Wolbachia属的特定细胞内细菌广泛存在于节肢动物和几种丝虫线虫中。媒介传播疾病的控制程序(登革热,Zika,疟疾)和抗生素的抗丝虫疗法是基于这种重要的内共生体。调查Wolbachia,然而,受到宿主细胞需求的阻碍。在这项研究中,通过qPCR表征了WolbachiawAlbB在无宿主细胞体外培养系统中生长的需求。来自白纹伊蚊C6/36昆虫细胞的细胞裂解物级分含有细胞膜和含有胎牛血清的培养基,被鉴定为Wolbachia无细胞复制所必需的。补充昆虫细胞裂解物的膜部分可使细胞外Wolbachia复制增加4.2倍。与在昆虫细胞内生长的Wolbachia相比,无昆虫细胞培养物中的复制速率较低。然而,内细菌能够复制长达12天,并感染未感染的C6/36细胞。用脂质II生物合成抑制剂磷霉素处理的无细胞Wolbachia具有扩大的表型,先前在C6/36细胞中观察到的细胞内沃尔巴克氏菌,表明细菌无法分裂。总之,我们开发了一种无细胞培养系统,其中Wolbachia复制长达12天,提供了一种体外工具来阐明这些内细菌的生物学,例如,通过使用可能不会进入C6/36细胞的化合物进行细胞分裂。更好地理解Wolbachia生物学,特别是宿主-共生体的相互作用,是在病媒控制计划中使用Wolbachia以及未来针对丝虫病的药物开发的关键。
Obligate intracellular endobacteria of the genus
Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating
Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for
Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to
Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cell-free Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which
Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases.