<em>In vitro</em> extracellular replication of <em>Wolbachia</em> endobacteria

Abstract

Obligate intracellular endobacteria of the genus <em>Wolbachia</em> 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 <em>Wolbachia</em>, however, is impeded by the need for host cells. In this study, the requirements for <em>Wolbachia</em> wAlbB growth in a host cell-free <em>in vitro</em> 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 <em>Wolbachia</em>. Supplementation with the membrane fraction of insect cell lysate increased extracellular <em>Wolbachia</em> replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to <em>Wolbachia</em> 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 <em>Wolbachia</em> treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular <em>Wolbachia</em> in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which <em>Wolbachia</em> replicate for up to 12 days, providing an <em>in vitro</em> 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 <em>Wolbachia</em> biology, and in particular host-symbiont interactions, is key to the use of <em>Wolbachia</em> in vector control programs and to future drug development against filarial diseases.