家蚕先天免疫的模式识别、信号传导和抗微生物肽基因表达调控机制研究
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摘要
家蚕是由野蚕驯化而来的泌丝昆虫,具有很高经济价值,然而我国每年却因蚕病造成近百亿左右的损失,严重影响到产业的可持续发展。研究表明,当微生物感染昆虫时,能诱导其产生不同的抗微生物肽以杀死入侵微生物。而这些抗微生物肽基因的表达主要通过Toll和Imd (immune deficiency)信号途径调控。我们通过对家蚕免疫相关基因中的短型肽聚糖识别蛋白3(BmPGRP-S3)和β-葡聚糖识别蛋白3、4(BmβGRP3、4)及Toll、Imd途径关键基因BmSpzl和Bmlmd的研究,初步探索家蚕体液免疫的模式识别、信号传导和抗微生物肽基因表达调控机制。获得主要结果如下：
1模式识别受体编码基因的克隆及表达谱分析
模式识别受体在体液免疫-Toll和Imd途径中行使模式识别功能,并调控激活下游信号因子和抗微生物肽基因的表达。我们对家蚕模式识别受体-肽聚糖识别蛋白(PGRPs)和β-葡聚糖识别蛋白(PGRPs)家族基因进行研究,克隆得到10个PGRPs和4个βGRPs,得到BmβGRP3、BmPGRP-S3、-S4和BmPGRP-L5的完整编码序列。βGRPs家族成员都具有信号肽,其中BmβGRP1-3成簇分布于11号染色体,且不具有典型的葡聚糖结合结构域,BmβGRP4独立分布于22号染色体,具有典型的葡聚糖结合结构域。PGRPs的长型和短型亚家族都具有典型的酰胺酶活性(amidase activity)结构域,短型亚家族具有信号肽,长型亚家族则不具有。5个长型亚家族基因成簇分布于1号染色体,5个短型亚家族基因中有2个分布于9号染色体和3个分布于16号染色体。人工饲料无菌饲育的大造品系5龄3天幼虫添食大肠杆菌(E.coli, Ec)、家蚕黑胸败血菌(B. bombysepticus, Bs)和家蚕白僵菌(B. bassiana, Bb),免疫诱导3、6、12和24h四个时间点的个体水平荧光定量PCR检测,发现PGRPs长型亚家族基因BmPGRP-L1、-L3和短型亚家族基因BmPGRP-S1-3均能被革兰氏阴性菌、革兰氏阳性菌和真菌诱导上调表达；βGRPs基因家族中的BmβGRP3、4也能被上述三种微生物诱导上调表达。组织表达谱分析表明,所检测的13个识别受体基因,在头部组织中,BmβGRP2,4、BmPGRP-L2和BmPGRP-S1,-S3-5均能被阴性菌、阳性菌和真菌诱导上调表达；在表皮、中肠和脂肪体中,只有少数识别受体基因被三类微生物诱导上调表达。
2 BmβGRP3功能研究
果蝇(Drosophila)基因组中有编码3个阴性菌结合蛋白的基因,其中GNBP1由Lys-PGN激活,GNBP3由真菌激活,GNBP1和GNBP3均在Toll信号途径上游起模式识别功能并调控该信号通路。家蚕BmβGRP1、2被证实具有结合β-1,3-葡聚糖的能力且激活家蚕细胞免疫应答,但并未发现它们具有在家蚕Toll或Imd信号途径上游行使模式识别的功能。
BmβGRP3的开放式阅读框(open reading frame, ORF)为1470bp,编码489个氨基酸,N-端有一个由17个氨基酸组成的信号肽,推测可能为分泌型蛋白,48和454位处的天冬氨酸是潜在的糖基化位点,蛋白分子量为53kDa,等电点6.25。BmβGRP3的氨基酸序列高度保留了结合β-1,3-葡聚糖的功能结构域,该结构域能结合三重螺旋结构的β-1,3-葡聚糖。家蚕BmβGRP3在个体水平能被革兰氏阴性菌、阳性菌和真菌诱导上调表达,其中在个体水平阳性菌和真菌诱导上调表达能力强于阴性菌。组织水平上,在表皮中能被真菌高量诱导上调表达,阳性菌对该基因基本无诱导表达能力；在脂肪体中能被阴性菌高量诱导上调表达,阳性菌和真菌诱导上调表达该基因的能力较弱；在中肠组织中,该基因能被阴性菌和阳性菌较明显地诱导上调表达,真菌对该基因基本无诱导表达能力,表明家蚕BmβGRP3在应对不同微生物诱导时有着不同的表达模式和组织特异性。RNA干涉抑制BmPGRP3表达相关研究表明其在家蚕Toll信号途径上游行使模式识别功能和激活调控该途径的作用；进一步研究发现BmβGRP3的抑制表达会降低Toll信号途径的信号因子BmSpzl等基因的表达和抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnbl的表达,表明Toll信号途径可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnbl的表达。另一方面,家蚕BmβGRP3的抑制表达不会影响到Imd信号途径信号因子Bmlmd和抗微生物肽基因BmCecropin-B、BmCecropin-D和gloverin2的表达,暗示其它免疫调控途径如Imd信号途径可能调控抗微生物肽基因BmCecropin-B、BmCecropin-D和gloverin2表达。
对家蚕Toll和Imd信号途径免疫因子BmSpz1、BmImd等基因的诱导表达分析结果表明三类微生物(革兰氏阴性菌、阳性菌和真菌)都能激活家蚕的Toll和Imd信号途径,这与模式生物果蝇的Toll和Imd信号途径有很大的不同。果蝇的Toll信号途径只能被拥有DAP-型肽聚糖的细菌和真菌免疫激活,其Imd信号途径能被革兰氏阴性菌和部分阳性菌免疫激活。推测家蚕在其长期的进化过程中获得了更有效的免疫应答机制。
3 BmβGRP4和BmPGRP-S3的原核表达和功能研究
本研究已初步证实BmβGRP3位于Toll信号途径上游并可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达。然而关于Imd信号途径的免疫激活和抗微生物肽的表达调控仍有待进一步研究。
我们期望通过对BmβGRP4和BmPGRP-S3进行研究,以阐明模式识别受体在家蚕体液免疫应答中的具体生物学功能。构建了BmβGRP4和BmPGRP-S3的原核表达载体,纯化得到BmβGRP4和BmPGRP-S3蛋白,并制备了多克隆抗体。革兰氏阴性菌、阳性茵和真菌免疫诱导18h,BmβGRP4和BmPGRP-S3的mRNA表达量和蛋白量都显著提高。RNA干涉抑制BmβGRP4表达造成BmPGRP-S3的mRNA表达量和蛋白表达量明显降低；RNA干涉抑制BmPGRP-S3表达对Bm(3GRP4的mRNA表达量和蛋白表达量无影响,推测BmβGRP4位于BmPGRP-S3上游行使功能。RNA干涉抑制BmβGRP4或BmPGRP-S3表达都造成Imd信号途径信号因子Bmlmd等基因和抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2表达量的降低,表明BmβGRP4和BmPGRP-S3位于Imd信号途径上游行使识别作用并激活该信号通路；Imd信号途径可能调控抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达。与此同时RNA干涉抑制BmβGRP4或BmPGRP-S3表达不影响Toll信号途径信号因子BmSpzl基因和抗微生物肽基因BmCecropin-A1、BmCecropin-E和BmAtt2的表达,该处理方式提高了Toll信号途径调控抗微生物肽基因BmEnb1的表达,暗示家蚕Toll和Imd信号途径可能各自独立地调控抗微生物肽基因的表达。
另外,BmβGRP4或BmPGRP-S3的RNA干涉抑制表达都会造成BmClip15、BmFrep3、BmGaletin3和BmSpnl2的表达量降低,BmFrep3属于纤维蛋白原基因(Fibrinogen-related proteins, FREPs),该基因家族是一类与无脊椎动物先天免疫相关的C-端含有纤维蛋白原结构域的基因,具有识别细菌和原虫的能力,家蚕有3个该家族基因；BmGaletin3属于半乳糖凝集素基因家族,是一种专门结合p-半乳糖苷糖并含有一个进化保守的碳水化合物结合结构域(CRDs),果蝇和按蚊的半乳糖凝集素被认为具有微生物识别或吞噬的功能；BmClip15和BmSpn12分别为丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂,它们参与先天免疫信号的级联调控,放大或减弱先天免疫系统识别的信号。由此推测BmβGRP4和BmPGRP-S3除调控Imd信号途径外还可能参与家蚕细胞免疫应答。
4 BmSpz1和BmImd的功能研究
通过对家蚕模式识别受体—BmβGRP3、BmβGRP4和BmPGRP-S3的研究,初步证明了它们在家蚕先天免疫应答反应中的功能。家蚕BmβGRP3调控激活Toll信号途径；BmβGRP4和BmPGRP-S3共同调控激活Imd信号途径；并且发现Toll和Imd信号途径可能分别调控家蚕抗微生物肽基因BmCecropin-A1,BmCecropin-E、BmAtt2、BmEnbl和BmCecropin-B、BmCecropin-D、BmMoricin、Bmgloverinl、2的表达。为了进一步验证家蚕Toll和Imd信号途径所调控的抗微生物肽基因,我们又分别对家蚕Toll和Imd信号途径的关键基因BmSpzl和Bmlmd进行研究,希望藉此揭示家蚕Toll和Imd信号途径免疫信号的传导机制和抗微生物肽基因的表达调控模式。
BmSpz1和Bmlmd均能被三类微生物(革兰氏阴性菌、阳性菌和真菌)诱导上调表达,Western blotting分析显示BmSpz1的前体和成熟体蛋白均被三类微生物诱导上调表达。RNA干涉抑制BmSpz1表达造成Toll信号途径信号因子BmTube、BmRel和抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2、BmEnb1表达量明显降低；同时RNA干涉抑制Bmlmd表达造成Imd信号途径信号因子BmDreed、BmRelish和抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin、Bmgloverinl,2表达量显著降低。
通过对BmSpzl和Bmlmd研究再次证实了家蚕Toll和Imd信号途径均能被三类微生物(革兰氏阴性菌、阳性菌和真菌)诱导激活；Toll信号途径可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达；Imd信号途径可能调控家蚕抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达。
5家蚕抗微生物肽基因表达调控机制
昆虫抗微生物肽是昆虫在抵抗微生物侵染过程中合成的一类小分子多肽,在昆虫先天免疫中起重要作用。家蚕基因组中含有比其它昆虫基因组更多的抗微生物肽基因,它们大多数以多基因家族的形式存在,具有协同表达特点。Yamakawa等分析了家蚕的7个抗微生物肽基因,发现它们含有一个或多个KB-like和GATA元件。另外家蚕的抗微生物肽基因还含有多个CAATW元件,可能对基因转录的起始频率起调节作用,同时家蚕抗微生物肽还具有很高的可诱导性和较好的抗微生物活性。到目前为止,我们仍不清楚家蚕抗微生物肽基因的表达调控机制,通过本文的研究,我们提出了一个可能的家蚕抗微生物肽基因表达调控模式。
综上所述,首先,Toll信号途径由其上游的模式识别受体BmβGRP3完成对入侵微生物的识别并顺序激活该途径的信号因子,如胞外的BmSpzl、跨膜的Toll受体和胞内的BmMyd88、BmTube等,致使BmRel由核外进入核内,结合家蚕抗微生物肽基因的表达调控区域(κB-like和GATA元件等),从而完成对家蚕抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达调控。其次,Imd信号途径由其上游的模式识别受体BmβGRP4和BmPGRP-S3完成对入侵微生物的识别并顺序激活该途径的信号因子,致使BmRelish由核外进入核内,结合家蚕抗微生物肽基因的表达调控区域(KB-like和GATA元件等),从而完成对家蚕抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达调控。同时家蚕Toll和Imd信号途径可能独立地完成相应抗微生物肽基因的表达调控。在本文中,我们给出了一个初步的家蚕抗微生物肽基因的表达调控机制,对该机制理的解还需要在本文的基础上进行更为深入、更为具体的研究来证明。
Bombyx mori, domesticated from wild silkworm, is a fusule insect with great economic value. However, the annual losses of China caused by silkworm disease are about millions, and seriously affect the sustainable development of the industry. Researches show that when insects are infected with microbes, various of antimicrobial peptides are induced and produced to kill the invading microorganisms. Thus the expression of these antimicrobial peptide genes is mainly regulated by Toll and Imd signal pathways. We focused on the immune-related genes such as short type peptidoglycan recognition protein 3(BmPGRP-S3) andβ-glucan recognition protein 3,4 (BmβGRP3,4), as well as the key genes BmSpzl and Bmlmd located in the Toll and Imd pathways, in order to explore the signal transduction of humoral immune and the expressional regulation mechanism of antimicrobial peptide genes in B. mori. The below are results obtained.
1. The cloning and expression analysis of genes coded by pattern recognition receptors
The pattern recognition receptors, which participate in pattern recognition in the upstream of humoral immune-Toll and Imd pathway, meanwhile regulate the activation of signal factors downstream and the expression of antimicrobial peptide genes. We do researches on the pattern recognition receptors of peptidoglycan recognition proteins andβ-glucan recognition proteins.10 PGRPs and 4βGRPs are cloned, and we obtain the complete coding sequences. All theβGRPs have signal peptide. BmPGRPl-3 are located in the chromosome 11 closely, the glucan-binding region is not detected, whereas BmPGRP4 is located in the chromosome 22 independently, including typical glucan-binding domain. For gene family of PGRPs, typical amidase activity domain is predicted regardless of long-and short- subfamily. Short type subfamily has signal peptide, but long type subfamily hasn't. Five genes of long type subfamily are located in chromosome 1 closely. Of short type subfamily,2 and 3 located in chromosome 9 and 16, respectively. RT-qPCR analysis of the silkworm larvae samples which were collected 3,6,12 and 24 hours after challenged with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana, respectively, showed that two long-type BmPGRP-L1,-L3 and three short-type BmPGRP-S1-3 could be up-regulated by gram-positive bacteria, gram-negative bacteria and fungi infection, respectively. Meanwhile, the induced expression profile among different tissues indicated that all the 13 receptor genes shared different expression patterns in head, integument, midgut and fat body challenged by gram-positive bacteria, gram-negative bacteria and fungi, respectively, many of them were induced in head, less were induced in integument and midgut, least in fat body.
2. Study on the function of BmβGRP3
There are three Gram-negative bacteria binding protein genes contained in the genome of Drosophila, two of them play a role in pattern recognition in the upstream of Toll signal pathway. It was confirmed that BmβGRP1 and BmβGRP2 can bindβ-1,3-glucan and active cellar immune response in B. mori. However, whether they participate in pattern recognition in upstream of Toll or IMD pathway is not detected.
BmβGRP3 has an open reading frame of 1470bp, encoding a protein with 489 residues. The 3'UTR contained a consensus polyadenylation signal (aataaa) upstream from the poly(A) tail. The deduced protein contained a putative signal peptide predicted by SignalP software in the N-terminal region. The calculated molecular mass of the mature peptide was about 53KD and theoretical pI was 6.25. To find pattern recognition receptors in the upstream of humoral immune pathway, we make focus on the study of BmβGRP3. We found the function region of bindingβ-1,3-glucan was conserved in amino acid sequence. BmβGRP3 can be upregulated (the level of individual and issue) by infecting with gram-positive, gram-negative bacteria and fungi, respectively. It shows that BmβGRP3 has different expression patterns and certain issue specificity responding to the induction of different miroorganisms. The RNAi knock-down BmβGRP3 indicates that it plays an important role in the innate immune response in B. mori. Further study suggests that Toll signal pathway can regulate the expression of some antimicrobial peptide genes. While RNAi knock-down BmβGRP3, the expressions of Toll pathway signal factors, such as BmSpzl and antimicrobial peptide genes CecAl、CecE、Att2 and Enbl decrease, which shows Toll signal pathway regulates the expression of antimicrobial peptide genes CecA1,CecE, Att2 and Enbl. On the other side, the expression of Bmlmd, IMD signal pathway signal factor, and antimicrobial peptide genes CecB, CecD and Glv2 was not affected, which suggests that there is other immune pathway, for example IMD signal pathway, may regulate the expression of antimicrobial peptide genes CecB, CecD and Glv2.
According to the induction expressions of BmSpzl and BmImd, Toll and Imd signal pathways can be activeted by gram-positive bacteria, gram-negative bacteria and fungi, respectively, which is significant different to Drosophila. The Toll signal pathway is only activated by gram-positive bacteria and fungi, the Imd signal pathway can be activated by gram-negative bacteria and certain gram-positive bacteria. It is reasonable that Bombyx mori obtain more efficient immune response mechanism when face to unique growth and development environment in long-standing evolution.
3. Prokaryotic expression and function analysis of BmβGRP4 and BmPGRP-S3
In this research, we found that BmβGRP3 is located the up-stream of Toll signal pathway and regulate the expression of some antimicrobial peptides genes, such as CecAl, CecE, Att2 and Enbl. However, it is yet to be studied further about the Imd pathway in immune activation and regulation of expression of antimicrobial peptides.
We look forward to studying on the BmβGRP4 and BmPGRP-S3, to clarify their specific biological functions in humoral immune response. We constructed BmβGRP4 and BmPGRP-S3 prokaryotic expression vectors, purified the two proteins, and obtained the polyclonal antibodies. The expression of BmβGRP4 and BmPGRP-S3 can be obviously increased by gram-negative bacteria, gram-positive and fungi induced, respectively, in both transcriptional and translational levels. RNAi knock-down BmβGRP4 causes that the expression of BmPGRP-S3 is repressed in both transcriptional and translational levels. However, RNAi knock-down BmPGRP-S3 does not affect the expression of BmβGRP4 in both transcriptional and translational levels. So we speculate that BmPGRP4 is located in the upstream of BmPGRP-S3 to exercise their functions. RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of signal transduction factor BmImd and antimicrobial peptides genes, including CecB, CecD, Moricin and Glv1,2, which indicates that the expressions of these antimicrobial peptide genes are mediated by Imd pathway. On the other hand, repressions of Toll pathway signal factor BmSpzl and antimicrobial peptides CecAl, CecE, Att2 and Enbl are not observed. Interestingly, the expression level of Enbl is even raised, which proves that Toll and Imd signal pathways independently mediate the expression of antimicrobial peptide genes.
Moreover, RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of BmClip15, BmFrep3, BmGaletin3 and BmSpn12. BmFrep3 is a fibrinogen-related protein family member which related with invertebrate innate immunity, it contains a fibrinogen domain in the C-terminal with the ability of bacteria and protozoan recognition. Three family members have been found in B.mori; BmGaletin3 belongs to Galactose lectin gene family, which contains a conserved carbohydrate recognition domain with the ability ofβ-galactosidase sugar binding. Galactose lectins are deemed to microbe recognition and phagocytosis. BmClip15 and BmSpnl2 are serine protease and serine protease inhibitor, respectively, which involved in cascade control of innate immune signal transduction by amplifying or reducing the signal. So we can speculate that BmPGRP4 and BmPGRP-S3 also may participate in the cellar immune response except for mediating Imd pathway.
4. Functional research of BmSpzl and Bmlmd
We identified the function of pattern recognition receptors-BmβGRP3、BmβGRP4 and BmPGRP-S3 in innate immune response in B.mori. BmβGRP3 mediates Toll pathway, Bm(3GRP4 and BmPGRP-S3 mediate Imd pathway. The expression of antimicrobial peptide genes CecAl, CecE, Att2, Enbl and CecB, CecD, Moricin, Glvl,2 are regulated via Toll and Imd pathway, respectively. In order to identify the antimicrobial peptide genes regulated via Toll and Imd pathways, and further confirming the transduction mechanism and antimicrobial peptide genes expression pattern, we performed RNAi knock-down BmSpzl and Bmlmd, which are the key genes in Toll and Imd pathways.
Both BmSpzl and BmImd can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. Western blotting analysis shows that the precursor and mature proteins of BmSpzl also can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. RNAi knock-down BmSpzl represses the expressions of signal factors, BmTube and BmRel in Toll pathway, and the antimicrobial peptide genes of BmCecropin-A1, BmCecropin-E, BmAtt2 and BmEnbl. Simultaneously, RNAi knock-down BmImd represses the expressions of signal factors, BmDreed, BmRelish in Imd pathway, and the antimicrobial peptide genes of BmCecropin-B, BmCecropin-D, BmMoricin and Bmgloverinl,2.
The fact that Toll pathway regulates antimicrobial peptide genes CecAl, CecE, Att2 and Enb1, the Imd pathway regulates antimicrobial peptide genes CecB, CecD, Moricin and Glvl,2 has been proved via studying on BmSpzl and Bmlmd. So we speculate that Toll and Imd pathway work separately in regulation of interrelated antimicrobial peptide genes.
5. The mechanism of antimicrobial peptide gene expression in B. mori
Antimicrobial peptides are kinds of minor polypeptides which are expressed during the insect self-defence, and which have played an important role in insect immunity. In the whole genomes, there are a lot of antimicrobial peptides have been expressed in the silkworm compared with other insects, and a great majority of them are exist in multi-gene family forms, most of them are coordinated expression. Yamakawa et al had analyzed 7 species of antimicrobial peptides in silkworm, which have one or more KB-like and GATA components. Moreover, some have CAATW components, which may be helpful in the starting frequency of gene transcription. In addition, the antimicrobial peptides in silkworm have much higher activity of bacteria induction and self-defence. But so far, the mechanism of antimicrobial peptide genes expression is still unclear. In this study, we propose a possible mechanism about antimicrobial peptide genes expression and regulation.
Firstly, Toll signal pathway is stimulated by pattern recognition receptor BmβGRP3 which recognizes the different pathogens and activates the other signal factors in this pathway, such as extracellular factor BmSpzl, transmembrane factor Toll receptor and intracellular factors BmMyd88、BmTube, to result in BmRel ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Toll pathway regulates the expression of those antimicrobial peptide genes:CecAl, CecE, Att2 and Enb1.
Secondly, Imd signal pathway is stimulated by pattern recognition receptors BmβGRP4 and BmPGRP-S3 which recognize the different pathogens and activate the related signal factors in Imd pathway, to result in BmRelish ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Imd pathway regulate the expression of those antimicrobial peptide genes:CecB, CecD, Moricin and Glvl,2. While Toll and Imd signal pathways independently mediate the expressional regulation of antimicrobial peptide genes. So here we only provide a preliminary mechanism of silkworm antimicrobial peptide genes expressional regulation, deeper and more specific understanding of the mechanisms need further studies to confirm.
1模式识别受体编码基因的克隆及表达谱分析
模式识别受体在体液免疫-Toll和Imd途径中行使模式识别功能,并调控激活下游信号因子和抗微生物肽基因的表达。我们对家蚕模式识别受体-肽聚糖识别蛋白(PGRPs)和β-葡聚糖识别蛋白(PGRPs)家族基因进行研究,克隆得到10个PGRPs和4个βGRPs,得到BmβGRP3、BmPGRP-S3、-S4和BmPGRP-L5的完整编码序列。βGRPs家族成员都具有信号肽,其中BmβGRP1-3成簇分布于11号染色体,且不具有典型的葡聚糖结合结构域,BmβGRP4独立分布于22号染色体,具有典型的葡聚糖结合结构域。PGRPs的长型和短型亚家族都具有典型的酰胺酶活性(amidase activity)结构域,短型亚家族具有信号肽,长型亚家族则不具有。5个长型亚家族基因成簇分布于1号染色体,5个短型亚家族基因中有2个分布于9号染色体和3个分布于16号染色体。人工饲料无菌饲育的大造品系5龄3天幼虫添食大肠杆菌(E.coli, Ec)、家蚕黑胸败血菌(B. bombysepticus, Bs)和家蚕白僵菌(B. bassiana, Bb),免疫诱导3、6、12和24h四个时间点的个体水平荧光定量PCR检测,发现PGRPs长型亚家族基因BmPGRP-L1、-L3和短型亚家族基因BmPGRP-S1-3均能被革兰氏阴性菌、革兰氏阳性菌和真菌诱导上调表达；βGRPs基因家族中的BmβGRP3、4也能被上述三种微生物诱导上调表达。组织表达谱分析表明,所检测的13个识别受体基因,在头部组织中,BmβGRP2,4、BmPGRP-L2和BmPGRP-S1,-S3-5均能被阴性菌、阳性菌和真菌诱导上调表达；在表皮、中肠和脂肪体中,只有少数识别受体基因被三类微生物诱导上调表达。
2 BmβGRP3功能研究
果蝇(Drosophila)基因组中有编码3个阴性菌结合蛋白的基因,其中GNBP1由Lys-PGN激活,GNBP3由真菌激活,GNBP1和GNBP3均在Toll信号途径上游起模式识别功能并调控该信号通路。家蚕BmβGRP1、2被证实具有结合β-1,3-葡聚糖的能力且激活家蚕细胞免疫应答,但并未发现它们具有在家蚕Toll或Imd信号途径上游行使模式识别的功能。
BmβGRP3的开放式阅读框(open reading frame, ORF)为1470bp,编码489个氨基酸,N-端有一个由17个氨基酸组成的信号肽,推测可能为分泌型蛋白,48和454位处的天冬氨酸是潜在的糖基化位点,蛋白分子量为53kDa,等电点6.25。BmβGRP3的氨基酸序列高度保留了结合β-1,3-葡聚糖的功能结构域,该结构域能结合三重螺旋结构的β-1,3-葡聚糖。家蚕BmβGRP3在个体水平能被革兰氏阴性菌、阳性菌和真菌诱导上调表达,其中在个体水平阳性菌和真菌诱导上调表达能力强于阴性菌。组织水平上,在表皮中能被真菌高量诱导上调表达,阳性菌对该基因基本无诱导表达能力；在脂肪体中能被阴性菌高量诱导上调表达,阳性菌和真菌诱导上调表达该基因的能力较弱；在中肠组织中,该基因能被阴性菌和阳性菌较明显地诱导上调表达,真菌对该基因基本无诱导表达能力,表明家蚕BmβGRP3在应对不同微生物诱导时有着不同的表达模式和组织特异性。RNA干涉抑制BmPGRP3表达相关研究表明其在家蚕Toll信号途径上游行使模式识别功能和激活调控该途径的作用；进一步研究发现BmβGRP3的抑制表达会降低Toll信号途径的信号因子BmSpzl等基因的表达和抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnbl的表达,表明Toll信号途径可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnbl的表达。另一方面,家蚕BmβGRP3的抑制表达不会影响到Imd信号途径信号因子Bmlmd和抗微生物肽基因BmCecropin-B、BmCecropin-D和gloverin2的表达,暗示其它免疫调控途径如Imd信号途径可能调控抗微生物肽基因BmCecropin-B、BmCecropin-D和gloverin2表达。
对家蚕Toll和Imd信号途径免疫因子BmSpz1、BmImd等基因的诱导表达分析结果表明三类微生物(革兰氏阴性菌、阳性菌和真菌)都能激活家蚕的Toll和Imd信号途径,这与模式生物果蝇的Toll和Imd信号途径有很大的不同。果蝇的Toll信号途径只能被拥有DAP-型肽聚糖的细菌和真菌免疫激活,其Imd信号途径能被革兰氏阴性菌和部分阳性菌免疫激活。推测家蚕在其长期的进化过程中获得了更有效的免疫应答机制。
3 BmβGRP4和BmPGRP-S3的原核表达和功能研究
本研究已初步证实BmβGRP3位于Toll信号途径上游并可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达。然而关于Imd信号途径的免疫激活和抗微生物肽的表达调控仍有待进一步研究。
我们期望通过对BmβGRP4和BmPGRP-S3进行研究,以阐明模式识别受体在家蚕体液免疫应答中的具体生物学功能。构建了BmβGRP4和BmPGRP-S3的原核表达载体,纯化得到BmβGRP4和BmPGRP-S3蛋白,并制备了多克隆抗体。革兰氏阴性菌、阳性茵和真菌免疫诱导18h,BmβGRP4和BmPGRP-S3的mRNA表达量和蛋白量都显著提高。RNA干涉抑制BmβGRP4表达造成BmPGRP-S3的mRNA表达量和蛋白表达量明显降低；RNA干涉抑制BmPGRP-S3表达对Bm(3GRP4的mRNA表达量和蛋白表达量无影响,推测BmβGRP4位于BmPGRP-S3上游行使功能。RNA干涉抑制BmβGRP4或BmPGRP-S3表达都造成Imd信号途径信号因子Bmlmd等基因和抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2表达量的降低,表明BmβGRP4和BmPGRP-S3位于Imd信号途径上游行使识别作用并激活该信号通路；Imd信号途径可能调控抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达。与此同时RNA干涉抑制BmβGRP4或BmPGRP-S3表达不影响Toll信号途径信号因子BmSpzl基因和抗微生物肽基因BmCecropin-A1、BmCecropin-E和BmAtt2的表达,该处理方式提高了Toll信号途径调控抗微生物肽基因BmEnb1的表达,暗示家蚕Toll和Imd信号途径可能各自独立地调控抗微生物肽基因的表达。
另外,BmβGRP4或BmPGRP-S3的RNA干涉抑制表达都会造成BmClip15、BmFrep3、BmGaletin3和BmSpnl2的表达量降低,BmFrep3属于纤维蛋白原基因(Fibrinogen-related proteins, FREPs),该基因家族是一类与无脊椎动物先天免疫相关的C-端含有纤维蛋白原结构域的基因,具有识别细菌和原虫的能力,家蚕有3个该家族基因；BmGaletin3属于半乳糖凝集素基因家族,是一种专门结合p-半乳糖苷糖并含有一个进化保守的碳水化合物结合结构域(CRDs),果蝇和按蚊的半乳糖凝集素被认为具有微生物识别或吞噬的功能；BmClip15和BmSpn12分别为丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂,它们参与先天免疫信号的级联调控,放大或减弱先天免疫系统识别的信号。由此推测BmβGRP4和BmPGRP-S3除调控Imd信号途径外还可能参与家蚕细胞免疫应答。
4 BmSpz1和BmImd的功能研究
通过对家蚕模式识别受体—BmβGRP3、BmβGRP4和BmPGRP-S3的研究,初步证明了它们在家蚕先天免疫应答反应中的功能。家蚕BmβGRP3调控激活Toll信号途径；BmβGRP4和BmPGRP-S3共同调控激活Imd信号途径；并且发现Toll和Imd信号途径可能分别调控家蚕抗微生物肽基因BmCecropin-A1,BmCecropin-E、BmAtt2、BmEnbl和BmCecropin-B、BmCecropin-D、BmMoricin、Bmgloverinl、2的表达。为了进一步验证家蚕Toll和Imd信号途径所调控的抗微生物肽基因,我们又分别对家蚕Toll和Imd信号途径的关键基因BmSpzl和Bmlmd进行研究,希望藉此揭示家蚕Toll和Imd信号途径免疫信号的传导机制和抗微生物肽基因的表达调控模式。
BmSpz1和Bmlmd均能被三类微生物(革兰氏阴性菌、阳性菌和真菌)诱导上调表达,Western blotting分析显示BmSpz1的前体和成熟体蛋白均被三类微生物诱导上调表达。RNA干涉抑制BmSpz1表达造成Toll信号途径信号因子BmTube、BmRel和抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2、BmEnb1表达量明显降低；同时RNA干涉抑制Bmlmd表达造成Imd信号途径信号因子BmDreed、BmRelish和抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin、Bmgloverinl,2表达量显著降低。
通过对BmSpzl和Bmlmd研究再次证实了家蚕Toll和Imd信号途径均能被三类微生物(革兰氏阴性菌、阳性菌和真菌)诱导激活；Toll信号途径可能调控抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达；Imd信号途径可能调控家蚕抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达。
5家蚕抗微生物肽基因表达调控机制
昆虫抗微生物肽是昆虫在抵抗微生物侵染过程中合成的一类小分子多肽,在昆虫先天免疫中起重要作用。家蚕基因组中含有比其它昆虫基因组更多的抗微生物肽基因,它们大多数以多基因家族的形式存在,具有协同表达特点。Yamakawa等分析了家蚕的7个抗微生物肽基因,发现它们含有一个或多个KB-like和GATA元件。另外家蚕的抗微生物肽基因还含有多个CAATW元件,可能对基因转录的起始频率起调节作用,同时家蚕抗微生物肽还具有很高的可诱导性和较好的抗微生物活性。到目前为止,我们仍不清楚家蚕抗微生物肽基因的表达调控机制,通过本文的研究,我们提出了一个可能的家蚕抗微生物肽基因表达调控模式。
综上所述,首先,Toll信号途径由其上游的模式识别受体BmβGRP3完成对入侵微生物的识别并顺序激活该途径的信号因子,如胞外的BmSpzl、跨膜的Toll受体和胞内的BmMyd88、BmTube等,致使BmRel由核外进入核内,结合家蚕抗微生物肽基因的表达调控区域(κB-like和GATA元件等),从而完成对家蚕抗微生物肽基因BmCecropin-A1、BmCecropin-E、BmAtt2和BmEnb1的表达调控。其次,Imd信号途径由其上游的模式识别受体BmβGRP4和BmPGRP-S3完成对入侵微生物的识别并顺序激活该途径的信号因子,致使BmRelish由核外进入核内,结合家蚕抗微生物肽基因的表达调控区域(KB-like和GATA元件等),从而完成对家蚕抗微生物肽基因BmCecropin-B、BmCecropin-D、BmMoricin和Bmgloverin1、2的表达调控。同时家蚕Toll和Imd信号途径可能独立地完成相应抗微生物肽基因的表达调控。在本文中,我们给出了一个初步的家蚕抗微生物肽基因的表达调控机制,对该机制理的解还需要在本文的基础上进行更为深入、更为具体的研究来证明。
Bombyx mori, domesticated from wild silkworm, is a fusule insect with great economic value. However, the annual losses of China caused by silkworm disease are about millions, and seriously affect the sustainable development of the industry. Researches show that when insects are infected with microbes, various of antimicrobial peptides are induced and produced to kill the invading microorganisms. Thus the expression of these antimicrobial peptide genes is mainly regulated by Toll and Imd signal pathways. We focused on the immune-related genes such as short type peptidoglycan recognition protein 3(BmPGRP-S3) andβ-glucan recognition protein 3,4 (BmβGRP3,4), as well as the key genes BmSpzl and Bmlmd located in the Toll and Imd pathways, in order to explore the signal transduction of humoral immune and the expressional regulation mechanism of antimicrobial peptide genes in B. mori. The below are results obtained.
1. The cloning and expression analysis of genes coded by pattern recognition receptors
The pattern recognition receptors, which participate in pattern recognition in the upstream of humoral immune-Toll and Imd pathway, meanwhile regulate the activation of signal factors downstream and the expression of antimicrobial peptide genes. We do researches on the pattern recognition receptors of peptidoglycan recognition proteins andβ-glucan recognition proteins.10 PGRPs and 4βGRPs are cloned, and we obtain the complete coding sequences. All theβGRPs have signal peptide. BmPGRPl-3 are located in the chromosome 11 closely, the glucan-binding region is not detected, whereas BmPGRP4 is located in the chromosome 22 independently, including typical glucan-binding domain. For gene family of PGRPs, typical amidase activity domain is predicted regardless of long-and short- subfamily. Short type subfamily has signal peptide, but long type subfamily hasn't. Five genes of long type subfamily are located in chromosome 1 closely. Of short type subfamily,2 and 3 located in chromosome 9 and 16, respectively. RT-qPCR analysis of the silkworm larvae samples which were collected 3,6,12 and 24 hours after challenged with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana, respectively, showed that two long-type BmPGRP-L1,-L3 and three short-type BmPGRP-S1-3 could be up-regulated by gram-positive bacteria, gram-negative bacteria and fungi infection, respectively. Meanwhile, the induced expression profile among different tissues indicated that all the 13 receptor genes shared different expression patterns in head, integument, midgut and fat body challenged by gram-positive bacteria, gram-negative bacteria and fungi, respectively, many of them were induced in head, less were induced in integument and midgut, least in fat body.
2. Study on the function of BmβGRP3
There are three Gram-negative bacteria binding protein genes contained in the genome of Drosophila, two of them play a role in pattern recognition in the upstream of Toll signal pathway. It was confirmed that BmβGRP1 and BmβGRP2 can bindβ-1,3-glucan and active cellar immune response in B. mori. However, whether they participate in pattern recognition in upstream of Toll or IMD pathway is not detected.
BmβGRP3 has an open reading frame of 1470bp, encoding a protein with 489 residues. The 3'UTR contained a consensus polyadenylation signal (aataaa) upstream from the poly(A) tail. The deduced protein contained a putative signal peptide predicted by SignalP software in the N-terminal region. The calculated molecular mass of the mature peptide was about 53KD and theoretical pI was 6.25. To find pattern recognition receptors in the upstream of humoral immune pathway, we make focus on the study of BmβGRP3. We found the function region of bindingβ-1,3-glucan was conserved in amino acid sequence. BmβGRP3 can be upregulated (the level of individual and issue) by infecting with gram-positive, gram-negative bacteria and fungi, respectively. It shows that BmβGRP3 has different expression patterns and certain issue specificity responding to the induction of different miroorganisms. The RNAi knock-down BmβGRP3 indicates that it plays an important role in the innate immune response in B. mori. Further study suggests that Toll signal pathway can regulate the expression of some antimicrobial peptide genes. While RNAi knock-down BmβGRP3, the expressions of Toll pathway signal factors, such as BmSpzl and antimicrobial peptide genes CecAl、CecE、Att2 and Enbl decrease, which shows Toll signal pathway regulates the expression of antimicrobial peptide genes CecA1,CecE, Att2 and Enbl. On the other side, the expression of Bmlmd, IMD signal pathway signal factor, and antimicrobial peptide genes CecB, CecD and Glv2 was not affected, which suggests that there is other immune pathway, for example IMD signal pathway, may regulate the expression of antimicrobial peptide genes CecB, CecD and Glv2.
According to the induction expressions of BmSpzl and BmImd, Toll and Imd signal pathways can be activeted by gram-positive bacteria, gram-negative bacteria and fungi, respectively, which is significant different to Drosophila. The Toll signal pathway is only activated by gram-positive bacteria and fungi, the Imd signal pathway can be activated by gram-negative bacteria and certain gram-positive bacteria. It is reasonable that Bombyx mori obtain more efficient immune response mechanism when face to unique growth and development environment in long-standing evolution.
3. Prokaryotic expression and function analysis of BmβGRP4 and BmPGRP-S3
In this research, we found that BmβGRP3 is located the up-stream of Toll signal pathway and regulate the expression of some antimicrobial peptides genes, such as CecAl, CecE, Att2 and Enbl. However, it is yet to be studied further about the Imd pathway in immune activation and regulation of expression of antimicrobial peptides.
We look forward to studying on the BmβGRP4 and BmPGRP-S3, to clarify their specific biological functions in humoral immune response. We constructed BmβGRP4 and BmPGRP-S3 prokaryotic expression vectors, purified the two proteins, and obtained the polyclonal antibodies. The expression of BmβGRP4 and BmPGRP-S3 can be obviously increased by gram-negative bacteria, gram-positive and fungi induced, respectively, in both transcriptional and translational levels. RNAi knock-down BmβGRP4 causes that the expression of BmPGRP-S3 is repressed in both transcriptional and translational levels. However, RNAi knock-down BmPGRP-S3 does not affect the expression of BmβGRP4 in both transcriptional and translational levels. So we speculate that BmPGRP4 is located in the upstream of BmPGRP-S3 to exercise their functions. RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of signal transduction factor BmImd and antimicrobial peptides genes, including CecB, CecD, Moricin and Glv1,2, which indicates that the expressions of these antimicrobial peptide genes are mediated by Imd pathway. On the other hand, repressions of Toll pathway signal factor BmSpzl and antimicrobial peptides CecAl, CecE, Att2 and Enbl are not observed. Interestingly, the expression level of Enbl is even raised, which proves that Toll and Imd signal pathways independently mediate the expression of antimicrobial peptide genes.
Moreover, RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of BmClip15, BmFrep3, BmGaletin3 and BmSpn12. BmFrep3 is a fibrinogen-related protein family member which related with invertebrate innate immunity, it contains a fibrinogen domain in the C-terminal with the ability of bacteria and protozoan recognition. Three family members have been found in B.mori; BmGaletin3 belongs to Galactose lectin gene family, which contains a conserved carbohydrate recognition domain with the ability ofβ-galactosidase sugar binding. Galactose lectins are deemed to microbe recognition and phagocytosis. BmClip15 and BmSpnl2 are serine protease and serine protease inhibitor, respectively, which involved in cascade control of innate immune signal transduction by amplifying or reducing the signal. So we can speculate that BmPGRP4 and BmPGRP-S3 also may participate in the cellar immune response except for mediating Imd pathway.
4. Functional research of BmSpzl and Bmlmd
We identified the function of pattern recognition receptors-BmβGRP3、BmβGRP4 and BmPGRP-S3 in innate immune response in B.mori. BmβGRP3 mediates Toll pathway, Bm(3GRP4 and BmPGRP-S3 mediate Imd pathway. The expression of antimicrobial peptide genes CecAl, CecE, Att2, Enbl and CecB, CecD, Moricin, Glvl,2 are regulated via Toll and Imd pathway, respectively. In order to identify the antimicrobial peptide genes regulated via Toll and Imd pathways, and further confirming the transduction mechanism and antimicrobial peptide genes expression pattern, we performed RNAi knock-down BmSpzl and Bmlmd, which are the key genes in Toll and Imd pathways.
Both BmSpzl and BmImd can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. Western blotting analysis shows that the precursor and mature proteins of BmSpzl also can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. RNAi knock-down BmSpzl represses the expressions of signal factors, BmTube and BmRel in Toll pathway, and the antimicrobial peptide genes of BmCecropin-A1, BmCecropin-E, BmAtt2 and BmEnbl. Simultaneously, RNAi knock-down BmImd represses the expressions of signal factors, BmDreed, BmRelish in Imd pathway, and the antimicrobial peptide genes of BmCecropin-B, BmCecropin-D, BmMoricin and Bmgloverinl,2.
The fact that Toll pathway regulates antimicrobial peptide genes CecAl, CecE, Att2 and Enb1, the Imd pathway regulates antimicrobial peptide genes CecB, CecD, Moricin and Glvl,2 has been proved via studying on BmSpzl and Bmlmd. So we speculate that Toll and Imd pathway work separately in regulation of interrelated antimicrobial peptide genes.
5. The mechanism of antimicrobial peptide gene expression in B. mori
Antimicrobial peptides are kinds of minor polypeptides which are expressed during the insect self-defence, and which have played an important role in insect immunity. In the whole genomes, there are a lot of antimicrobial peptides have been expressed in the silkworm compared with other insects, and a great majority of them are exist in multi-gene family forms, most of them are coordinated expression. Yamakawa et al had analyzed 7 species of antimicrobial peptides in silkworm, which have one or more KB-like and GATA components. Moreover, some have CAATW components, which may be helpful in the starting frequency of gene transcription. In addition, the antimicrobial peptides in silkworm have much higher activity of bacteria induction and self-defence. But so far, the mechanism of antimicrobial peptide genes expression is still unclear. In this study, we propose a possible mechanism about antimicrobial peptide genes expression and regulation.
Firstly, Toll signal pathway is stimulated by pattern recognition receptor BmβGRP3 which recognizes the different pathogens and activates the other signal factors in this pathway, such as extracellular factor BmSpzl, transmembrane factor Toll receptor and intracellular factors BmMyd88、BmTube, to result in BmRel ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Toll pathway regulates the expression of those antimicrobial peptide genes:CecAl, CecE, Att2 and Enb1.
Secondly, Imd signal pathway is stimulated by pattern recognition receptors BmβGRP4 and BmPGRP-S3 which recognize the different pathogens and activate the related signal factors in Imd pathway, to result in BmRelish ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Imd pathway regulate the expression of those antimicrobial peptide genes:CecB, CecD, Moricin and Glvl,2. While Toll and Imd signal pathways independently mediate the expressional regulation of antimicrobial peptide genes. So here we only provide a preliminary mechanism of silkworm antimicrobial peptide genes expressional regulation, deeper and more specific understanding of the mechanisms need further studies to confirm.
引文
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