Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections
Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections
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DOI：
10.1007/s10493-008-9159-5
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发表时间：
2008-12-01
影响因子：
2.2
通讯作者：
Sonenshine, Daniel E.
中科院分区：
文献类型：
--
作者：
Kocan, Katherine M.;de la Fuente, Jose;Sonenshine, Daniel E.
抗微生物肽，包括防御素，是蜱固有免疫系统的组成部分，已被证明对革兰氏阴性和革兰氏阳性细菌提供保护。Varisin是在革螨变异中发现的一种防御素，主要在血细胞中产生，但转录水平也在中肠和其他蜱虫细胞中表达。在本研究中，我们研究了缬氨酸在蜱对革兰氏阴性牛病原体边缘无形体免疫中的作用。通过RNA干扰（RNA interference, RNAi），给雄性蜱注射varisin dsRNA，然后让其进食并感染实验感染的小牛，从而沉默了varisin基因的表达。real - time RT-PCR证实了varisin在血细胞、中肠和唾液腺中的沉默表达。我们预计，沉默varvarin会增加蜱中的边缘A.感染，但结果表明，细菌数量，由A.边际msp4定量PCR测定，在沉默varvarin的蜱中显著减少。此外，用于RNAi的蜱的边缘蜱菌落在形态上与注射洗脱缓冲液的对照蜱不同。菌落形状不规则，在中肠细胞细胞质中有游离的边缘芽孢杆菌。一些蜱被发现全身感染了一种微生物，这种微生物可能与缬素的沉默有关。这似乎是第一个通过RNAi沉默蜱中防御蛋白表达的报告，导致减少边缘单胞杆菌感染。
Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.