Henkle-Dührsen K, Eckelt V H, Wildenburg G, Blaxter M, Walter R D
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Mol Biochem Parasitol. 1998 Oct 30;96(1-2):69-81. doi: 10.1016/s0166-6851(98)00109-1.
Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O. volvulus nuclear genome. The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression system. The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1). The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT. CAT was detected in the endobacteria in the hypodermis of adult male and female O. volvulus, O. ochengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae. O. volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords.
在盘尾丝虫抗氧化酶研究的背景下，从盘尾丝虫成虫λZapII cDNA文库中分离出编码过氧化氢酶（CAT）的DNA克隆。对其核苷酸和编码的氨基酸序列进行分析后发现，它们来源于细胞内细菌，而非盘尾丝虫的核基因组。发现内共生细菌的CAT基因位于一个基因簇中，后面跟着一个铁蛋白基因和一个核酸外切酶基因。内共生细菌的CAT基因编码一种能够解毒H2O2的功能性酶，这在大肠杆菌表达系统中产生活性重组蛋白得到了证明。纯化的54 kDa蛋白在很宽的pH范围内具有CAT活性，比活性为103,000±3000 U mg(-1)。内共生细菌CAT的光谱表明它是一种含铁血红素的蛋白质，在405 nm处有一个Soret带。为了研究盘尾丝虫中细胞内细菌的系统发育，使用通用细菌引物通过聚合酶链反应从总基因组DNA中扩增出16S rRNA基因的一段。对盘尾丝虫来源的16S rRNA序列进行系统发育分析表明，这种内共生细菌属于立克次氏体目一个独特的沃尔巴克氏体分支。使用针对重组内共生细菌CAT产生的多克隆抗体，对含有不同盘尾丝虫种类的盘尾丝虫瘤和活检组织进行免疫组织化学染色。在成年雄性和雌性盘尾丝虫、奥氏盘尾丝虫、吉氏盘尾丝虫和带形盘尾丝虫皮下组织的内共生细菌中检测到了CAT。在内共生细菌的卵母细胞以及包括子宫内微丝蚴和皮肤微丝蚴在内的所有胚胎阶段也检测到了这种酶。因此，盘尾丝虫含有类似沃尔巴克氏体的内共生菌，它们通过卵传递，并对侧索的皮下组织表现出特殊的亲和力。
