The influence of enzyme EII of the cellobiose-phosphotransferase system on the virulence of Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)
Introduction
Streptococcus agalactiae (also referred as group B Streptococcus, GBS) is an important and prevalent pathogen in tilapia, leading to high morbidity and mortality in the global tilapia aquaculture industry every year (Chen et al., 2012; Chideroli et al., 2017). GBS, like many pathogenic bacteria, encodes a myriad of virulence factors that are attributed to its ability to cause severe invasive disease and tissue damage, including the pore-forming toxins (cfb and cylE), the immune evasion factors (cps, sodA, ponA and cspA), the adhesion, colonization and invasion factors (fbsA, fbsB, srr-1, bibA, sip, pavA, bca, spb1and hlyB), as well as other virulence factors (ccpA and psaA) (Arturo and Liise-anne, 2001; Landwehr-Kenzel and Henneke, 2014; Shabayek and Spellerberg, 2018). Regulation of gene expression in response to the external environment is important for GBS adherence and invasion of host cells, and the evasion of host immunity (Maisey et al., 2008). The most common signal transduction systems in bacteria are the two component systems (TCSs) and the phosphorylation cascade of the phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs), which change the expression of virulence factors to enable the organism to adapt to the environmental signal (Abranches et al., 2006; Bourret and Silversmith, 2010). The genome sequence of GBS has 21encoded pairs of TCS, in which RgfC/A, CiaH/R, Sak188/189, DltR/S, CsrR/S (also known as CovR/S), and BgrR/S are involved in regulating gene expression in GBS in response to changes in the external environment (Lin et al., 2009; Rozhdestvenskaya et al., 2010; Vasilyeva et al., 2015).
GBS is also a pathogen of humans, other fish species, and other animal species such as frogs, hamsters, mice, chickens, camels, dogs, horses, cats, bottlenose dolphins and monkeys (Elliott et al., 1990; Elliott et al., 1990; Johri et al., 2006). It has also been identified as a food contaminant, mainly in pastries and seafood products (Mee-Marquet et al., 2009). GBS can survive and develop a wide variety of environments due to its significant adaptive abilities (Flechard and Gilot, 2014). The capacity of GBS to import a wide range of carbon sources may explain its high adaptive capacity. For example, GBS expresses 17 sugar-specific PTS-EII complexes, four sugar-specific ABC transporters, three glycerol permeases, and one glycerol-phosphate permease. These PTSs have been supposed to import cellobiose, β-glucoside, trehalose, mannose, lactose, fructose, mannitol, N-acetylgalactosamine, or glucose (Glaser et al., 2002). The typical bacterial PTS is composed of the PEP-dependent protein kinase enzyme I (EI), the histidine-containing protein (HPr), and substrate-specific enzyme II (EII) complexes. The EII complexes are sugar-specific and are responsible for the concomitant phosphorylation and internalization of many different sugars, and consist of two soluble domains EIIA and EIIB involved in phosphotransfer and the membrane-bound transporter domain EIIC (Bettenbrock et al., 2007). In a PTS pathway, an incoming sugar is translocated across the cytoplasmic membrane and concomitantly phosphorylated by EII (Heravi et al., 2011). The energy for the EI translocation is provided by PEP, and is then transferred to HPr, which then transfers a phosphoryl group to EIIA, then on to EIIB, and finally to the incoming sugar which is transported across the internal membrane via EIIC (Wu et al., 2016).
PTSs can sense and respond to external stimuli as well as the internal metabolic status of the cell (Aboulwafa and Saier Jr., 2013). In addition to sugar uptake, some PTSs have been associated with growth rate, biofilm formation, the expression of virulence genes, and virulence (Loo et al., 2003; Lun and Willson, 2005; Weaver et al., 2000). In Escherichia coli, the glucose-PTS (glu-PTS) controls carbon catabolite repression (CCR) and regulates the expression of the rpoS gene, which encodes the sigma factor that is a central regulator in the transcriptional control of such genes (Ueguchi et al., 2001). In Listeria monocytogenes, mannose-PTS (man-PTS) permease was related to virulence gene expression and virulence (Liu et al., 2017). Moreover, in Klebsiella pneumoniae, the EIIC of the cellobiose-PTS (cel-PTS) is not only involved in sugar transport and metabolism, but also contributes to biofilm formation and virulence through the metabolism of cellobiose (Wu et al., 2012). Previous studies have indicated that the PTSs are involved in the positive regulation of streptococcal virulence. McAllister et al. (2012) demonstrated that the deletion of the cel-PTS gene in virulent S. pneumonia of different serotypes leads to its decreased colonization and virulence in mice.
We previously reported that deletion of cel-EIIB of cel-PTS in S. agalactiae THN0901, impaired biofilm formation and its invasion and colonization abilities, and that its virulence is attenuated but not significantly (Xu et al., 2019). Also that the EIIB of cel-PTS is highly expressed in the low-virulence strain S. agalactiae TFJ0901, and is expressed to a lesser extent in the high-virulence strain S. agalactiae THN0901 (Li et al., 2014). Moreover, in comparative genomics studies of high and low virulence strains in S. iniae, it was found that the EIIB gene of the fru-PTS system was absent in the low virulence strain (Pridgeon et al., 2013).We hypothesized that there may be differences between the low virulence strain S. agalactiae TFJ0901 and the high virulence strain S. agalactiae THN0901 may be present. Importantly, in Bacillus subtilis, the trehalose and sucrose transporters lack the specific EIIA domains, and phosphate is transferred from the EIIA domain of glu-EII to its own EIIB domain, while glucose competes with the phosphorylation of the EIIB domain, thereby reducing the transport of trehalose and sucrose (Dahl, 1997; Sutrina et al., 1990).
Therefore, in order to avoid an additional EIIB, or cross-talk between other PTS components, interfering with the effect of cel-EII complexes on the virulence mechanism of S. agalactiae, we designed the deletion of the cel-EII genes instead of the EIIB gene to further investigate the influence of the cel-PTS system on the virulence of S. agalactiae and the differences in the pathogenesis mechanism of high and low virulence S. agalactiae. In this study, we constructed the △cel-EII mutants by homologous recombination, to evaluate their pathogenicity using the biological characteristics, virulence, and the expression levels of some PTSs, TCSs, and virulence genes in order to provide new avenues for the study of the pathogenic mechanism of S. agalactiae in tilapia.
Section snippets
Bacteria, plasmids, cells and culture protocols
The bacteria, plasmids and cells used in this work are listed in Table 1. S. agalactiae THN0901 (a natural high virulence strain) and TFJ0901 (a natural low virulence strain) were isolated from two tilapia farms infected with GBS in the Hainan and Fujian Provinces, China, respectively (Su et al., 2017). They were cultured on brain-heart broth (BHI) (Huankai, China) and incubated at 28 °C. Escherichia coli DH5a was cultured on Luria-Bertani broth (LB) (Sangon, China) and incubated at 37 °C. The
Construction and verification of the △cel-EII mutant
The △cel-EII mutants were generated using homologous recombination of cel-EII complexes with the cat gene (Fig. 1A), as verified using PCR analysis (Fig. 1B/C) and DNA sequencing. Briefly, PCR was used to confirm that cel-EII complexes were replaced by the cat gene using EII-UP/DOWN (2626 bp) and Cat-F/R as primers. The qPCR analysis showed that the relative expression levels of cel-EIIA, cel-EIIB, and cel-EIIC were no expressed in △cel-EII-THN0901 (Fig. 10A) and △cel-EII-TFJ0901 (Fig. 10D).
Discussion
Phosphotransferase systems are complex and control sugar phosphorylation and transport in bacteria. The cel-PTS is one of the PTS systems in S. agalactiae, and its EII complexes are sugar-specific. The PTS consists of three domains (IIA, IIB and IIC) (Wu et al., 2012). Previous studies have shown that after the deletion of either EIIA, EIIB or EIIC genes, the growth rate of a bacterium is weakened (Ake et al., 2011; Liu et al., 2017). Similarly, the growth rate of the Δcel-EIIB was
Author statement
Youlu Su: Conceptualization, Resources, Investigation, Funding acquisition, Project administration, Writing-original draft, Writing-review and editing, Validation. Yundan Xie: Methodology, Software, Validation, Formal analysis, Writing-Original draft, Writing-review and editing. Baotun Wang, Juan Feng, Wei Li, Biao Jiang, Chun Liu and Yanhua Huang: Supervision, Visualization, Data curation, Validation. All authors read and contributed to the manuscript.
Declaration of Competing Interest
I wish to submit an original research article for publication in Aquaculture, titled “The influence of enzyme EII of the cellobiose-phosphotransferase system on the virulence of Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)”. The paper was coauthored by Yundan Xie, Baotun Wang, Juan Feng, Wei Li, Biao Jiang, Chun Liu, Yanhua Huang, Youlu Su. This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal.
Acknowledgments
This work was supported by grants from the Key Project of Department of Education of Guangdong Province (2019KZDXM043).
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