Cytochrome c Gene in Procambarus clarkii Inhibits WSSV Infection by Regulating the Apoptosis Pathway

Abstract

Apoptosis is programmed cell death and is regulated by a series of related genes. It is of great significance in resistance to pathogen invasion and maintaining homeostasis in the environment. The release of cytochrome c (Cytc) from the mitochondria into the cytoplasm is a key step in the initiation of apoptosis. Increasing evidence from investigation of Cytc in cell apoptosis and immunity shows that it can participate in cell apoptosis induced by virus infection. For example, white spot syndrome virus (WSSV) stimulation can induce Cytc gene expression in Litopenaeus vannamei hepatopancreas and hemocytes, and the apoptosis of Epinephelus akaara hepatocytes induced by red-spotted grouper nervous necrosis virus (RGNNV) is related to the release of Cytc. However, the role of Cytc-mediated apoptosis in Procambarus clarkii WSSV infection has not yet been reported. Therefore, in this study, the full length of the the cytochrome c gene of P. clarkii (PcCytc) was cloned, and the role of PcCytc in P. clarkii was analyzed. Its expression in various tissues of P. clarkii proved that WSSV infection can induce the expression of PcCytc. The mechanism of PcCytc involvement in cell apoptosis during WSSV infection was also explored using RNA interference technology, to gain a deeper understanding of the potential role of apoptosis-related factors in the immune response of P. clarkii.In this study, PcCytc was cloned using RACE technology, with a total length of 897 bp, including the 163 bp 5′-UTR, 419 bp 3′-UTR, and 315 bp open reading frame; it encoded 104 amino acids. The structure prediction showed that PcCytc contained a conserved Cytochrom_C domain, proving that it is related to energy production and tends to be conserved in evolution.The results of the quantitative PCR showed that the PcCytc gene was expressed in all tissues of P. clarkii. The expression was lowest in the stomach and higher in the gills, intestines, and muscles, which showed, respectively, 9.46, 8.65 and 7.88 times greater PcCytc expression than that in the stomach. PcCytc showed relatively high expression in tissues with high energy consumption, such as the intestines and muscles, which is consistent with previous studies in Penaeus vannamei. The highest expression level was observed in the gills of the main immune and respiratory tissues of P. clarkii, indicating that PcCytc may be involved in the related biological processes. Based on the above results, we speculate that PcCytc may play different functions in different tissues.WSSV infection experiments showed that the expression level of PcCytc in the tested hepatopancreas, intestines, and muscle tissues increased after virus infection, and reached the highest value at 24 h (P < 0.01), after which it began to decrease until it returned to a normal level at 96 h; the overall performance was an induced expression pattern. This showed that PcCytc is involved in the process of WSSV infection. In addition, considering that PcCytc can participate in ATP production as a key element in the mitochondrial respiratory chain, the low expression of PcCytc leads to energy deficiency. We speculate that once the virus disrupts the energy metabolism of the host cell, the host may compensate for the loss by upregulating the expression of PcCytc.RNAi technology revealed the role of PcCytc in the process of WSSV infection. At 24 and 48 h after WSSV infection, the WSSV copies of the PcCytc RNAi group were significantly increased compared to the uninterrupted group (P < 0.01), and at 72 h were still significantly increased (P < 0.05). These results indicate that PcCytc plays an important role in inhibiting the replication of WSSV in P. clarkii and delays the infection process. To further confirm whether PcCytc mainly inhibits WSSV infection through the apoptotic pathway, we tested the expression changes of some important apoptosis-related genes (bcl-2, bax, and caspase-3). Among them, caspase-3 is an effector protein that regulates cell apoptosis, and its expression directly reflects the result of cell apoptosis. The ratio of bcl-2/bax is considered to be an indicator of the process of cell apoptosis; an increase in the ratio indicates that apoptosis has been affected. Inhibition (a decrease in the ratio) indicates that apoptosis was promoted. The test results were as follows: compared with the PBS group, the expression of bcl-2, bax, and caspase-3 genes of P. clarkii in the WSSV group was up-regulated to varying degrees, with a very significant difference in values (P < 0.01). This shows that WSSV can cause hemolymph apoptosis in P. clarkii, which is consistent with observations in mud crab and shrimp. In addition, the expression of caspase-3 in the dsCytc injection group was significantly downregulated (P < 0.01), indicating that apoptosis was inhibited after interfering with PcCytc. The value of bcl-2/bax in the dsCytc injection group was significantly increased (P < 0.01), which supported this conclusion.In summary, our results indicate that PcCytc can inhibit WSSV infection by regulating the apoptotic pathway. The results of this study provide new insights into the immune response of P. clarkii to WSSV infection.

Keywords

• procambarus clarkii
• cytochrome c gene
• apoptosis
• wssv infection
• rna interference