斯氏按蚊肽聚糖识别蛋白S2调节共生菌稳态的功能分析

doi:10.12140/j.issn.1000-7423.2023.04.001

摘要/Abstract

摘要：

目的研究斯氏按蚊肽聚糖识别蛋白S2（PGRP-S2）的功能及其对按蚊体内共生菌稳态的影响。方法将羽化后2~4日龄雌性斯氏按蚊分为对照组（喂食10%蔗糖溶液）、感染血组（叮咬原虫血症为4%~8%的伯氏疟原虫ANKA感染小鼠）和健康血组（叮咬健康小鼠），每组60只，饱血24 h后分离中肠和其余组织，用Trizol法提取RNA，实时荧光定量PCR（RT-qPCR）检测pgrp-s2基因的相对转录水平。将羽化后0~1日龄雌性斯氏按蚊分为对照组（喂食10%蔗糖溶液）与抗生素处理组（喂食含有青霉素、链霉素和庆大霉素的10%蔗糖溶液），每组30只，喂养5 d后分离中肠和其余组织，RT-qPCR检测pgrp-s2基因的相对转录水平。将雌性斯氏按蚊分为pgrp-s2敲低组和绿色荧光蛋白基因（gfp）对照组，每组60只，分别注射pgrp-s2的双链RNA（dsRNA）和gfp基因dsRNA（69 nl/只），2 d后每组取蚊虫提取RNA，RT-qPCR检测pgrp-s2的相对转录水平，提取DNA并使用16S rRNA特异性引物PCR检测蚊虫体内共生菌总量。分别取30只pgrp-s2敲低组和gfp对照组按蚊，用10⁷个/ml摩氏摩根氏菌液浸湿棉球喂养5 d后，提取RNA，RT-qPCR检测获得共生菌16S rRNA相对总量和摩氏摩根氏菌的相对数量；提取pgrp-s2敲低组和gfp对照组按蚊中肠组织RNA，转录组测序后进行聚类分析、京都基因与基因组百科全书（KEGG）功能注释和富集分析。使用SMART网站预测分析PGRP-S2氨基酸序列后利用CLC Main Workbench软件进行比对。克隆按蚊pgrp-s2基因，利用昆虫杆状病毒表达系统（pFastbacⅠ）在SF9细胞中表达PGRP-S2重组蛋白，Western blotting检测蛋白表达情况。取10、20、40 μg/ml纯化后的PGRP-S2重组蛋白溶液（以蛋白缓冲液为对照）分别与40 μg赖氨酸（Lys）型肽聚糖和二氨基庚氨酸（DAP）型肽聚糖孵育，每间隔12 h测定相对吸光度（A₅₄₀值）以判定肽聚糖的降解情况，验证PGRP-S2的酰胺酶活性。结果 RT-qPCR结果显示，吸血后24 h，感染血组、健康血组和对照组按蚊中肠pgrp-s2的相对转录水平分别为1 590.0 ± 665.2、126.8 ± 100.4和15.84 ± 6.92，感染血组高于对照组（t = 2.38，P < 0.05）；对照组按蚊中肠的pgrp-s2的相对转录水平高于其余组织（1.71 ± 0.51）（t = 2.04，P < 0.05）。抗生素处理组按蚊中肠pgrp-s2的相对转录水平为0.33 ± 0.18，低于对照组的117.9 ± 54.5（t = 2.16，P < 0.05）。pgrp-s2敲低组按蚊体内共生菌16S rRNA相对总量为3 653 ± 2 023，低于gfp对照组的14 982 ± 3 892（t = 2.58，P < 0.05）；摩氏摩根氏菌饲喂后，pgrp-s2敲低组按蚊体内摩氏摩根氏菌的相对数量为571 517 ± 61 258，低于gfp对照组的919 754 ± 123 397（t = 2.53，P < 0.05）。转录组分析结果显示，pgrp-s2敲低可以上调按蚊免疫相关的Toll/Imd通路、mTOR通路、FoxO通路基因，下调脂肪酸代谢、三羧酸循环等代谢相关基因。10、20、40 μg/ml PGRP-S2重组蛋白与DAP型肽聚糖孵育48 h后，相对A₅₄₀值分别为0.49 ± 0.07、0.40 ± 0.10和0.44 ± 0.07，均低于对照的0.90 ± 0.09（t = 3.53、3.65、3.97，均P < 0.05）；但与Lys型肽聚糖孵育48 h后，相对A₅₄₀值分别为0.52 ± 0.03、0.62 ± 0.03和0.65 ± 0.04，与对照（0.64 ± 0.05）的差异均无统计学意义（t = 1.95、0.31、0.11，均P > 0.05）。结论斯氏按蚊体内pgrp-s2主要在中肠表达，且表达水平受共生菌调控。PGRP-S2重组蛋白可以降解DAP型肽聚糖，具有酰胺酶活性，可通过负调节免疫反应和调节代谢反应调控按蚊体内共生菌水平。

关键词: 斯氏按蚊, 肽聚糖识别蛋S2, 共生菌, 真核表达, 酰胺酶活性

Abstract:

Objective To study the function of Anopheles stephensi peptidoglycan recognition protein S2 (PGRP-S2) and its role in regulating the homeostasis of symbiotic microbiota. Methods The female An. stephensi mosquitoes aged 2-4 days after eclosion were divided into control group (by biting the 10% sucrose solution), infected blood group (by biting the Plasmodium berghei ANKA-infected mice with 4%-8% parasitemia) and healthy blood group (by biting healthy mice), 60 mosquitoes in each group, the midgut and carcass were isolated after 24 h of full blood meal. The RNA was extracted by TRIzol, and relative transcription level of the pgrp-s2 gene was detected by real-time fluorescence quantitative PCR (RT-qPCR). Female An. stephensi mosquitoes 0-1 day after eclosion were divided into control group (fed with 10% sucrose solution) and antibiotic treatment group (fed with 10% sucrose solution containing penicillin, streptomycin and gentamicin), 30 mosquitoes in each group, midgut and carcass were isolated after fed for 5 days to detect the relative transcription level of pgrp-s2 gene by RT-qPCR. Female An. stephensi mosquitoes were divided into pgrp-s2 knockdown group and green fluorescent protein (gfp) control group, 60 mosquitoes in each group, each group were injected with pgrp-s2 double-stranded RNA (dsRNA) or green fluorescent protein gene dsRNA (69 nl/mosquito) respectively. After 2 days, mosquito RNA was extracted and RT-qPCR was used to detect the relative transcription level of pgrp-s2, mosquito DNA was extracted and PCR was used to detect the total amount of symbiotic bacteria in the mosquitoes using 16S rRNA specific primers. RNA was extracted from 30 An. stephensi mosquitoes of the knockdown group and control group respectively, which were fed with cotton balls soaked with 10⁷/ml Morganella morganii for 5 days, and subsequently, the relative total amount of 16S rRNA specific symbiotic bacteria and the relative number of M. morganii were detected by RT-qPCR; the RNA extracted from the midgut tissues of the knockdown group and gfp control group mosquitoes was use for transcriptome sequencing, cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analysis. The PGRP-S2 amino acid sequence was predicted and analyzed using SMART website, and aligned using CLC Main Workbench software. The pgrp-s2 gene was cloned from An. stephensi mosquito cDNA, and the PGRP-S2 recombinant protein was expressed in SF9 cells using the insect baculovirus expression system (pFastbacI), and the protein expression was verified by Western blotting. The purified PGRP-S2 recombinant protein solution of 10, 20 and 40 μg/ml (protein buffer as control) was separately incubated with 40 μg of Lys-type peptidoglycan or DAP-type peptidoglycan, and the relative absorbance value (A₅₄₀) was detected every 12 h to check the degradation of peptidoglycan and verify the amidase activity of PGRP-S2. Results The RT-qPCR results showed that 24 h after the blood meal, the An. stephensi mosquitoes midgut pgrp-s2 relative transcription levels in the infected blood group, healthy blood group and control group were 1 590.0 ± 665.2, 126.8 ± 100.4 and 15.84 ± 6.92, respectively. The pgrp-s2 relative transcription levels of the infected blood group was higher than that of the control group (t = 2.38, P < 0.05); the pgrp-s2 of An. stephensi midgut relative transcription level of the control group was higher than that of the carcass (1.71 ± 0.51) (t = 2.04, P < 0.05). The pgrp-s2 of An. stephensi midgut relative transcription level of antibiotic treatment group was 0.33 ± 0.18, which was lower than that of the control group (117.9 ± 54.5) (t = 2.16, P < 0.05). The relative total amount of 16S rRNA of symbiotic bacteria in the pgrp-s2 knockdown group was 3 653 ± 2 023, which was lower than 14 982 ± 3 892 in the gfp control group (t = 2.58, P < 0.05); after feeding with M. morganii, the relative number of M. morganii in the An. stephensi of pgrp-s2 knockdown group was 571 517 ± 61 258, which was lower than 919 754 ± 123 397 of GFP control group (t = 2.53, P < 0.05). Transcriptome analysis results showed that pgrp-s2 knockdown could up-regulate An. stephensi Toll/Imd pathway, mTOR pathway, FoxO pathway and immune-related genes, while down-regulate metabolism-related genes such as fatty acid metabolism and tricarboxylic acid cycle. After incubation of PGRP-S2 recombinant protein at concentrations of 10, 20 and 40 μg/ml with DAP-type peptidoglycan for 48 h, the relative A₅₄₀ values were 0.49 ± 0.07, 0.40 ± 0.10 and 0.44 ± 0.07, respectively, which were lower than 0.90 ± 0.09 of control (t = 3.53, 3.65, 3.97; all P < 0.05); after incubation with Lys-type peptidoglycan for 48 h, the relative A₅₄₀ was 0.52 ± 0.03, 0.62 ± 0.03 and 0.65 ± 0.04, respectively, and there was no difference from 0.64 ± 0.05 of control (t = 1.95, 0.31, 0.11; all P > 0.05). Conclusion pgrp-s2 is mainly expressed in the midgut of An. stephensi, and the expression level is regulated by symbiotic bacteria. PGRP-S2 recombinant protein can degrade DAP-type peptidoglycan, having amidase activity, and may regulate the level of symbiotic bacteria in An. stephensi by negatively regulating immune responses and regulating metabolic responses.

Key words: Anopheles stephensi, Peptidoglycan recognition protein S2, Symbiotics, Eukaryotic expression, Amidase activity

中图分类号:

R384.111

王之谦, 王敬文, 宋秀梅. 斯氏按蚊肽聚糖识别蛋白S2调节共生菌稳态的功能分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 397-403.

WANG Zhiqian, WANG Jingwen, SONG Xiumei. Function analysis of Anopheles stephensi peptidoglycan recognition protein S2 in regulating homeostasis of symbiotic microbiota[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(4): 397-403.

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