Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling

Abstract

Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

Figure 1. Stage specific distribution of ES in the adults, microfilariae, L3 larvae and L3 larvae molting to L4 of Brugia malayi.

Proteins identified are represented as a Venn diagram, illustrating the overlap in proteins detected in the adult male, adult female, microfilarial and larval stages of B.malayi. The numbers indicate the total number of proteins detected in each of the stages. The complete listing of each of these proteins along with the peptides identified are given in Table S2, Table S3, Table S4, Table S5, and Table S6.

Figure 2. Functional profiles of Brugia malayi ES proteins.

Pie-chart representing the percentage of proteins identified from each of the stages, plotted based on their functional classification. Only a single annotation was assigned to a given protein. All unknown and hypothetical proteins have been classified as uncharacterized. Note: Metabolism includes amino acid, carbohydrate, nuclear and energy metabolism. A complete list is given with additional annotation and embedded links in the Final Secretome-Web (http://exon.niaid.nih.gov/transcriptome/Bm-secretome/Brugia-Secretome-Web.zip) version. AMES: Adult male ES; AFES: Adult female ES; MFES: Microfilariae ES; L3-ES: L3 larval ES; L3-MES:L3 larval molting ES.

Figure 3. Stage specific functional profiles of Brugia malayi ES proteins.

Pie-charts representing percentage of proteins identified from each of the stages, plotted based on their functional classification. Only a single annotation was assigned to a given protein. All unknown and hypothetical proteins have been classified as uncharacterized. Note: Metabolism includes amino acid, carbohydrates, nuclear and energy metabolism). The complete list is given with additional annotation and embedded links in the Final Secretome-Web (http://exon.niaid.nih.gov/transcriptome/Bm-secretome/Brugia-Secretome-Web.zip) version. AMES: Adult male ES; AFES: Adult female ES; MFES: Microfilariae ES; L3-ES: L3 larval ES; L3-MES:L3 larval molting ES.

Figure 4. Relative abundance of ES proteins of Brugia malayi.

The most abundant proteins identified (relative abundance of peptides) of the Brugia malayi ES of adult females, adult males microfilariae, and L3 larvae (pre- and during molting) are illustrated as a heat map. Relative abundance scale are coded (spectral values) based on number of peptides identified per protein with separate scales for each stage on the bottom of the figure. AMES: Adult male ES; AFES: Adult female ES; MFES: Microfilariae ES; L3-ES: L3 larval ES; L3-MES:L3 larval molting ES.

Figure 5. Distribution and functional classification of Wolbachia ES proteins.

Proteins identified in the ES were searched against the Wolbachia database and a total of 91 were identified in the ES of Brugia malayi. The pie-chart represents the percentage of Wolbachia proteins identified from all of the stages, plotted on as a function of their classification. Only a single annotation was assigned to a given protein. All unknown and hypothetical proteins have been classified as uncharacterized. (Note: Metabolism includes amino acid, carbohydrates, nuclear and energy metabolism.)