Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

National Institute Of Allergy And Infectious Diseases

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Abstract

In the past year we have closed fully genome gaps for Loa loa, W. bancrofti, and S. stercoralis. We now have telomere-to-telemere genomic data for these organisms. We have utilized the genomic data as the backdrop for performing a large number of proteomic and transcriptomic studies. To understand better the developmental programs that underscore the transition between the mosquito-derived infective stage larvae (L3) to mammalian adapted L3s and to L4s following a molt, and the initial week of adaptation to the human host, we adapted an in vitro system that allowed for L3 development and subsequent molting to the L4. Using microarray and proteomic assessments at multiple times through this 9 day process we have not only identified those genes/pathways that are critical for the L3/L4 transition but we have also demonstrated by both pharmacologic inhibition (cysteine protease inhibition) and RNAi (of the critical CPLs) the critical role played by cysteine proteases in the early development of mammalian adapted L3s to L4s. We have recently performed shotgun mass spectroscopy on both human sera of patients with defined filarial infections, excretory/secretory (E/S) products of Loa loa microfilariae, all stages of the O. vovlulus worm, and appropriate controls to identify parasite derived biomarkers of active infection. This has led to identification of molecular targets that have been used d to configure quantitative immunoassays for the rapid detection of active infection for O. volvulus and Loa loa. We have exploited informatic pipelines to identify tandomly and/or interspersed repeats within the genomes of Angiostrongylus cantonensis, Loa loa, Wuchereria bancrofti, Strongyloides stercoralis, T.cruzi, and the various Schistoma spp. We have configured qPCR assays for each of these identified targets and have improved the limits of detection in validated assays. Some of these have been shown to be useful for the diagnosis of these infections in appropriate patient samples. A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by 50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.