 3 controls levels of transcription of fimbrial genes remain unknown.…”

Insights Into Mechanisms of Biofilm Formation in Acinetobacter baumannii and Implications for Uropathogenesis

“…They observed that strains carrying CRISPR systems were enriched for biofilm-associated genes (>70 vs. <2% non-CRISPR strains), suggesting a link between CRISPR immunity and biofilm formation (Mangas et al, 2019). Previous research has shown that Cas3 endonuclease is involved in the control of biofilm formation in both gram-positive and gram-negative bacteria (Tang et al, 2019;Cui et al, 2020).…”

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

“…In addition to adaptive immunity, noncanonical functions have been suggested for the components of CRISPR-Cas systems (Cas proteins, crRNAs and other CRISPR-associated RNAs) that are implicated in the control of bacterial pathogenesis and physiology [ 163 , 164 , 165 , 166 , 167 ]. The molecular mechanisms of CRISPR-Cas-mediated control of gene expression remain in many cases to be explored, but important links of these regulatory processes with virulence, biofilm formation and envelope stress response could be emphasized in several pathogens, i.e., Francisella novicida , Neisseria meningitidis, Campylobacter jejuni, Legionella pneumophila , L. monocytogenes, and P. aeruginosa [ 164 , 166 , 168 ]. Interestingly, a recent work suggests targeting of quorum-sensing systems by CRISPR-Cas in Salmonella to mediate virulence and biofilm formation regulation [ 168 ].…”