AUTHOR=Behrmann Lara Vanessa , Meier Kirstin , Vollmer Jennifer , Chiedu Chukwuebuka Chibuzo , Schiefer Andrea , Hoerauf Achim , Pfarr Kenneth 

TITLE=In vitro extracellular replication of Wolbachia endobacteria

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1405287

DOI=10.3389/fmicb.2024.1405287

ISSN=1664-302X

ABSTRACT=<p>Obligate intracellular endobacteria of the genus <italic>Wolbachia</italic> 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 <italic>Wolbachia</italic>, however, is impeded by the need for host cells. In this study, the requirements for <italic>Wolbachia w</italic>AlbB growth in a host cell-free <italic>in vitro</italic> culture system were characterized via qPCRs. A cell lysate fraction from <italic>Aedes albopictus</italic> C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of <italic>Wolbachia</italic>. Supplementation with the membrane fraction of insect cell lysate increased extracellular <italic>Wolbachia</italic> replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to <italic>Wolbachia</italic> 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 <italic>Wolbachia</italic> treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular <italic>Wolbachia</italic> in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which <italic>Wolbachia</italic> replicate for up to 12 days, providing an <italic>in vitro</italic> 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 <italic>Wolbachia</italic> biology, and in particular host-symbiont interactions, is key to the use of <italic>Wolbachia</italic> in vector control programs and to future drug development against filarial diseases.</p>