Ferrara Taíse Fernanda da Silva, Schneider Vanessa Karine, Kishi Luciano Takeshi, Carmona Adriana Karaoglanovic, Alves Marcio Fernando Madureira, Belasque-Júnior Jose, Rosa José César, Hunter Wayne Brian, Henrique-Silva Flávio, Soares-Costa Andrea
Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil.
PLoS One. 2015 Dec 30;10(12):e0145132. doi: 10.1371/journal.pone.0145132. eCollection 2015.
Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.
黄龙病（HLB）是影响柑橘类植物的最具破坏性的病害之一。其致病因子与韧皮部受限细菌亚洲韧皮杆菌（CLas）以及柑橘木虱（该病害的传播媒介）有关，柑橘木虱传播与HLB相关的细菌。可以通过抑制细菌或传播媒介来控制该病害。在HLB病害的控制策略中，广泛使用的一种策略是控制病害传播媒介柑橘木虱的酶。昆虫柑橘木虱属于半翅目，其肠道中经常含有半胱氨酸肽酶。这类酶的重要性促使我们在柑橘木虱转录组中寻找酶，以建立控制HLB的替代策略。在本研究中，我们报道了从柑橘木虱中鉴定和表征一种组织蛋白酶B样半胱氨酸肽酶（DCcathB）。DCcathB在毕赤酵母中重组表达，分子量约为50 kDa。该酶水解荧光底物Z-F-R-AMC（Km = 23.5 μM）和组织蛋白酶B的选择性底物Z-R-R-AMC（Km = 6.13 μM）。重组酶被半胱氨酸蛋白酶抑制剂E64（IC50 = 0.014 μM）和甘蔗CPI-4（Ki = 0.05 nM）以及选择性组织蛋白酶B抑制剂CA-074（IC50 = 0.095 nM）抑制。RT-qPCR分析显示，DCcathB在若虫和成虫中的表达比在卵中大约高9倍。此外，该酶在肠道中的表达分别比其余组织和头部高175倍和3333倍，这表明DCcathB可能是控制HLB的一个靶点。
