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.

Keywords: RNA-Seq; SPI-1-T3SS; Salmonella virulence; biofilm formation; cas3; quorum sensing; type I-E CRISPR-Cas system.

Figure 1

Schematic of the Salmonella type I-E clustered interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) operon. Salmonella has two CRISPR loci, CRISPR1 and CRISPR2, both encoded on the minus strand. There are eight cas genes that are located upstream of CRISPR1, shown as colored boxed arrows. Type I system signature gene, cas3, is shown (red). The cas1 and cas2 genes are universal, and present in all CRISPR-Cas systems (orange). The remaining cas genes are type I-E-dependent (green). The CRISPR locus comprises a leader region and repeats (black cross lines) that are separated by unique spacers (vertical colored lines). crRNA (CRISPR RNA) consists of a spacer flanked by partial repeats.

Figure 2

Impact of cas3 on the growth and biofilm formation of Salmonella. (A,B) Deletion of cas3 has no effect on bacterial growth. (C) Biofilm formation of cas3 WT, Δcas3, and Δcas3/p-cas3 after 48 h of incubation at 37 °C under static conditions quantified by crystal violet. (D) Biofilm formation in LB (Luria-Bertani) broth was detected and quantified by crystal violet after 96 h of incubation at 28 °C. NTC, non-treated control; Bars, mean ± SD; n = 3; ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA plus Dunnet’s multiple comparisons test).

Figure 3

Deficiency of cas3 in Salmonella weakens the bacterial virulence to host cells. (A–E) cas3 deletion decreased the invasion of Salmonella to different cells and the cas3 WT and Δcas3/p-cas3 strain showed a higher count of intracellular bacteria at different time points compared to the Δcas3 strain. This figure shows the CFU (Colony-Forming Unit) of each strain in the cells at different times after infection and the results were based on the CFU assays obtained from three independent experiments. (F,G) Measurement of murine alveolar macrophage cell MH-S death by propidium iodide (PI) uptake. (F) The sample of uninfected MH-S cells was regarded as a control. The MH-S cells of trial groups were infected by cas3 WT, Δcas3, and Δcas3/p-cas3 strains, respectively, for 2 h and assayed by PI uptake. The percentage of dead cells (Q3 region) is shown, respectively, and the statistical results of the three strains are shown as a column diagram (G). (H) LD₅₀ (50% lethal dose) of Salmonella for chickens following oral infection. Bars, mean ± SD; n = 3; ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA plus Dunnet’s multiple comparison test).

Figure 4

Gene ontology (GO) annotations of differentially expressed genes (DEGs) and classification graph of GO terms for DEG. GO includes the molecular function (MF), cellular component (CC), and biological process (BP).

Figure 5

Comparison of the expression level of some key genes acquired from RNA-Seq and RT-qPCR, including cas operon genes (A), lsr (LuxS-regulated) operon genes (B), and some of the SPI-1-T3SS genes (Salmonella pathogenicity island 1 genes related to the type three secretion system) (C).

Figure 6

Proposed mechanisms of the CRISPR-Cas system in regulating biofilm-forming-related genes and SPI-1 (Salmonella pathogenicity island 1) expression by degrading lsrF encoded by lsr operon. Cas3 may target and downregulate the expression of lsrF, and then delay the degradation of p-AI-2. Accordingly, activated AI-2 is increased, which then inhibits the activity of LsrR protein to increase the expression of the lsr operon. However, the expression of lsrF remains inhibited by this time. Finally, the expression of the biofilm-forming-related genes and SPI-1-T3SS genes are increased, and the biofilm formation and invasion of Salmonella to epithelial cells are activated.