Host-pathogen interactions in filarial worm infections

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

Neglected tropical diseases caused by parasitic nematodes such as the filarial parasite Brugia malayi, a causative agent of lymphatic filariasis, also known as elephantiasis, remain a leading cause of morbidity and a significant health burden in the developing world. The continued lack of effective vaccines and the limited outcomes of mass drug administration efforts highlight the need for non-traditional therapeutic approaches. Such approaches require a deeper understanding of the underlying biology of parasitism and a clear definition of the host-parasite interface. This project is divided into two related components: one aspect focuses on the interaction of Brugia malayi worms with their mammalian host, and the other on the interaction of the worm with its Wolbachia bacterial endosymbiont. Brugia malayi-host interactions: Filarial nematodes are known to modulate the immune system of their host to sustain their persistence. Galectins, a class of proteins that bind beta-galatoside sugars, are known to play a role in immunomodulation. B. malayi expresses Bma-lec-2, a homologue of the human galectin-9, which has been shown to interact with the adaptive immune system of its host. Our study focuses on the extent Bmalec-2 interacts with host proteins, with specific emphasis on interactions with the innate immune system. Using mass spectrometry and co-immuno precipitation using a recombinant Bmal-lec-2 and human serum, we identified 10 human proteins that potentially interact with Bma-lec-2. These candidates were predominantly glycoproteins. Some of the proteins identified (vitronectin, clusterin, C4BP) are inhibitors of the complement system. We are planning on confirming these interactions using biolayer interferometry (BLI). We are confirming the M/S results by repeating the co-immunoprecipitation with the recombinant protein and with the native Bma-lec-2 that was immunoprecipitated from a protein lysate of 48 female adult worms. Using confocal microscopy, we localized Bma-lec-2 in secretory organs of adult female and male worms. In adult female worms and microfilariae, Bma-lec-2 co-localized with Alix, an accessory protein of the endosomal sorting complex required for transport and that is involved in the biogenesis of extracellular vesicles. Electron microscopy showed that Bma-lec-2 was on the surface of filarial extracellular vesicles, and associated with vitronectin, a host protein that can be used as a "trojan horse" by pathogens for internalization into endothelial cells. We are testing agonists of vitronectin to confirm interactions and internalization. Wolbachia-parasite interactions: According to recent findings, parasites secrete their miRNAs in mammalian host biofluids and therefore may cause pathology through these regulatory molecules. We initiated studies where we intend to validate how Brugia miRNAs are involved in causing pathology of lymphatic filariasis in human. We are studying 2 Brugia miRNAs: one is a Brugia-specific miRNA and another is a nematode-specific miRNA. We are testing mimics of these miRNAs in in vitro experiments with human lymphatic endothelial cells (hLECs). We obtained transcriptomic data of LECs treated with miRNA-mimics and controls. The data have been used to determine effects of these parasite miRNAs on human cells. We plan to use molecular and cellular methods including confocal microscopy to validate the effects of miRNAs on LECs and discover their role in developing pathology of filariasis. The project also evaluates how Wolbachia impact the regulation of host (filarial nematode) genes. We analyzed the expression of parasite miRNAs (regulatory molecules) in control and anti-wolbachia treated (using doxycycline) adult females to find parasite miRNA expression dependent on Wolbachia activity. We selected 2 miRNAs for further study. We observed that the expression of the selected miRNAs is important for Wolbachia survival in Brugia worms. Additionally, we are expanding our research into addressing questions on how parasite and endosymbiont obtain essential metabolites for their survival, and how they evade the hosts intracellular immune defenses. Wolbachia are common intracellular bacteria found in arthropods and filarial nematodes. The association between filarial worms and their mutualistic and obligatory endosymbionts is different from that of Wolbachia with insects: in worms they appear to provide crucial elements for fitness and survival, whereas the insects are mostly viable without Wolbachia. We predicted 73 chokepoints, i.e. parasite and Wolbachia enzymes that are essential for symbiosis. We screened 6 drugs that target 4 chokepoints in the in vitro model and one drug (that targets pyruvate kinase, a parasite glycolytic enzyme) was selected for further validation by our collaborator Dr. Hubner (Germany) who has an in vivo model of Litomosoides infections where he observed a significant reduction in Wolbachia in Litomosoides parasites after drug treatment. Wolbachia is an intracellular bacteria that can be used as a natural tool to inhibit arbovirus transmission by mosquitoes. We have been using a mosquito cell line (C6/36, Aedes albopictus) infected with Wolbachia to study the mechanisms underlying such inhibition. We observed that Wolbachia redirect host resources from host cholesterol metabolism resulting in reduction of cholesterol and lipids in C6/36 cells. This interferes with Zika virus infection. We confirmed that the role of Wolbachia in modifying metabolism in C6/36 cells suppresses ZikV replication. We presented these results in a manuscripts that is currently under review at Microbiology Spectrum (American Society for Microbiology Journals). More recently we developed in collaboration with Dr. Giacomello (SciLife Labs) a spatial transcriptomics approach on small tissues such as a section of B. malayi adult female worms. In situ we are able to determine the expression of Brugia genes in different tissues of the worm. Moreover, using a co-expression technique we were able to compare expression of Brugia genes in Wolbachia vs Wolbachia-free worm tissues in situ. This technique allows us to further investigate mechanisms of endosymbiosis between bacteria and worm tissues/cells. This project led to a manuscript recently accepted for publication in Nature Communications. Probing the mechanisms of interaction of the filarial worm Brugia malayi with its bacterial symbiont, Wolbachia, and of the worm with its host will give insight into the unique biology of this family of important pathogens. This could better inform novel therapeutic strategies.