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