Transcriptome analysis of heat resistance regulated by quorum sensing system in Glaesserella parasuis

Abstract

The ability of bacteria to resist heat shock allows them to adapt to different environments. In addition, heat shock resistance is known for their virulence. Our previous study showed that the AI-2/luxS quorum sensing system affects the growth characteristics, biofilm formation, and virulence of Glaesserella parasuis. The resistance of quorum sensing system deficient G. parasuis to heat shock was obviously weaker than that of wild type strain. However, the regulatory mechanism of this phenotype remains unclear. To illustrate the regulatory mechanism by which the quorum sensing system provides resistance to heat shock, the transcriptomes of wild type (GPS2), ΔluxS, and luxS complemented (C-luxS) strains were analyzed. Four hundred forty-four differentially expressed genes were identified in quorum sensing system deficient G. parasuis, which participated in multiple regulatory pathways. Furthermore, we found that G. parasuis regulates the expression of rseA, rpoE, rseB, degS, clpP, and htrA genes to resist heat shock via the quorum sensing system. We further confirmed that rseA and rpoE genes exerted an opposite regulatory effect on heat shock resistance. In conclusion, the findings of this study provide a novel insight into how the quorum sensing system affects the transcriptome of G. parasuis and regulates its heat shock resistance property.

Keywords: Glaesserella parasuis; heat shock resistance; molecular mechanism; quorum sensing; transcriptome.

Figure 1

(A) Venn diagram of differentially expressed genes. “A” means differentially expressed genes between GPS2 and ΔluxS groups; “B” means differentially expressed genes between ΔluxS and C-luxS groups; “C” means differentially expressed genes between GPS2 and C-luxS groups. (B) Volcano plot of differentially expressed genes between ΔluxS and GPS2 groups. Red dots represent up-regulated genes; green dots represent down-regulated genes; and blue dots represent non-differentially expressed genes.

Figure 2

GO and KEGG pathway enrichment analysis of differentially expressed genes in ΔluxS and GPS2 groups. The numbers of genes that are up- (A) and down-regulated (B) in ΔluxS versus GPS2 were categorized according to role categories with GO enrichment analysis. KEGG pathway enrichment analysis of ΔluxS versus GPS2 up-regulated (C) and down-regulated (D) differentially expressed genes. The size of each point shows the number of genes in the pathway.

Figure 3

RT-qPCR verification. Eight up-regulated genes (A) and eight down-regulated genes (B) in RNA-seq results were selected for verification. The experimental data were calculated using the 2^−ΔΔCt method. All above assays were performed thrice in triplicate. Bars represent the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, and ***p <  0.001.

Figure 4

RT-qPCR verification of heat shock-related genes in Glaesserella parasuis. (A) The differential expression of rseA, rpoE, rseB, degS, clpP, and htrA genes in GPS2, ΔluxS, and C-luxS strains. All above assays were performed thrice in triplicate. Bars represent the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. (B) PCR identification of the rseA mutant strain by amplification of the rseA-UKD fragment, kanamycin resistance cassette sequence fragment, and rseA gene fragment. (C) PCR identification of the rpoE mutant strain by amplification of the rpoE-UKD fragment, kanamycin resistance cassette sequence fragment, and the rpoE gene fragment. M: DL 2000 Mark, 1: rseA or rpoE mutant strain, P: positive control, and N: negative control. (D) The differential expression of rseA, rpoE, rseB, degS, clpP, and htrA genes in GPS2, ΔrseA, and ΔrpoE strains. All above assays were performed thrice in triplicate. Bars represent the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

The survival rates of GPS2, ΔluxS, C-luxS, ΔrseA, and ΔrpoE strains under heat shock conditions. All above assays were performed thrice in triplicate. Bars represent the mean ± standard deviation of three independent experiments. **p < 0.01, and ***p < 0.001.

Figure 6

Schematic drawing of the σ^E signaling pathway. As stress sensor proteins, DegS protease is activated by the binding of the OMP C-terminus, and RseB is relieved from RseA by the accumulation of LPS in the periplasm. RseA is sequentially digested by DegS and ClpP, thereby releasing σ^E in the cytoplasm. Finally, σ^E along with RNA polymerase can induce the transcription of the htrA gene and other heat shock-relate genes.