A Glutathione Peroxidase Gene from Litopenaeus vannamei Is Involved in Oxidative Stress Responses and Pathogen Infection Resistance

Abstract

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes' involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.

Keywords: Antimicrobial peptide genes; Litopenaeus vannamei; glutathione peroxidase; oxidative stress.

Figure 1

LvGPX3 domain distribution and phylogenetic analysis of the GPXs. (A) Schematic representation of the structural motifs of LvGPX3; (B) Phylogenetic tree of PGXs. HsGPX, Homo sapiens glutathione peroxidase (GenBank accession No. BAA00525.1); PtGPX3, Pan troglodytes glutathione peroxidase 3 precursor (GenBank accession No. NP_001108629.1); OoGPX3, Orcinus orca glutathione peroxidase 3 (GenBank accession No. XP_004280386.2); OaGPX3, Ovis aries glutathione peroxidase 3 (GenBank accession No. XP_014951639.1); CmGPX3, Chelonia mydas glutathione peroxidase 3 isoform X2 (GenBank accession No. XP_027689463.1); GgGPX3, Gallus gallus glutathione peroxidase 3 precursor (GenBank accession No. NP_001156704.1); XlGPX3, Xenopus laevis glutathione peroxidase 3 homeolog precursor (GenBank accession No. NP_001085319.2); PmGPX3, Petromyzon marinus glutathione peroxidase 3-like (GenBank accession No. XP_032814057.1); TcGPX, Tribolium castaneum glutathione peroxidase (GenBank accession No. NP_001164309.1); ApGPX, Acanthaster planci glutathione peroxidase-like (GenBank accession No. XP_022106637.1); NlGPX3, Nilaparvata lugens glutathione peroxidase 3-like (GenBank accession No. XP_022184461.1); ZnGPX6, Zootermopsis nevadensis glutathione peroxidase 6-like isoform X2 (GenBank accession No. XP_021936526.1); LpGPX, Limulus polyphemus glutathione peroxidase-like isoform X1 (GenBank accession No. XP_013772703.1); MeGPX, Metapenaeus ensis glutathione peroxidase (GenBank accession No. ACB42236.1); PcGPX, Pomacea canaliculata glutathione peroxidase-like (GenBank accession No. XP_025090868.1); PtrGPX6, Portunus trituberculatus Glutathione peroxidase 6 (GenBank accession No. MPC16807.1); PmGPX, Penaeus monodon selenium-dependent glutathione peroxidase (GenBank accession No. AQW41378.1); LvGPXL, Penaeus vannamei glutathione peroxidase-like (GenBank accession No. XP_027224546.1).

Figure 2

Expression profile of LvGPX3 and secretory expression of LvGPX3. (A) Total RNA extracted from different tissues were reversely transcribed into cDNAs to serve as templates. Relative expression levels of LvGPX3 were normalized to LvEF1α. The results are based on three independent experiments and expressed as mean values ±S.D. (B) The location of the proteins was visualized with a Leica laser scanning confocal microscope. (C) S2 cells were transfected with pAC-LvGPX3 expression plasmids. At 48 h post transfection, the cells were harvested and the V5-LvGPX3 was detected by Western blot assay using anti-V5 antibody. CM, (Culture media); CL, (Cell lysates).

Figure 3

LvGPX3 is regulated by oxidative stress response pathway. (A) Schematic diagram of LvGPX3 promoter regions in the luciferase reporter gene assay is constructed. +1 denotes the transcription initiation site for LvGPX3 gene. Luc denotes the firefly luciferase reporter gene. The putative AREs are indicated by oval boxes. The mutated AREs are indicated with triangle boxes. (B) Activation of the LvGPX3 promoter by LvNrf2. The relative luciferase activity of pGL3B-LvGPX3, pGL3B-LvGPX3mARE1, pGL3B-LvGPX3mARE2 and pGL3B-LvGPX3mARE were detected. The bars indicate mean values ±S.D. of luciferase activity (n = 3). (C) The mRNAs were collected at 0, 3, 6, 12, 18, 24, 36, 48 and 72 h after β-glucan injection. The relative expression of LvGPX3 upon β-glucan injection was normalized with LvEF1α and compared to time zero. The bars represent the mean values ±S.D. of three replicates. The statistical significance was calculated using Student’s t-test (** indicates p< 0.01).

Figure 4

Knockdown of expression of LvGPX3 increased cumulative mortality of shrimps under oxidative stress. Shrimp (n = 50) were injected intramuscularly with dseGFP (control) or dsLvGPX3. At 48 h after the initial injection, shrimps were injected with β-glucan or PBS. Cumulative mortality was recorded every 4 h. The mortality levels among different treatments were analyzed by Kaplan–Meier plot (log-rank X² test). Significant differences in L. vannamei mortality were marked with asterisks (** indicates p < 0.01).

Figure 5

Expression of LvGPX3 in hemocytes of immune-challenged shrimps. The mRNAs were collected at 0, 3, 6, 9, 12, 24, 30, 36, 48, 72 and 96 h after WSSV infection (A), or at 0, 4, 8, 12, 24, 36, 48, 72 and 96 h after V. alginolyticus infection (B). The relative expression of LvGPX3 upon WSSV or V. alginolyticus infection was normalized with LvEF1α and compared to time zero. The bars represent the mean values ±S.D. of three replicates. The statistical significance was calculated using Student’s t-test (* indicates p < 0.05; ** indicates p < 0.01).

Figure 6

Cumulative mortality following treatment with dsLvGPX3 plus immune challenged. (A) RT-PCR analysis was carried out for determining knockdown effect. The internal control was LvEF1α. Samples were taken 48 h after dsRNA injection. For cumulative mortality test, shrimps (n = 50) were injected intramuscularly with dseGFP (control) or dsLvGPX3. At 48 h after the initial injection, shrimps were infected with (B) WSSV or (C) V. alginolyticus. Cumulative mortality was recorded every 4 h. Differences in mortality levels between treatments were analyzed by Kaplan–Meier plot (log-rank X² test). Significant differences in L. vannamei mortality were marked with asterisks (** indicates p < 0.01).

Figure 7

LvGPX3 was important for enhancing AMP expression under immune challenge. (A) The promoter activities of Pen4, Mtk and Drs were measured via dual luciferase reporter gene assays. LPS was added to S2 cells at a concentration of 5 μg/mL 6 h before cells were harvested. The bars indicate mean values ± S.D. of the luciferase activity (n = 3). (B) Upon LPS treatment, inhibiting LvGPX3 decreased the expression of Pen2a (a), Pen3a (b), and ALF1 (c) but not Crustin. For LPS injection, each shrimp was injected with LPS at 36 h after dsRNA injection with a concentration of 240 μg LPS dissolved in 50 μL PBS [21]. The bars represent the mean values ± S.D. of three replicates. The statistical significance was calculated using Student’s t-test (* indicates p < 0.05, ** indicates p < 0.01).