A Proteomic Analysis of the Body Wall, Digestive Tract, and Reproductive Tract of Brugia malayi

Abstract

Filarial worms are parasitic nematodes that cause devastating diseases such as lymphatic filariasis (LF) and onchocerciasis. Filariae are nematodes with complex anatomy including fully developed digestive tracts and reproductive organs. To better understand the basic biology of filarial parasites and to provide insights into drug targets and vaccine design, we conducted a proteomic analysis of different anatomic fractions of Brugia malayi, a causative agent of LF. Approximately 500 adult female B. malayi worms were dissected, and three anatomical fractions (body wall, digestive tract, and reproductive tract) were obtained. Proteins from each anatomical fraction were extracted, desalted, trypsinized, and analyzed by microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry. In total, we identified 4,785 B. malayi proteins. While 1,894 were identified in all three anatomic fractions, 396 were positively identified only within the digestive tract, 114 only within the body wall, and 1,011 only within the reproductive tract. Gene set enrichment analysis revealed a bias for transporters to be present within the digestive tract, suggesting that the intestine of adult filariae is functional and important for nutrient uptake or waste removal. As expected, the body wall exhibited increased frequencies of cytoskeletal proteins, and the reproductive tract had increased frequencies of proteins involved in nuclear regulation and transcription. In assessing for possible vaccine candidates, we focused on proteins sequestered within the digestive tract, as these could possibly represent "hidden antigens" with low risk of prior allergic sensitization. We identified 106 proteins that are enriched in the digestive tract and are predicted to localize to the surface of cells in the the digestive tract. It is possible that some of these proteins are on the luminal surface and may be accessible by antibodies ingested by the worm. A subset of 27 of these proteins appear especially promising vaccine candidates as they contain significant non-cytoplasmic domains, only 1-2 transmembrane domains, and a high degree of homology to W. bancrofti and/or O. volvulus.

Fig 1. Anatomy of adult female B. malayi.

Tissues and structures dissected for proteomic analysis include body wall (red labels), reproductive tract [blue labels], and digestive tract (green labels). Illustrated by Wensi Sheng.

Fig 2. Dissection process of adult female B. malayi.

Top left and bottom left show break in the body wall and extrusion of digestive and reproductive tracts. Top right and bottom right: Body wall is in process of being slid away from digestive and reproductive tracts. Magnification: top left: 40x, bottom left: 100x, top right: 30x, bottom right: 20x.

Fig 3. Venn diagram of proteins identified within each anatomic fraction of adult female Brugia malayi based on 2 peptide minimum for identification.

Fig 4. Association of transporter proteins with the digestive tract as measured by gene set enrichment analysis (GSEA).

P-value = 0.005. The enrichment score is represented by the green line. Proteins were rank ordered according to their number of spectral counts within the digestive tract and are depicted in the heat map (red = more abundant, blue = less abundant). Black vertical lines represent each of the proteins associated with transporter function. D = Digestive tract, R = Reproductive tract, and B = Body wall.