Glucose 6-phosphate transport regulatory protein UhpA regulates the virulent genes in Edwardsiella piscicida

Abstract
 Edwardsiella piscicida (E. piscicida) is an important zoonotic pathogen, which infects animals by colonizing the intestine. Glucose 6-phosphate (Glu6P) was an important carbohydrate in intestine and could be used as a regulate signal. Here we identify a virulence-regulating pathway named Glu6P transport regulatory protein UhpA, which affects the virulent genes of hemolysins, flagellar, T3SS, T6SS and metabolism related genes how to promote E. piscicida infect the host. The results showed that the metabolism related gene expression of cysteine synthase (orf 1134) and sulfate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (P < 0.05), the gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (P < 0.05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (P < 0.05), the gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold 1.28-fold and 1.23-fold higher than the ones in the wild strains (P < 0.05). Besides, the survival rate of fish challenged with E. piscicida EIB202 and ΔuhpA was 50% and 30% respectively. These suggested that although the uhpA gene deletion decreased the metabolic level and the hemolysins related gene expression in E. piscicida, the uhpA gene could down regulate the key virulent gene expression to decrease the pathogenicity of E. piscicida in fish.


Introduction
Edwardsiella piscicida (E. piscicida) is a rod-shaped Gram-negative, facultatively anaerobic, noncapsulated, motile, intracellular bacteria, which belong to an Enterobacteriaceae, Edwardsiella species, the old name was called as E. tarda (1,2). This bacterium could infect sh and move through the food chain to infect amphibians, reptiles, and birds, and humans, which particularly infects sh and causes large economic losses (3,4). E. piscicida infected host meanly through the intestine because it provides nutrition for bacteria (5,6). The microbiotas rely on intestine nutrient availability and invading pathogens compete for these resources and regulate its virulent gene expression to enhance its pathogenicity (6)(7)(8).
The pathogenesis of E. piscicida include many virulence factors such as the production of hemolysis (9,10), lament structural protein of agellar (11), possession of type Ш secretion system (T3SS) (2,9), and type VI secretion system (T6SS) (12), production of growth metabolism (13,14), ability to adhere, invade, survive and replicate in host cells (2). Additionally, most of them were regulated by the two-component signal transduction system (TCS), such as UhpA/UhpB-UhpC, which enables the cell to acquire phosphorylated sugars from its environment that can be used as carbon or energy sources (15). Although uhpA gene deletion affects Glucose 6-phosphate (Glu6P) usage in E. piscicida and UhpA could decrease its pathogenicity in zebra sh (15), UhpA how to regulate the main virulent genes of hemolysis, agellar, T3SS, T6SS and metabolism are still not available.
The study aims to nd out the uhpA gene in E. piscicida on how to affect the main virulent gene expression and pathogenicity of E. piscicida. Elucidate the key virulent gene expression of hemolysis, agellar, T3SS, T6SS and metabolism in the uhpA gene mutant strain and the wild type strain of E. piscicida. We believe that understanding this mechanism of E. piscicida would be helpful to explain the key role of UhpA in the pathogenicity of E. piscicida.

The uhpA gene deletion down-regulates the gene expression of metabolism
To evaluate the function of the uhpA gene in E. piscicida how to affect the metabolism related genes. The gene expression was carried out in the uhpA mutant strain and the wildtype strain, and the results were shown in (Fig. 1). Results demonstrated that the metabolism related gene expression of cysteine synthase (orf 1134) and sulfate transporter (ychM) in the mutant strains was 0.76-fold and 0.68-fold lower than the ones in the wild strains (P < 0.05). This suggested that the uhpA gene deletion decreased the metabolic level E. piscicida.

The uhpA gene deletion down-regulates the gene expression of hemolysis
Hemolysins are lipids and proteins that cause lysis of red blood cells by destroying their cell membrane. The primary function of hemolysins is that of hemolysis, the result of two key hemolysis related gene expressions was shown in (Fig. 2). Results showed that the gene expression of ethA and ethB in the mutant strains was 0.80-fold and 0.72-fold lower than the ones in the wild strains (P < 0.05). This suggested that the uhpA gene deletion decreased the hemolysis related gene expression in E. piscicida.

The uhpA gene deletion up-regulates the gene expression of agellar
The primary function of a agellum is that of locomotion, the result of two agellar related gene expressions was shown in (Fig. 3). Results showed that the gene expression of iC and gN in the mutant strains was 1.51-fold and 1.21-fold higher than the ones in the wild strains (P < 0.05). This suggested that the uhpA gene deletion increased the agellar related gene expression in E. piscicida.

2.4
The uhpA gene deletion up-regulates the gene expression of T3SS and T6SS The result of T3SS and T6SS related gene expressions was shown in (Figs. 4 and 5). Results showed that the gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the mutant strains was 1.27fold, 1.13-fold 1.28-fold and 1.23-fold higher than the ones in the wild strains (P < 0.05). This suggested that the uhpA gene deletion increased the T3SS and T6SS related gene expression in E. piscicida.

2.5
The uhpA gene deletion of E. piscicida attenuates virulence in sh E. piscicida EIB202 caused mortalities in tilapia at rst day after being orally injected infection, whereas the death occurred at 1-3 days after infection with the mutant strain E. piscicida ΔuhpA (Fig. 6). The survival rate of E. piscicida EIB202 and ΔuhpA groups was 50% and 30% respectively. None of the sh in the control group died and pure cultures of E. piscicida strains were recovered from the kidney and liver of dead sh.

Discussion
Host-bacterial interactions are very complex, the fundamental relationship between them are competition between host immunity and bacterial pathogenicity (6,(16)(17)(18). For example, host immunity after vaccination was greater than the invading ability of bacteria; sh could not be dead appearing (4,19,20). As we know E. piscicida is an important zoonotic pathogen, although Edwardsialla has been known as a serious pathogen of aquatic animals for a long time, its pathogenicity mechanisms are yet to be fully elucidated. The pathogenesis of E. piscicida appears to be multifactorial virulence factors. One of them, the UhpA was regulated by the transport protein UhpT, to transport a broad range of phosphorylated sugars (15). In this system, the Glu6P has been used as a broad speci city of sugar phosphates (21), which decreased the pathogenicity of E. piscicida in zebra sh (15). Although UhpA decreased its pathogenicity has been reported, UhpA how to regulate the main virulent gene are still not available.
Cysteine synthase coded by open reading frame 1134 in E. piscicida genome, have been reported to participate in the regulation of ethanol utilization and production of antioxidant respectively (13). With the growing knowledge on the physiology of dissimilatory sulfate, one of them coded by ychM gene has been identi ed in bacteria including E. piscicida (14,22). The virulence element hemolysis (Eth) system, which comprised EthA and EthB, widely distributed in this bacterium, which is essential for the sh invasion in vivo and in vitro (9,10). In this study, our results showed that the metabolism related gene and the hemolysis related gene expression were lower than the ones in the wild strains. This suggested that the uhpA gene deletion decreased the metabolic level and hemolysis ability in E. piscicida.
On the other hand, a agellar protein encoded by gN and iC plays an important role in the adhesion and the proptosis in E. piscicida respectively (11,23,24). Meanwhile, EsrA and EsrB are responsible for T3SS regulation in E. piscicida (2,9). Also, EvpB and EvpC proteins regulated iron-restricted conditions in vitro had a crucial role in the T6SS mediated pathogenesis of E. piscicida (12). The result in this study showed that the agellar related gene, T3SS and T6SS related gene expressions were higher than the ones in the wild strains. Importantly, the survival rate of sh infected with E. piscicida EIB202 was higher than sh infected with ΔuhpA. In general, the uhpA gene deletion more effect on the agellar protein, T3SS and T6SS, enhanced the pathogenicity of E. piscicida. In summary, the pathogenicity of the E. piscicida mutant strain ΔuhpA increased because the key virulent gene upregulated after the uhpA gene deletion.

The Expression Of Main Virulent Genes
The E. piscicida EIB202 and ΔuhpA were grown in TSB cultures at 28℃. Total RNA was extracted at 0.5 OD 600 using a total RNA isolation system (TaKaRa company, Dalian, China). To verify that the RNA was free of DNA contamination, PCR ampli cation was performed using the RNA as a template. The primers for virulent genes are shown in (Table 1). The RT-PCR and qPCR was carried out performing our previous research (8,26,27). The tilapia was prepared as our previous paper and has been approved by the Animal Ethics Committee of Shandong Agricultural University (8), the mean weight of the tilapia was 73.09 ± 3.07 g, and the cultured water's temperature was 28.0 ± 1.0 °C. These sh were recognized as disease-free animals to assess the virulence of E. piscicida strains (EIB202 and ΔuhpA). Fish from each group were orally infected with 100 µL PBS containing bacterial cells at 10 10 CFU/mL according to the previous research.
The number of dead sh was calculated observed over for 14 days.

Statistical Analysis
Statistical signi cance was determined by ANOVA analysis. Differences were considered signi cant at P < 0.05.

Declarations Con ict of interest
The authors declare that they have no con ict of interest.

Availability of data and material
All data generated or analyzed during this study are included in this published article. Figure 1 The difference of transcript levels of the cysteine synthase and sulfur metabolism related genes in E.