Brugia malayi is a parasitic nematode that causes lymphatic filariasis in man. Transmission of the parasite depends upon a complex sequence of migration and development of the parasitic larvae within the mosquito vector. The role of microfilarial chitinase in transmission of brugian filariasis will be studied. Microfilarial chitinase was identified by a transmission-blocking monoclonal antibody, MF1. The appearance of the chitinase during microfilarial development coincides with the onset of the larva's ability to infect the mosquito vector. Chitinase enzyme activity in native and recombinant molecules will be examined, and known inhibitors of chitinase (allosamidin and related compounds) will be tested for their effects in vitro. It is hypothesized that the microfilarial chitinase is composed of two separate domains: one with enzymatic activity, and one that functions to localize the secreted enzyme by binding to the cuticular surface of the larva. This will be tested by proteolytic dissociation of the native molecule, and by mutagenesis of the cloned recombinant. Three possible roles for the chitinase during larval infection of the mosquito are postulated: 1) chitinase degrades a worm-derived structure that would otherwise inhibit development and moulting; 2) the enzyme degrades a mosquito-derived structure that blocks larval migration; or 3) the enzyme releases ligands that block defensive agglutinins elaborated by the mosquito. To distinguish among these alternatives, infective microfilariae will be fed to susceptible mosquitoes in the presence of inhibitors of chitinase, and phenotypically chitinase- negative microfilariae will be fed in the presence of oligosaccharides or recombinant enzyme. the progress of infection will be monitored in each case to ascertain the time and site of action of chitinase during the natural infection process. The two isozymes that comprise the MF1 antigen, p70 and p75, will be compared at the level of protein structure and function. Differences in primary sequence and post-translational modifications will be analyzed. The existence of two separate genes, or differentially processed messages, will be investigated. These studies will elucidate the role of this enzyme in parasite-vector interactions, and may thereby clarify the molecular mechanisms that operate during transmission. Furthermore, understanding the enzyme's structure and activity may point to novel control strategies for nematode parasites and the diseases they cause.
