extracellular replication of endobacteria.

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