Research on the Molecular Mechanism of Crayfish prx 6 in the Process of Defending against Staphylococcus aureus Infection

doi:10.13560/j.cnki.biotech.bull.1985.2023-1205

Abstract

Abstract:

【Objective】 To unravel the molecular intricacies associated with the immune modulatory role of the crayfish prx 6 gene in response to Staphylococcus aureus infection and to expound its function in the orchestration of innate immunity in crayfish, this investigation was carried out. 【Method】 The study pivoted on the prx 6 gene and harvested total RNA from diverse unstimulated crayfish tissues. The RT-qPCR was utilized to pinpoint the tissue-specific distribution patterns of this gene. Following immune provocation by S. aureus, the prx 6 gene expression dynamics within crayfish’s immune-centric tissues were monitored. The research further endeavored to gauge the relative expression of immune executor genes, specifically, Pc-crustin 3, Pc-crustin 4, Pc-ALF 9, and Pc-lectin 1 in the hepatopancreas after RNAi-mediated silencing of prx 6, in the wake of S. aureus infection. Additionally, a quantitative appraisal of the bacterial burden in crayfish hemolymph was undertaken. 【Result】 The investigations underscored an amplified expression of the prx 6 gene within the hepatopancreas as compared to other tissues. Post S. aureus infection, the prx 6 gene expression showcased substantial augmentation in the hepatopancreas, hemocytes, and intestinal tissues, with a preliminary surge of expression detected in gill tissues. In terms of survival rates, a pronounced decrement in crayfish vitality was observed after prx 6 knockdown, relative to dsGFP + S. aureus controls. To elucidate the key factors linked to escalated mortality, the expression strata of antimicrobial peptide genes integral to bacterial defense were quantified. The outcomes revealed a significant downturn in the expression of Pc-crustin 3, Pc-crustin 4, Pc-ALF 9, and Pc-lectin 1 within the hepatopancreas, seemingly tied to prx 6 repression. Intriguingly, assessment of hemolymph bacterial loads unveiled a substantial elevation in bacterial load in the prx 6 knockdown group as set against controls. 【Conclusion】 The prx 6 gene in crayfish plays a pivotal role in the antibacterial innate immune response through modulating the expressions of antimicrobial peptide genes and enhancing the clearance capabilities of bacteria from the hemolymph.

Key words: innate immunity, PRX 6, RNA interference, antimicrobial peptides, Staphylococcus aureus

JIN Bo-yang, QIN Shi-yu, ZHANG Ming-da, LI Qian-qian, WEN Jing, SHEN Xiu-li, DU Zhi-qiang. Research on the Molecular Mechanism of Crayfish prx 6 in the Process of Defending against Staphylococcus aureus Infection[J]. Biotechnology Bulletin, 2024, 40(7): 314-322.

Figures/Tables 8

Table 1 RT-qPCR primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
prx 6-RT-F	CGGATCACTGGAGGGTCA AACA
prx 6-RT-R	GCA ATTTTCATCCTCGGCATCA
prx 6-iF	GCGTAATACGACTCACTATAGGATGGTTAACTTAGGCGAT
prx 6-iR	GCGTAATACGACTCACTATAGGGTCTATGGCTCTAAGAAT
GFP-iF	GCGTAATACGACTCACTATAGGCGAGCTGGACGGCGACGTAAAC
GFP-iR	GCGTAATACGACTCACTATAGGCTTGAAGTTCACCTTGATGCC
18S RNA-RT-F	TCTTCTTAGAGGGATTAGCGG
18S RNA-RT-R	AAGGGGATTGAACGGGTTA
Pc-ALF 9-RT-F	AGTGGCGTCATACAGGAAGGGG
Pc-ALF 9-RT-R	CCAAAGGATGGCGAGAAATAGT
Pc-crustin 3-RT-F	TACGTCTTGCCCTCGTCTTA
Pc-crustin 3-RT-R	CAGCGTCCTCCTCTTTGTAATC
Pc-crustin 4-RT-F	CTCTGACTGCCAGGTCTTT
Pc-crustin 4-RT-R	TGCGAGCTGTGATGGTTAG
Pc-lectin 1-RT-F	GGGGAGGGCTGCTACTACTTG
Pc-lectin 1-RT-R	CGTGCCACCCACCCATAAG

Fig. 1 Expression patterns of prx 6 gene in normal tissues

Fig. 2 Expression patterns of prx 6 after S. aureus stimulation Relative expression of prx 6 in tissues after S. aureus infection. Different lowercase letters showed significant differences（P<0.05）. The same letters indicate insignificant differences（P>0.05）. The same below

Fig. 3 Survival rates of P. clarkii statistics after S. aureus injection in RNAi assay **** indicates highly significant different with control group（P<0.000 1）

Fig. 4 Results for relative expression analysis of Amps genes in crayfish hepatopancreas after S. aureus challenge in RNAi assay 18S RNA is used as inner control

Fig. 5 Bacterial clearance experiments results in crayfish hemolymph in RNAi assay The hemolymph of crayfish from 1×PBS, dsPrx 6 + S. aureus and dsGFP + S. aureus groups was coated on the agar plate. Bacterial abundance at 12 and 24 h was detected

Fig. 6 Statistics results of bacterial colonies Individual colonies in the LB plates were counted and cakulated using the formula

Fig. 7 Theoretical speculation for prx 6 affecting the expression of Amps genes to defend against S. aureus infection After S. aureus infection, crayfish can further modulate the transcription and expression of genes related to antimicrobial peptides by activating prx 6, thereby facilitating the secretion of antimicrobial peptides against foreign pathogens

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