Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition

Abstract

Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatographytandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo-and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.

The barber's pole worm CAP protein superfamily — A basis for fundamental discovery and biotechnology advances

“…Surprisingly, most (82%) of the curated 45 CAP protein sequences inferred here had not been detected previously by proteomic analysis (Yatsuda et al, 2003). The relatively large number of transcripts encoding various CAP proteins in H. contortus compares with findings for Ancylostoma caninum, A.…”

“…Within group 2, three predicted proteins (Hc-CAP-22, -23 and -24) that shared homology to Ac-ASP-7 (Datu et al, 2008;Osman et al, 2012) formed a separate clade to the exclusion of Hc-CAP-25. Group 4 double-domain proteins also formed separate clusters according to homology amongst published CAP proteins; Hc-CAP-34 and Hc-CAP-35, which had homology to Hc40 (Rehman and Jasmer, 1998;Yatsuda et al, 2003) formed a separate clade to double-domain proteins which shared sequence homology to ASP-1 (i.e. Hc-CAP-36 to Hc-CAP-41) and VAP-2 (i.e.…”

“…In addition, cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily members (Gibbs et al, 2008), typified by SCP-like extracellular domains (InterProScan codes: IPR014044 and IPR001283), are also prominent in ES products. Although the two CAP proteins Hc24 and Hc40 have been identified amongst ES molecules of H. contortus (see Schallig et al, 1997;Rehman and Jasmer, 1998;Yatsuda et al, 2003), there appear to be many more related molecules in this worm, according to recent evidence . This information supports the proposal of a diversified and specific involvement of CAP proteins during H. contortus infection and in host interplay.…”

“…1A). Specifically, within group 1, 12 predicted proteins with close homology to Na-ASP-2 and Hc24 (Yatsuda et al, 2003;Asojo et al, 2005a) grouped separately (pp = 0.59-1.00) to the exclusion of CAP proteins with homology to C. elegans LON-1 protein (Hc-CAP-15 and -16) and GLIPR-1 (Hc-CAP-17) (cf. Shaye and Greenwald, 2011).…”

cTrans: Generating polypeptide databases from cDNA sequences

“…As polymorphism might exist between different species at the amino acid level even for the most conserved proteins, the cross-species match approach is compromised when the mass spectra data are affected by the polymorphism [1,2]. Researchers have proposed to query the EST raw sequence databases or the EST translation databases as an alternative in species without full genome sequence information to overcome this shortcoming [1,[3][4][5][6]. MS fingerprinting searches against EST raw sequences can be performed using MASCOT [7], ProFound [8] or Protein-Prospector [9] by directly specifying certain parameters; and identification using MS/MS data against EST raw sequences can be performed using search programs such as SEQUEST (http://fields.…”