UC Santa Cruz

Filarial nematodes are human parasites that infect millions of people across the globe and cause debilitating diseases such as Elephantiasis and African River Blindness. These worms share a symbiotic relationship with Wolbachia, an obligate, alpha-proteobacteria endosymbiont, and rely on these bacteria for survival and proper embryogenesis. Taking advantage of this crucial symbiosis, efforts to identify drugs that kill the adult worm by targeting Wolbachia have proven to be promising. Here, I describe the discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. These potent quinazolines were identified by pairing a primary cell-based high-throughput imaging screen with a secondary ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. To better understand the relationship between Wolbachia and its worm host, adult Brugia pahangi were exposed to varying concentrations of common antibiotics in vitro and assessed for Wolbachia numbers in the germline tissue. Surprisingly, we found that worms treated with higher concentrations of antibiotics had higher Wolbachia titers, and antibiotics given at low concentrations reduced Wolbachia titers. This counterintuitive dose response is known as the “Eagle effect” and the presence of this effect in Wolbachia suggests a common underlying mechanism that allows diverse bacterial and fungal species to persist despite exposure to high concentrations of antimicrobial compounds. To our knowledge this is the first report of this phenomenon occurring in an intracellular endosymbiont, Wolbachia, in its filarial host. While several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. We investigated the long-term effects of the antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Clusters of densely packed Wolbachia within the worm’s ovarian tissues were observed by confocal microscopy. The number, size, and Wolbachia density of these clusters were not diminished despite large doses of rifampicin antibiotic. This finding suggests that these clusters may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. Lastly, I define the cellular characteristics of these clusters, which fit the definition of endosymbiont bacteriocytes, and I identify drugs that target them. Nascent bacteriocytes arise in newly formed sheath cells adjacent to the distal tip cell of the Brugia pahangi germline. They dramatically enlarge but do not appear to disrupt the integrity of the sheath cells. We determined that the Wolbachia within bacteriocytes are either in a quiescent form or replicating at a very low rate. These Wolbachia-based bacteriocytes are present in Brugia malayi, one of the nematode species which cause human filariasis, as well as B. pahangi. Screens of known antibiotics and other drugs revealed two drugs, Fexinidazole and Corallopyronin A, have strong anti-bacteriocyte efficacy.