Wolbachia depletion blocks transmission of lymphatic filariasis by preventing chitinase-dependent parasite exsheathment

Abstract

Lymphatic filariasis is a vector-borne neglected tropical disease prioritized for global elimination. The filarial nematodes that cause the disease host a symbiotic bacterium, Wolbachia, which has been targeted using antibiotics, leading to cessation of parasite embryogenesis, waning of circulating larvae (microfilariae [mf]), and gradual cure of adult infection. One of the benefits of the anti-Wolbachia mode of action is that it avoids the rapid killing of mf, which can drive inflammatory adverse events. However, mf depleted of Wolbachia persist for several months in circulation, and thus patients treated with antibiotics are assumed to remain at risk for transmitting infections. Here, we show that Wolbachia-depleted mf rapidly lose the capacity to develop in the mosquito vector through a defect in exsheathment and inability to migrate through the gut wall. Transcriptomic and Western blotting analyses demonstrate that chitinase, an enzyme essential for mf exsheathment, is down-regulated in Wolbachia-depleted mf and correlates with their inability to exsheath and escape the mosquito midgut. Supplementation of in vitro cultures of Wolbachia-depleted mf with chitinase enzymes restores their ability to exsheath to a similar level to that observed in untreated mf. Our findings elucidate a mechanism of rapid transmission-blocking activity of filariasis after depletion of Wolbachia and adds to the broad range of biological processes of filarial nematodes that are dependent on Wolbachia symbiosis.

Keywords: Wolbachia; antibiotic treatment; lymphatic filariasis; transmission blocking.

Fig. 1.

Box and whisker plots illustrating L3 recovery rates 12 d after blood feeding the mosquito vector using a variety of treatment regimens. (A) Recovery rates of L3s from mosquitoes after mf were treated for 2 (n = 100 for control and treated groups), 4 (n = 108 and 100), or 6 (n = 100 for both groups) wk with tetracycline in vivo. Statistically significant reductions were observed for all three treatment durations compared to matched controls (*P < 0.05 for 2 and 4 wk, ***P < 0.001 for 6 wk). (B and C) Recovery rates of L3s from mosquitoes after in vivo treatment with tetracycline for 6 wk, with both repeats showing a statistically significant reduction in L3 recovery rate (***P < 0.001). For B, n = 20 for both control and treated groups, and C shows n = 200 for both control and treated groups. Due to the large number of mosquitoes to process, results for Fig. 1C were processed over 2 d, with no statistically significant differences observed between like-for-like treatments on the different days. (D) Recovery rate of L3s from mosquitoes after in vivo treatment with a suboptimal dose of rifampicin, a member of a different antibiotic class to tetracycline, again showing statistically significant reduction in L3 recovery rates (***P < 0.001); n = 50 for both groups. All statistical significance tests here were performed using the Mann–Whitney U test.

Fig. 2.

Bar graphs of GO enrichment analysis of transcriptomics data. The graphs are separated into three segments based on the three main GO categories. Bars are colored on an increasing intensity of statistical significance, with intense reds indicating high significance. (A) Top 15 statistically significant GO terms enriched within the set of down-regulated B. malayi genes. (B) All top statistically significant GO terms enriched within the set of up-regulated B. malayi genes.

Fig. 3.

Western blots of chitinase from mf either treated or untreated with antibiotics. Note how untreated mf showed a greater concentration of chitinase protein than treated mf. Samples were normalized for protein amounts using actin as a control.

Fig. 4.

Box and whisker plots comparing mf exsheathment, midgut tallies, and midgut escape rates based on different treatments and within different conditions. (A) Percentage of mf that have successfully exsheathed in vitro after chemical inducement, with control mf showing a statistically significantly higher exsheathment rate (n = 40 replicates, 50 mf per replicate, ***P < 0.001). (B) Percentage of mf that have successfully exsheathed 2 h after uptake into the mosquito vector via a blood meal, with median exsheathment rates of 44.0% and 6.3% for control and treated groups, respectively (n = 23 replicates, ***P < 0.001). (C) Total counts of mf that remained within the mosquito midgut, showing a statistically significant increase in number of treated mf present compared to control (n = 23, ***P < 0.001), with medians of 14 and 30 mf per midgut for control and treated groups, respectively (***P < 0.001,). (D) Percentage of mf that successfully escape from the mosquito midgut after tetracycline treatment showing a statistically significant reduction (n = 5, ***P < 0.001), with median escape rates of 12.7% and 0.2% for control and treated groups, respectively. (E) Percentage of mf that successfully escape the mosquito midgut after suboptimal treatment with rifampicin showing a statistically significant reduction (n = 8, ***P < 0.001), with median escape rates of 17.9% and 2.9% for control and treated groups respectively. All statistical tests were performed used a negative binomial generalized linear model.

Fig. 5.

Box and whisker plots comparing mf exsheathment rates after chemical inducement in vitro after in vivo treatment with the anti-Wolbachia compound AWZ1066 with or without supplementation with recombinant chitinases. Supplementation of untreated mf with recombinant chitinase showed either a statistically significant increase (with S. griseus chitinase) or no significant changes in exsheathment rate (T. viride chitinase). Treated mf were noted to have significantly reduced exsheathment rates when no chitinases were supplemented (***P < 0.001). However, treated mf exsheathment rates were effectively recovered after supplementation with chitinases compared to untreated mf, with no significant differences in exsheathment rate between control and treated mf after like-for-like chitinase supplementation (P > 0.5). The experiment used n = 10 replicates per condition, with 100 mf per replicate; NS, not significant.