CRISPR-dependent endogenous gene regulation is required for virulence in piscine Streptococcus agalactiae

Abstract

The clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) system is a prokaryotic defence against invading mobile genetic elements, such as bacteriophages or exogenous plasmids. Beyond this, this system has been shown to play an important role in controlling the virulence of some bacterial pathogens. Streptococcus agalactiae strain GD201008-001, a causative agent of septicemia and meningitis in tilapia, contains a single type II CRISPR-Cas system with Cas9 as a signature protein. In this study, we found that the deletion of CRISPR significantly reduced adhesion, invasion, cytotoxicity and haemolysis, and caused severely attenuated virulence in the piscine S. agalactiae strain. RNA-Seq identified 236 endogenous genes regulated by CRISPR, with 159 genes upregulated and 77 genes downregulated. The resulting change in gene transcription by CRISPR was much more pronounced than that by cas9 in this bacterium, indicating CRISPR-mediated endogenous gene regulation was mostly independently of cas9. Subsequent studies showed that CovR/S two-component system was transcriptionally upregulated due to CRISPR deletion, which repressed the expression of the cylE gene coding for a cytolytic toxin, and thus decreased the activity of β-haemolysin/cytolysin. However, upregulation of CovR/S was not the contributor to the attenuation phenotype of ΔCRISPR. Further, we demonstrated that CRISPR is capable of repressing the expression of Toll-like receptor 2 (TLR2)-activating lipoprotein Sag0671 and thus dampens the innate immune response. This study revealed that the CRISPR system of S. agalactiae exhibited extraordinary potential capability in the regulation of endogenous transcripts, which contributes to bacterial innate immune evasion and virulence.

Keywords: CRISPR; CovR/S; Streptococcus agalactiae; lipoprotein Sag0671; virulence.

Figure 1.

The biological characteristics of the WT and ΔCRISPR strains. (A) Growth curves of the WT and ΔCRISPR strains in THB medium. (B) Adhesion ability of the WT and ΔCRISPR strains to bEnd3 cells. (C) Invasion ability of the WT and ΔCRISPR strains in bEnd3 cells. (D) Cytotoxicity of the WT and ΔCRISPR strains to RAW264.7 macrophage cells. The data are shown as the means ± SD from three independent experiments. *P < 0.05 or ***P < 0.001.

Figure 2.

Survival percentage and bacterial distribution in different organs of mice infected with the WT, ΔCRISPR and CΔCRISPR strains. (A) The survival of the mice was monitored every 4 h. At 16 h post-infection, spleen (B), blood (C) and brain (D) were harvested to count the number of CFUs. Bars represent the means for six infected mice. The data are shown as the means ± SD. ***P < 0.001.

Figure 3.

CRISPR promotes BBB penetration. BALB/c mice were intraperitoneally injected with 100 CFU of the WT, ΔCRISPR and CΔCRISPR strains. At 3, 9 and 15 h post-infection, the brains were collected. The level of BBB penetration was assessed by quantitative measurement of E. coli M5 loads per gram of brain. ***P < 0.001.

Figure 4.

Comparative transcriptome analysis between the WT and ΔCRISPR strains. (A) Volcano plot. The X-axis is the log2 of the linear fold change (ΔCRISPR/WT), and the Y-axis is the negative log10 of the Benjamini-Hochberg corrected t-test P value. Up- and down-regulated genes are indicated in shades of red and green, respectively. (B) Venn diagram showing the numbers of differentially abundant genes between ΔCRISPR and Δcas9. (C) The transcription levels of covR/S in the WT, ΔCRISPR, CΔCRISPR and Δcas9 strains. *P < 0.05 or **P < 0.01.

Figure 5.

CovR/S is required for the CRISPR-mediated Δ-haemolysin activity decrease. (A) The haemolytic activity of the WT, ΔCRISPR, ΔcovR/S and ΔCRISPR-covR/S strains. (B) The transcription level of cylE in the WT, ΔCRISPR, ΔcovR/S and ΔCRISPR-covR/S strains. ***P < 0.001.

Figure 6.

CovR/S is required for the CRISPR-mediated adhesion decrease. Adhesion of the WT, ΔCRISPR, ΔcovR/S and ΔCRISPR-covR/S strains to bEnd.3 cells. At 2 h post-infection, cells were washed and lysed for measurement of the number of CFU. Data are presented as the means ± SD of three independent experiments. **P < 0.01 or ***P < 0.001.

Figure 7.

The effect of CovR/S on the CRISPR-mediated attenuation of virulence. (A) Survival percentage of mice infected by the WT, ΔCRISPR, ΔcovR/S and ΔCRISPR-covR/S strains. (B-D) The bacterial distribution in different organs of mice infected by the WT, ΔCRISPR, ΔcovR/S and ΔCRISPR-covR/S strains. *P < 0.05 or ***P < 0.001.

Figure 8.

S. agalactiae-induced cytokine expression in RAW264.7 macrophages. (A-B) The expression levels of IL-1β, IL-6 and TNF-α in W264.7 macrophages infected with WT, ΔCRISPR, Δsag0671, WT+psag0671 and ΔCRISPR-sag0671 strains. (C-D) The effect of antagonist C29 on the expression levels of IL-1β, IL-6 and TNF-α in W264.7 macrophages. RAW264.7 cells were grown in DMEM containing 15% foetal bovine serum in 24-well tissue culture plates. For inactivated TLR2 signalling, the cells were incubated with 100 μg/mL antagonist C29 for 1 h. RAW264.7 macrophages were infected with the WT, ΔCRISPR, WT + psag0671 Δsag0671 and ΔCRISPR-sag0671 strains at a MOI of 1:1. Extracellular bacteria were killed by antibiotics, and cells were harvested at 8 and 16 h. The expression levels of IL-1β, IL-6 and TNF-α were measured by qRT-PCR. Data are presented as the means ± SD of three independent experiments. **P < 0.01 or ***P < 0.001.