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Proc Natl Acad Sci U S A. Jun 7, 2011; 108(23): 9649–9654.
Published online May 23, 2011. doi:  10.1073/pnas.1011481108
PMCID: PMC3111283
Microbiology

Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia

Abstract

Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as “hypothetical,” the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well.

Keywords: filaria, nematode

Disease associated with infection by Brugia malayi (Bm) and Wuchereria bancrofti, the two major causative organisms of human lymphatic filariasis, is the second leading cause of morbidity/disability worldwide, in large part because of the parasites’ ability to alter the structural and functional integrity of the lymphatics, leading to lymphedema and elephantiasis. Invasion, establishment of infection within the host and development are essential processes within the complex parasite life cycle (SI Appendix, Fig. S1), with many of the parasitic stages being targets for therapeutic intervention or vaccines. Each of the filarial life cycle stages has characteristics that are shared and others that are stage-specific.

Filarial infections are often characterized by a series of discrete host responses directed at the parasite and its endosymbiont Wolbachia that evolve during the course of infection. Because proteins are usually the effectors of most biological functions, proteomic data enable a more direct understanding of these important processes compared with those inferred from genomic studies. Absolute quantification of genome-wide expressed proteins is not yet within our reach for most eukaryotes. However, spectral counts of massive MS-based data (e.g., observed frequencies of each peptide) allow for relative quantification. Proteomic data also allow for clearer genomic curation by improving annotation and the identification of translational sites, stop codon read-throughs, frame shifts, and predicted orphan genes. These data also can help delineate the expression status of known and predicted/hypothetical genes.

Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. Previously, we and others have described the secretomes of Bm (13). We report here large-scale proteomic analyses of almost all the major mammalian stages of Bm, resulting in the identification of more than 62% of the products predicted from the Bm draft genome (4). We also report the identification of the majority of the expressed proteins of the Bm–Wolbachia (wBm), the obligate endosymbiont of Bm that also appears to express proteins in a stage-specific manner.

Results

Overview of Bm Proteome.

To assemble a high-density proteome map of Bm, proteins from the adult male (AM) and adult female (AF) parasites, microfilariae (MF), L3 larvae (L3), and the immature (i.e., uterine) MF (UTMF) were extracted. After having been digested into tryptic peptides, each stage was analyzed independently by using reverse-phase liquid chromatography–tandem MS (RPLC-MS/MS). The spectra were searched against the genomic databases for Bm and its endosymbiont Wolbachia (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 AM, AF, MF, L3 larvae, and UTMF, respectively (SI Appendix, Table S1). Combining these data with those from a study performed previously on the Bm secretome (1) (that included 122 proteins not found in the current analyses) and 164 additional proteins (based on peptide matches that identified more than one protein; SI Appendix, Table S2) resulted in the definitive identification of a total of 7,103 proteins of the 11,610 proteins (~61%) predicted from the genome (4) [Fig. 1A and SI Appendix, Table S2; Brugia Proteome Database (http://exon.niaid.nih.gov/transcriptome/brugia/Brugia_Proteome.zip)].

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 ...

Genomic analysis predicted that 4,956 (42.7%) of the 11,610 potential proteins were hypothetical proteins; the present study was able to confirm 2,336 (47.1%) of these 4,956 predicted proteins as bona fide proteins. Interestingly, 594 of these 2,336 hypothetical proteins are classified as “conserved hypothetical proteins.” Although the function of these “conserved” proteins is not completely known, approximately 30% of these could be assigned probable functions based on a conserved sequence motif or subtle similarities to other characterized functional and structural features. Moreover, there appears to be some stage-specific enrichment/abundance of many of the conserved hypothetical proteins (SI Appendix, Fig. S2) that may fill in the gaps believed to be missing in specific metabolic pathways or that may act as mediators with activities that have not been recognized previously (reviewed in ref. 5).

Stage-Specific Expression.

Among the identified proteins from each of the stages (Fig. 1A), 31% (2,255 and 7,267) were expressed exclusively in one of the five stages analyzed. Indeed, only 15% were common to all the stages. Moreover, within a given parasite stage, the percentages of the total number of proteins identified in each stage that was specific for that stage ranged from 9.3% to 18.7%. Although the function of most of these stage-specific proteins is largely unknown, their stage specific expression is an indicator of the developmental regulation of particular processes, only some of which are defined at the present time.

To corroborate and extend the analysis of the stage-specific expression in a subset of the proteomic data, the proteomic profiles of the AM and AF Bm (using a female:male ratio of spectral counts) were compared with transcriptional profiling (fold change, female:male). A significant relationship (r = 0.4657; P < 0.0001; SI Appendix, Fig. S3) between gene expression and protein production was observed. Interestingly, apart from the expected sperm-associated proteins, AM parasites appear to expend more energy on the production of cytoskeletal proteins [e.g., myosins (Bm1_40715), troponins (Bm1_35060), and intermediate filament proteins (Bm1_45215)], whereas the AF worm protein production was skewed toward proteins [e.g., major filarial sheath proteins (Bm1_19100), CHD4 (Bm1_47050), and cullin (Bm1_45370)] involved in embryogenesis or the production of MF.

Amino Acid Composition, Codon Use, and CAI.

The LC-MS/MS–based proteomic analysis resulted in the identification of proteins across a broad pI range. Comparison of the pI and MW of all the predicted proteins (SI Appendix, Fig. S4, blue) with that of the proteins identified by LC-MS/MS (SI Appendix, Fig. S4, red) suggests a close overlap between the two sets of data (Fig. S4), but the larger proteins were more readily detected by LC-MS/MS than those with lower molecular weight (MW). Indeed, the median length of the proteins detected using LC-MS/MS was 353 residues, whereas that of the nondetected proteins (inferred from the genome) was 168 residues (SI Appendix, Fig. S5). The LC-MS/MS identification of a greater number of higher-MW proteins (compared with lower-MW proteins) could be a result of Bm using multidomain proteins in its parasitic lifestyle or because smaller proteins generate fewer tryptic peptides available for identification. Although the latter hypothesis seems to be supported by the increasing number of peptides detected in relation to size (SI Appendix, Fig. S6), analysis of the number of peptides identified as a proportion of the total theoretical tryptic peptides (SI Appendix, Fig. S7) does not suggest that there was a differential detectability of the peptides across a wide range of MWs. The presence of relatively large proteins in the proteomes of parasitic organisms compared with free-living species may also be related to parameters such as temperature, environment, and other stresses (6). With only 65% of the detected proteins encoded by complete ORFs [defined by the presence of an ATG start and a stop codon (Bm draft genome (4); Brugia Proteome Database)], this suggests that either there was an incomplete/partial annotation of the Bm genome or that Brugia can read through in-frame stop codons (TGA and TAG) to code for selenocysteine and pyrrolysine.

The “butterfly” pattern distribution with large acidic and basic “wings” of the Bm proteome is a phenomenon that has been observed in a number of genomes (7). This bimodality in pI distribution may be related to the difficulty in maintaining protein structural stability and solubility near physiological/cytoplasmic pH or the preponderance of highly acidic and basic residues (i.e., D, E, K, T, R) versus the ones with pKa values close to 7 (i.e., H, C). Indeed only approximately 3% of the genome encodes for neutral proteins (based on calculated pI). Interestingly, the MW/pI plot for Caenorhabditis elegans, the only other nematode for which this type of analysis has been done, does not exhibit a clearly distinct butterfly pattern (8), suggesting that this distribution may have relevance to how parasitic (e.g., Bm) and nonparasitic, free-living nematodes differ biologically.

Codon usage tables (SI Appendix, Table S4) were generated by using the 25 most abundant proteins (SI Appendix, Table S5), and the codon adaptation index (CAI) was analyzed. CAI scores not only indicate that the vast majority of the proteins have moderate expression levels (CAI > 0.5 and < 0.7), but also highlights the identification of proteins with relatively low abundance (CAI < 0.5; SI Appendix, Fig S8A). Moreover, when the CAI was analyzed by stage, there were no obvious differences among the different stages (SI Appendix, Fig. S8B). Analysis indicates that, of the 16 predicted proteins (comprising entirely hypothetical proteins) with a CAI value greater than 0.9, only one was detectable in the proteome. This could also be attributed to the fact that all the predicted proteins in the CAI range of 0.9 to 1.0 were small proteins or peptides. Despite this, the proportion of peptides identified (relative to the number of proteins within a given CAI range) increased with increasing CAI values (SI Appendix, Fig. S8C).

Amino Acid Repeats.

Amino acid composition, especially single amino acid repeats (SAARs) and tandem repeats (TRs) are an important feature of proteomic analyses. The most prominent (>5 aa) repeats were Gln, Ser, Asp, Ala, Pro, Thr, and Glu (SI Appendix, Fig. S9 and Tables S6 and S7). SAARs account for 12% to 14% of the amino acid content of proteomes from eukaryotes, archaea, and bacteria, but are not present in every protein class (e.g., they are absent from metabolic enzymes and heat shock proteins) (911). SAARs also tend to appear primarily in the flexible regions and loops of transcription factors and protein kinases (12, 13).

Similar to that of Plasmodium knowlesi and Plasmodium vivax, the filarial genome is highly A- and T-rich (~70%) (4, 14). In comparison, the ORFome is closer to 60% A and T-rich. Under conditions of codon frequencies that control the dependency of repeat expansion, an A–T-rich genome should show relatively equal distributions of lysine, asparagine, phenylalanine, and isoleucine. Although the A–T content certainly influences the number of Lys and Asn repeats, the underrepresentation of Phe and Ile (SI Appendix, Fig. S9) indicates a selective determinant at play.

TRs are common in structural proteins such as collagens, keratins, and antifreeze proteins. Approximately 250 InterPro entries have been characterized as repeats that do not fold into a globular domain of their own, such as ankyrins, keltch, TRP, and armadillo repeats. The occurrence of TRs in proteins from parasitic organisms such as Plasmodium spp. (15), Leishmania spp. (16), and Trypanosome spp. (17, 18), is widespread. Although their exact function is still unclear, it has been suggested that they are involved in protein–protein interactions, binding to host-cell receptors or other processes. A relevant feature concerning protein repeats is the presence of substantial humoral response that barely confer a protective immunity (19, 20), which leads to the speculation that these could be decoy or immunomodulatory moieties (15, 18, 21, 22). Although the Bm-predicted proteome (11,610 proteins) has an overall occurrence of TRs of approximately 15% (i.e., using a threshold of six or more residues occurring twice or more in a given protein; SI Appendix, Table S7), it does not appear that the presence of TR in proteins is related to the humoral responses they engender. Only a few known immunologically active proteins (LL20, Chromadorea ALT, major microfilarial sheath protein) were found to contain repetitive domains, the functions of which are still speculative (20).

Functional Classification.

The identified proteins were classified into functional categories based broadly on the KOG classification of C. elegans (www.wormbase.org), with some adaptations. Similar to our previous classification (1), hypothetical, uncharacterized conserved, and unknown proteins were grouped as uncharacterized, whereas metabolic processes of carbohydrates, amino acids, lipids, nuclear, and energy were grouped within a single category termed metabolism. Of all the proteins identified to date as part of the Brugia proteome characterization, 45% of the proteins have no known function (Fig. 1B). To account for the relative abundance of specific functional processes, the proteomic data from each stage were normalized to fructose 1,6 bisphosphate aldolase (Bm1_15350). Stage-specific analysis of the functional annotation (as percentage of total proteins identified per stage) reveals that none of the stages was biased toward any particular functional process (SI Appendix, Fig. S10). However, enrichment analysis suggests that immunologically relevant (Fig. 2 and SI Appendix, Fig. S11) and extracellullar matrix (ECM)-related proteins (SI Appendix, Fig. S12) are enriched in the microfilaria and L3 stages compared with the other stages studied (SI Appendix, Table S8). Some of these immunologically relevant proteins have been described previously (2333) and relate to those molecules that are highly immunogenic in human infections or that have activity on the mammalian host immune response (through mimicry or other mechanisms). These data corroborate a number of previous studies that demonstrate stage-specific expression and/or serologic reactivity of ALT-2 and the larval allergens (in the L3 stage) (34) and BmR1 (35), BmMIF (36), TGF-β homologue (30), SXP-1 (37), galectins (38), and microfilarial sheath proteins (in the microfilarial stage) (39). Particular sets of ECM-associated proteins were common and highly enriched in the both the L3s (SI Appendix, Fig. S12A) and in the MF (SI Appendix, Fig. S12B).

Fig. 2.
Stage-specific enrichment in Bm proteome. Microfilarial stage-specific enrichment of immunologically relevant Bm proteins. GSEA analysis was performed on proteins ranked based on their relative abundance in each stage. The green curve shows the enrichment ...

Approximately 73% of the proteins had matches to the InterPro, Prosite, ProDom, or Pfam databases. Among the top 25 protein domains detected in the proteome, the most abundant were domains associated with protein kinases (PF00069), WD (PF00400), zinc finger C2H2 type (PF00096), RNA recognition motif (PF00076), and collagen triple helix (PF01391)-containing proteins. Although this type of analysis examines the relative diversity rather than the overall activity within a given protein family domain, we have been able to demonstrate that MFs have a marked enrichment of C2H2 domain-containing zinc finger proteins, proteins that generally bind DNA or act as transcription factors (SI Appendix, Fig. S13). The infective L3 larvae similarly contained a large number of collagen protein family members, whereas Ser/Thr phosphatase and DnaJ protein families were prominent within the AM proteome.

Gene sets specific to the L3 (293) and UTMF (905) and those common with the AF indicates possible roles as developmental proteins, whereas the core genes (n = 1,093) implicate necessary components required at all stages of the lifecycle. Further in-depth functional analysis of subsets of proteins should help in identifying target molecules that are crucial for the development of the parasite and establishment of infection.

Wolbachia.

The genome of wBm is represented by a single circular chromosome that is approximately 66% A- and T-rich with an extremely low density of predicted functional genes (40). Proteomic analysis of the various stages of Bm resulted in the identification of 557 of the 805 wBm-predicted proteins [based on peptides matching a single Wolbachia protein, “unique peptides”; SI Appendix, Table S9; and Wbm Proteome Database (http://exon.niaid.nih.gov/transcriptome/brugia/Wbm_Proteome.zip)], many of which are expressed in a stage-specific manner (Fig. 3A). Among the most abundantly (relatively) detected wBm proteins were the outer surface protein WSP (Wbm0432), probable outer membrane protein (Wbm0010), outer membrane protein-pal like (Wbm0152) protein, chaperonin GroEL, HSP60 (Wbm0350), and the molecular chaperone DnaK, HSP70 (Wbm0495; Fig. 3B). A total of 96 of the 166 hypothetical proteins could be validated as bona fide proteins, of which Wbm0253 and Wbm0603 happen to be among the proteins that were identified with the most abundant peptide counts (Fig. 3B). Interestingly, the ribosomal protein S18 (Wbm0501) that was found in the excretory–secretory products of MF (1) was not detected in any of the somatic proteomes. Functionally, genes involved in translation, ribosomal structure, and biogenesis were the second most abundant (12%) after uncharacterized/hypothetical proteins (27%; Fig. 3C). Immunologically, the major responses to Wolbachia following infection with L3 larvae have been to the WSP protein, a Wolbachia protein that has been suggested to be expressed in a stage-specific manner (41). Stage-specific analysis of this endosymbiont also indicates a bias toward proteins involved in posttranslational modifications, protein turnover, and chaperones in the adult worms compared with the other stages. On a comparative basis, there seems to be low numbers of Wolbachia in the immature stages of the MF (i.e., UTMF), or this stage has Wolbachia that are metabolically less active (Fig. 3D).

Fig. 3.
Overview and functional features of Wolbachia proteome. (A) Venn diagrams illustrating the overview of Wolbachia proteins identified from each stage of the filarial lifecycle. The total number of proteins identified (N = 557) includes the excretory-secretory ...

The presence of complete pathways for nucleotide and heme biosynthesis in Wolbachia and their partial absence in the filarial parasites, coupled with the observation of a loss of viability and reproductive capacity of the filarial organisms following elimination of Wolbachia by antibiotics (4244), suggest that the endosymbiont provides crucial signals and pathway components critical for parasite survival. Interestingly, all the members of the heme biosynthetic pathway, except for the ferrochelatase (Wbm0719) and the protoporphyrinogen oxidase, which is absent from the Wolbachia genome, were detected in the present proteomic analysis. Although wBm is devoid of a cell wall, the functional cellular machinery for the synthesis of lipid-II exists (45). Proteomic analysis identified each of the components involved in lipid-II synthesis.

Conclusion

The primary challenge in systems biology is to understand and integrate genomic, transcriptomic, proteomic, and metabolomic data. Attaining complete proteomic coverage of any organism is not inherently possible because of the limits of the technology as well as the dynamic nature of any proteome. Although depleting the most abundant proteins can enable lower abundance proteins to be identified, it is still impossible to know a priori the complete set of genes that are being translated within any cell under a specific set of conditions. Recent studies points out that the core proteome represents only a small fraction of the full proteome (20.7% in yeast and 7.6% in humans) (46).

Proteomic analysis by LC-MS/MS does not detect the protein expression levels with similar sensitivities as seen with isotope labeling. Nevertheless, it provides empirical evidence of protein expression and allows for high-throughput comparisons. Differences in protein recovery from the various stages could have resulted in the proteins being under- or oversampled. The immature uterine microfilarial stage protein recovery (from pooled samples) was relatively higher compared with the adult worms, MF, and L3 larvae. Therefore, the conclusions on the stage-specific identification of the proteins should be considered tentative. To account for the protein recovery from various stages, the data were normalized to fructose 1,6 bisphosphate aldolase as a common housekeeping gene product. This approach provides provisional evidence for relative protein abundance and the presence or absence of a particular protein in any given stage.

Nevertheless, the proteome maps of Bm and its endosymbiont wBm provide a detailed and stage-specific picture that complements genome annotation and gene prediction. Biological function arises in part from the concerted actions of interacting proteins in specialized networks. The library of stage-specific polypeptides now allows expansion to high-resolution surveys of metabolic or regulatory pathways, and thus the binary interaction networks that will help in understanding host–parasite interactions.

Materials and Methods

Parasites and in Vitro Culture.

Adult Bm male (BmAM) and female (BmAF) parasites, MF (BmMF), and the L3 larvae were obtained from the Filariasis Research Reagent Repository Center (Athens, GA). The immature forms of MF (i.e., UTMF) shed by the AFs in vitro were collected every 24 h. The animal procedures were conducted in accordance with the animal care and use committee guidelines at the National Institutes of Health and the University of Georgia.

Protein Isolation.

The parasite stages were lysed in lysis buffer, dialyzed, desalted, and digested with trypsin. Strong cation-exchange liquid chromatography fractionation of tryptic peptides was performed.

Nanobore RPLC-MS/MS.

Fractions collected from the strong cation-exchange column were pooled, lyophilized, and reconstituted in 20 μL 0.1% TFA before analysis by nanobore RPLC-MS/MS, by using an Agilent 1100 Nanoflow LC system coupled online with a linear ion trap–Fourier transform mass spectrometer.

LC-MS/MS Data Analysis.

Proteins were identified by searching the LC-MS/MS data using SEQUEST against the Bm database downloaded from The Institute for Genomic Research and the Wolbachia database from New England Biolabs. Methionine oxidation and phosphorylation on serine, threonine, and tyrosine were included as dynamic modifications in the database search. Only tryptic peptides with as many as two missed cleavage sites that met the criteria (delta correlation ≥ 0.08 and charge state-dependent cross-correlation scores ≥ 1.9 for [M+H]1+, ≥ 2.2 for [M+2H]2+, and ≥ 3.1 for [M+3H]3+) were considered legitimately identified. Further evaluation of the peptide identifications were also performed by searching a subset of the data against a decoy reversed database generated from the sequences in The Institute for Genomic Research database. Functional analysis and annotations were carried out by using various bioinformatic tools (SI Appendix). Peptides were assigned to proteins only if they could be matched to a single protein (termed unique peptides). Peptides that matched more than one protein (as in protein families or related proteins) were noted as nonunique peptides. Proteins identified based on matches to nonunique peptides (except for being enumerated and listed in SI Appendix, Table S2) were not included in any other analyses.

Gene Set Enrichment Analysis.

Gene Set Enrichment Analysis (GSEA), a method for analyzing molecular profiling data, examines the clustering of a predefined group of genes or proteins (gene set) across the entire database to determine whether the gene set has biased expression in one condition (or stage) versus another (47). For this analysis, the entire list of Brugia proteins was sorted on their relative abundance (i.e., spectral counts). The distribution of proteins from an a priori defined set throughout this ranked list was then determined by using GSEA. Sets of genes encoding for proteins in each functional category (Fig. 1B and SI Appendix) were analyzed by using GSEA for specific enrichment of genes/proteins (SI Appendix).

Supplementary Material

Supporting Information:

Acknowledgments

This project was funded primarily by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), and in part with federal funds from the National Cancer Institute, NIH, under Contract HHSN261200800001E.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. P.B.R. is a guest editor invited by the Editorial Board.

Data deposition: Detailed database and extensive annotation of the genome-wide proteins identified from Brugia malayi and its endosymbiont Wolbachia is available for download from the National Institutes of Health server mentioned in the manuscript.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011481108/-/DCSupplemental.

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