Secreted proteomes of different developmental stages of the gastrointestinal nematode Nippostrongylus brasiliensis

Abstract

Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.

Fig. 1.

Silver stained analytical 2-DE gel of N. brasiliensis adult excretory/secretory proteins. Numbered circles indicate the spots that were analyzed by mass spectrometry. Annotation of the individual spots can be found in Table I.

Fig. 2.

Overlap between L3 and adult excretory/secretory proteins. A, Venn diagram showing the common proteins between L3 ESP, adult ESP, and adult NEx N. brasiliensis. Abbreviations used: ESP- excretory/secretory products from N. brasiliensis; NEx- whole worm extract from N. brasiliensis. B, 2D-DIGE image comparing L3 ESP (green spots) and adult ESP (red spots) from N. brasiliensis.

Fig. 3.

Gene Ontology analysis. Bar graph showing the most abundantly represented gene ontology molecular function terms in excretory/secretory proteins derived from N. brasiliensis L3 A, and adult B.

Fig. 4.

Pfam analysis. Bar graph showing the most represented protein families (Pfam) in the excretory/secretory proteins of N. brasiliensis L3 A, and adult B, the predicted proteome from L3 C, and adult D, and the predicted proteome containing a signal peptide from L3 E, and adult F. Colored bars represent the families present in more than two panels.

Fig. 5.

Comparison of the protein abundance in the L3 excretory/secretory proteins (ESP), adult ESP and adult adult worm extract (NEx) samples. A, The 50 most abundant proteins in L3 ESP ranked by total spectrum count (left) compared with the same proteins in adult ESP (center) and adult NEx (right). Maximum values are colored in red and minimum in blue. B, The 50 most abundant proteins in adult ESP ranked by total spectrum count (center) compared with the same proteins in L3 ESP (left) and adult NEx (right). The 50 most abundant proteins in adult NEx ranked by total spectrum count (right) compared with the same proteins in L3 ESP (left) and adult ESP (center).

Fig. 6.

Phylogenetic relationships of astacins based on Bayesian Inference (BI). The posterior probability supporting each clade is indicated. The accession numbers of each protein are shown.

Fig. 7.

Phylogenetic relationships of SCP/TAPS proteins based on Bayesian Inference (BI). The posterior probability supporting each clade is indicated. The accession numbers of each protein are shown.

Fig. 8.

Similarity analysis. Relationships of ESP from Brugia malayi, Heligmosomoides polygyrus and Nippostrongylus brasiliensis were compared with the ESP predicted from the genome of Necator americanus and displayed in a Simitri plot (37).