Lara Flavio Alves, Pohl Paula C, Gandara Ana Caroline, Ferreira Jessica da Silva, Nascimento-Silva Maria Clara, Bechara Gervásio Henrique, Sorgine Marcos H F, Almeida Igor C, Vaz Itabajara da Silva, Oliveira Pedro L
Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.
Centro de Biotecnologia e Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
PLoS One. 2015 Aug 10;10(8):e0134779. doi: 10.1371/journal.pone.0134779. eCollection 2015.
In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.
在蜱虫中，血液消化在细胞内进行，血红蛋白的蛋白水解消化发生在一种特殊类型的溶酶体即消化泡中，随后血红蛋白的血红素部分转移到一个积累大量血红素聚集体的特殊细胞器，即血球体。在本研究中，我们研究了血红素类似物荧光金属卟啉以及双甲脒（一种最常用于控制牛蜱感染的杀螨剂）被微小扇头蜱（牛蜱属）中肠细胞摄取的情况。这两种化合物在体外均被中肠细胞摄取并积累在血球体内。两种分子的转运对环孢素A（CsA）敏感，环孢素A是一种著名的ATP结合盒（ABC）转运蛋白抑制剂。罗丹明123是一种荧光探针，也是一种公认的ABC底物，它同样以CsA敏感的方式被导向血球体。使用针对PgP - 1型ABC转运蛋白保守结构域的抗体，我们能够在消化泡膜上对PgP - 1进行免疫定位。对双甲脒抗性和敏感的两种微小扇头蜱菌株的比较显示，抗性菌株比敏感菌株更有效地解毒双甲脒和锡原卟啉IX，这一过程对CsA也敏感。与敏感菌株相比，含有ABC转运蛋白特征的转录本在双甲脒抗性菌株中的表达增加了2.5倍。RNA干扰诱导该ABC转运蛋白的下调导致金属卟啉在消化液泡中积累，中断了血红素向血球体的运输。这一证据进一步证实该转录本编码一种血红素转运蛋白。这是关于吸血生物中血红素运输的首次报道。虽然血球体的主要生理功能是解毒血红素并减弱其毒性，但我们认为蜱虫利用这种杀螨剂解毒途径可能代表了一种新的抗农药分子机制。
