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

Abstract

<i>Salmonella</i> 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 <i>Salmonella</i> 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 <i>cas3</i> in <i>Salmonella enterica</i> serovar Enteritidis. Compared to the <i>cas3</i> gene wild-type (<i>cas3</i> WT) <i>Salmonella</i> strain, <i>cas3</i> deletion upregulated the <i>lsrFGBE</i> genes in <i>lsr</i> (<i>luxS regulated</i>) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and <i>Salmonella</i> pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the <i>cas3</i> deletion mutant (Δ<i>cas3</i>). The bacterial invasive and intracellular capacity of Δ<i>cas3</i> 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 <i>cas3</i> 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 <i>Salmonella</i> and potentially other bacteria.