Abstract
Pseudomonas plecoglossicida is a facultative pathogen that is associated with diseases of multiple fish, mainly at 15–20°C. Although fish disease caused by P. plecoglossicida has led to significant economic losses, the mechanisms of the temperature-dependent virulence are unclear. Here, we identify potential pathogenicity mechanisms and demonstrate the direct regulation of several virulence factors by temperature with transcriptomic and proteomic analyses, quantitative real-time PCR (qRT-PCR), RNAi, pyoverdine (PVD) quantification, the chrome azurol S (CAS) assay, growth curve measurements, a biofilm assay, and artificial infection. The principal component analysis, the heat map generation and hierarchical clustering, together with the functional annotations of the differentially expressed genes (DEGs) demonstrated that, under different growth temperatures, the animation and focus of P. plecoglossicida are quite different, which may be the key to pathogenicity. Genes involved in PVD synthesis and in the type VI secretion system (T6SS) are specifically upregulated at the virulent temperature of 18°C. Silencing of the PVD-synthesis-related genes reduces the iron acquisition, growth, biofilm formation, distribution in host organs and virulence of the bacteria. Silencing of the T6SS genes also leads to the reduction of biofilm formation, distribution in host organs and virulence. These findings reveal that temperature regulates multiple virulence mechanisms in P. plecoglossicida, especially through iron acquisition and T6SS secretion. Meanwhile, integration of transcriptomic and proteomic data provide us with a new perspective into the pathogenesis of P. plecoglossicida, which would not have been easy to catch at either the protein or mRNA differential analyses alone, thus illustrating the power of multi-omics analyses in microbiology.
Highlights
The correlation between temperature and bacterial disease is of escalating concern because the virulence of an increasing number of pathogens is found to be regulated by temperature (Igbinosa and Okoh, 2008; Vezzulli et al, 2010; Hashizume et al, 2011; Lam et al, 2014)
Compared to the samples cultured at 12◦C, our results revealed significant up-regulation of PVDS1, PVDS2, PVDS3, PVDS4, PVDS5, tbdr, hcp, icmF, and dotU in NZBD9 grown at 18◦C by 4.06, 3.03, 3.95, 3.03, 2.27, 2.23, 2.14, 2.29, and 3.03-fold, respectively (Table S3)
Compared to the samples cultured at 12◦C, our quantitative real-time PCR (qRT-PCR) results revealed significant up-regulation of PVDS1, PVDS2, PVDS3, PVDS4, PVDS5, tbdr, hcp, icmF, and dotU in NZBD9 grown at 18◦C by 1.88, 3.23, 1.73, 1.56, 1.33, 2.56, 2.50, 1.43, and 4.00-fold, respectively (Figure 3D)
Summary
The correlation between temperature and bacterial disease is of escalating concern because the virulence of an increasing number of pathogens is found to be regulated by temperature (Igbinosa and Okoh, 2008; Vezzulli et al, 2010; Hashizume et al, 2011; Lam et al, 2014). Temperature regulation can be achieved at DNA, RNA, or protein level, and several virulence factors could respond to temperature, the exact mechanisms of regulation still need to be investigated in many instances (Lam et al, 2014). Omics analyses, for instance, genomics, transcriptomics, and proteomics, are emerging in the investigation of mechanisms involved in temperaturerelated infections. The genome, transcriptome, and proteome are not isolated biological entities, and multi-omics data should be concomitantly used and integrated to map mechanisms of temperature-related infections (Manzoni et al, 2016). Integration of transcriptomic and proteomic approaches could provide the multidimensional collection of a core set of genes that probably involved in temperature-regulated bacterial virulence (Miao et al, 2015)
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