Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia Sasisekhar Bennurua,1, Zhaojing Mengb, José M. C. Ribeiroc, Roshanak Tolouei Semnania, Elodie Ghedind, King Chanb, David A. Lucasb, Timothy D. Veenstrab, and Thomas B. Nutmana aLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; bLaboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC–Frederick, Frederick, MD 21702; cLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; and dCenter for Vaccine Research, Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Paul B. Rainey, Massey University, Auckland, New Zealand, and accepted by the Editorial Board May 1, 2011 (received for review August 5, 2010) Global proteomic analyses of pathogens have thus far been limited stop codon read-throughs, frame shifts, and predicted orphan to unicellular organisms (e.g., protozoa and bacteria). Proteomic genes. These data also can help delineate the expression status of analyses of most eukaryotic pathogens (e.g., helminths) have been known and predicted/hypothetical genes. restricted to specific organs, specific stages, or secretomes. We Proteomic analyses of most eukaryotic pathogens (e.g., hel- report here a large-scale proteomic characterization of almost all minths) have been restricted to specific organs, specific stages, or the major mammalian stages of Brugia malayi, a causative agent secretomes. Previously, we and others have described the secre- of lymphatic filariasis, resulting in the identification of more than tomes of Bm (1–3). We report here large-scale proteomic anal- 62% of the products predicted from the Bm draft genome. The yses of almost all the major mammalian stages of Bm, resulting in analysis also yielded much of the proteome of Wolbachia, the the identification of more than 62% of the products predicted obligate endosymbiont of Bm that also expressed proteins in from the Bm draft genome (4). We also report the identification a stage-specific manner. Of the 11,610 predicted Bm gene prod- of the majority of the expressed proteins of the Bm–Wolbachia ucts, 7,103 were definitively identified from adult male, adult fe- (wBm), the obligate endosymbiont of Bm that also appears to male, blood-borne and uterine microfilariae, and infective L3 express proteins in a stage-specific manner. larvae. Among the 4,956 gene products (42.5%) inferred from the genome as “hypothetical,” the present study was able to con- Results fi fi rm 2,336 (47.1%) as bona de proteins. Analysis of protein fam- Overview of Bm Proteome. To assemble a high-density proteome ilies and domains coupled with stage-specific expression highlight map of Bm, proteins from the adult male (AM) and adult female the important pathways that benefit the parasite during its de- (AF) parasites, microfilariae (MF), L3 larvae (L3), and the im- fi velopment in the host. Gene set enrichment analysis identi ed mature (i.e., uterine) MF (UTMF) were extracted. After having extracellular matrix proteins and those with immunologic effects been digested into tryptic peptides, each stage was analyzed in- fi as enriched in the micro larial and L3 stages. Parasite sex- and dependently by using reverse-phase liquid chromatography– fi fi stage-speci c protein expression identi ed those pathways re- tandem MS (RPLC-MS/MS). The spectra were searched against fi lated to parasite differentiation and demonstrates stage-speci c the genomic databases for Bm and its endosymbiont Wolbachia expression by the Bm endosymbiont Wolbachia as well. (wBm). A total of 72,318 unique peptides were matched to 6,981 proteins (3,653, 3,688, 3,135, 2,672, and 4,843 proteins) from filaria | nematode AM, AF, MF, L3 larvae, and UTMF, respectively (SI Appendix, Table S1). Combining these data with those from a study per- isease associated with infection by Brugia malayi (Bm) and formed previously on the Bm secretome (1) (that included 122 DWuchereria bancrofti, the two major causative organisms of proteins not found in the current analyses) and 164 additional human lymphatic filariasis, is the second leading cause of mor- proteins (based on peptide matches that identified more than bidity/disability worldwide, in large part because of the parasites’ one protein; SI Appendix, Table S2) resulted in the definitive ability to alter the structural and functional integrity of the identification of a total of 7,103 proteins of the 11,610 proteins lymphatics, leading to lymphedema and elephantiasis. Invasion, (∼61%) predicted from the genome (4) [Fig. 1A and SI Appen- establishment of infection within the host and development are dix, Table S2; Brugia Proteome Database (http://exon.niaid.nih. essential processes within the complex parasite life cycle (SI gov/transcriptome/brugia/Brugia_Proteome.zip)]. MICROBIOLOGY Appendix, Fig. S1), with many of the parasitic stages being targets Genomic analysis predicted that 4,956 (42.7%) of the 11,610 for therapeutic intervention or vaccines. Each of the filarial life potential proteins were hypothetical proteins; the present study cycle stages has characteristics that are shared and others that are stage-specific. Filarial infections are often characterized by a series of dis- Author contributions: S.B. and T.B.N. designed research; S.B., Z.M., J.M.C.R., K.C., and crete host responses directed at the parasite and its endosym- D.A.L. performed research; J.M.C.R., E.G., T.D.V., and T.B.N. contributed new reagents/ biont Wolbachia that evolve during the course of infection. analytic tools; S.B., Z.M., J.M.C.R., R.T.S., E.G., T.D.V., and T.B.N. analyzed data; and S.B. Because proteins are usually the effectors of most biological and T.B.N. wrote the paper. fl functions, proteomic data enable a more direct understanding of The authors declare no con ict of interest. these important processes compared with those inferred from This article is a PNAS Direct Submission. P.B.R. is a guest editor invited by the Editorial Board. genomic studies. Absolute quantification of genome-wide ex- Data deposition: Detailed database and extensive annotation of the genome-wide pro- pressed proteins is not yet within our reach for most eukaryotes. teins identified from Brugia malayi and its endosymbiont Wolbachia is available for However, spectral counts of massive MS-based data (e.g., ob- download from the National Institutes of Health server mentioned in the manuscript. served frequencies of each peptide) allow for relative quantifi- 1To whom correspondence should be addressed. E-mail: [email protected]. cation. Proteomic data also allow for clearer genomic curation by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. improving annotation and the identification of translational sites, 1073/pnas.1011481108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011481108 PNAS | June 7, 2011 | vol. 108 | no. 23 | 9649–9654 Downloaded by guest on September 26, 2021 Fig. 1. (A) Venn diagrams illustrating the overview of the Brugia proteome. The total numbers of proteins definitively identified (N = 7,103) include the excretory-secretory products and the somatic proteins. In addition, the 164 proteins for which a definitive identification could not be made are depicted separately as nonunique. (B) Functional annotation of the total Brugia proteome. Pie chart representing the percentage of proteins within each functional category as a function of the total proteome. Only a single annotation was assigned to a given protein. All unknown and hypothetical proteins have been classified as uncharacterized. Note that metabolism includes amino acid, carbohydrate, nuclear, and energy metabolism. A complete list is given with ad- ditional annotation and embedded links in the Brugia Proteome Database (http://exon.niaid.nih.gov/transcriptome/brugia/Brugia_Proteome.zip). was able to confirm 2,336 (47.1%) of these 4,956 predicted proteins identified by LC-MS/MS (SI Appendix, Fig. S4, red) proteins as bona fide proteins. Interestingly, 594 of these 2,336 suggests a close overlap between the two sets of data (Fig. S4), hypothetical proteins are classified as “conserved hypothetical but the larger proteins were more readily detected by LC-MS/ proteins.” Although the function of these “conserved” proteins is MS than those with lower molecular weight (MW). Indeed, the not completely known, approximately 30% of these could be median length of the proteins detected using LC-MS/MS was 353 assigned probable functions based on a conserved sequence residues, whereas that of the nondetected proteins (inferred motif or subtle similarities to other characterized functional and from the genome) was 168 residues (SI Appendix, Fig. S5). The structural features. Moreover, there appears to be some stage- LC-MS/MS identification of a greater number of higher-MW specific enrichment/abundance of many of the conserved hypo- proteins (compared with lower-MW proteins) could be a result thetical proteins (SI Appendix, Fig. S2) that may fill in the gaps of Bm using multidomain proteins in its parasitic lifestyle or believed to be missing in specific metabolic pathways or that may because smaller proteins generate fewer tryptic peptides avail- act as mediators with activities that have not been recognized able for identification. Although the latter hypothesis seems to previously (reviewed in ref. 5). be supported by the increasing number of peptides detected in relation to size (SI Appendix, Fig. S6), analysis of the number of fi fi Stage-Speci c Expression. Among the identi ed proteins from peptides identified as a proportion of the total theoretical tryptic each of the stages (Fig. 1A), 31% (2,255 and 7,267) were fi peptides (SI Appendix, Fig. S7) does not suggest that there was expressed exclusively in one of the ve stages analyzed.