Proteomic analysis of adult S. mansoni gut contents.

During development and egg production, adult intravascular schistosome parasites consume up to 33,000 red cells per hour [1] and significant volumes of blood plasma with its rich source of host proteins. The schistosome gut is a major interface between parasite and host. It is lined by a syncitial epithelium called the gastrodermis. Gastrodermal cells have the ultrastructural appearance of secretory cells with an active Golgi apparatus, a well-developed granular endoplasmic reticulum and numerous vesicles [2]. The lumen of the gut must therefore contain both ingested host cells and plasma proteins as well as secretory products from the parasite [3]. As the gut is blind-ended, schistosome worms regurgitate gut contents by rhythmic peristalsis. A male adult worm requires four hours to fully exchange the content of its caeca, and a female 3 hours [1]. Identifying the major ingested host proteins would be an important first step in defining how schistosomes degrade host proteins and absorb protein fragments. At the same time defining the proteins released by the gastrodermal cells would give clues to both schistosome metabolism and the interaction of the parasite with the host immune system. We therefore isolated the regurgitant from adult schistosomes that had developed in golden hamsters (Mesocricetus auratus). Approximately one hundred, mixed-sex, six-week-old worms, perfused from one hamster, were induced to regurgitate gut contents by osmotic shock in distilled water [4, 5]. Figure 1 shows the appearance of the female gastrodermis before and after this procedure. The extensive hematin deposits associated with the gastrodermal lamellae (Figure 1A) are absent subsequent to regurgitation, and there is a greater proportion of lipid droplets within the lumen (Figure 1B). The underlying muscle layer and tegumental cytons are vacuolated due to osmotic shock, but the gastrodermis itself is intact, including much of the delicate villus-like lamellae. There is no evidence of lysis of gastrodermis or significant disruption of tegumental surface that would contribute to the protein content of the regurgitant. This is indeed reflected in the character of the most abundant proteins identified in Table 1. Figure 1 Ultrastructural appearance of gut lumen (top of photo) and gastrodermis (nucleated syncytium) of adult female S. mansoni before (A) and after (B) regurgitation. Note vacuolization of muscle and parenchyma from osmotic shock (short arrow, lower left), ... Table 1 Host and schistosome proteins identified in adult S. mansoni GIC by mass spectrometry analysis. Figure 1C shows a one dimensional (1D) SDS-PAGE of 30 micrograms of schistosome regurgitant. The major bands of this SDS-PAGE gel were excised, reduced with DTT, alkylated with iodoacetamide, and subjected to in-situ tryptic digestion with side-chain protected porcine trypsin (Promega) for 4 h at 37°C; the resulting peptide mixture was then extracted according to the protocol: http://ms-facility.ucsf.edu/ingel.html. The resulting peptides were fractionated on a homemade nanocolumn (C12, 75 µm × 150 mm); utilizing an Ultimate Pump/Famos autosampler system (Dionex/LC Packings), that operated at a flow rate of ~ 300 mL/min, with a water/acetonitrile/0.1% formic acid mobile phase; developing a 5%–45% solvent B gradient over 35 min. The eluent was analyzed by LC/MS/MS on a QSTAR quadruple-orthogonal acceleration-time-of-flight hybrid mass spectrometer (MDS Sciex) in a data-dependent fashion, i.e. 1 sec mass acquisitions were followed by 3 sec CID experiments on computer-selected multiply charged ions. Utilizing the appropriate Analyst script all the CID data were submitted for database search with an in-house Mascot search engine. The full NCBI database was searched; one missed cleavage was permitted; Cys-carbamidomethylation was considered a fixed modification; while Met-oxidation, N-terminal pyroglutamic acid formation from Gln and protein N-acetylation were permitted as variable modifications; the mass accuracy was 100 ppm for the precursor ions and 0.2 Da for the MS/MS fragments. Only proteins identified from multiple unique sequences were accepted. Table 1 lists the major proteins identified in S. mansoni gut regurgitant. Host hemoglobin (both the tetramer and alpha/beta subunits) was readily identified. Host immunoglobulin (IgG) and serum albumin were also major protein components in regurgitant. The degradation of these proteins by schistosome gut proteases has been reported [4–8]. In addition to the identification of ingested host proteins, the major S. mansoni-derived proteins identified several key biological interactions occurring in the gut. First, hemoglobin degradation would lead to release of heme iron that in turn would be bound by ferritin-1. In addition, as free heme can be toxic, the presence of the antioxidants, thioredoxin and superoxide dimutase, suggests that the worm deals with heme toxicity at the site of heme release. Two proteins identified in schistosome regurgitant suggest a relationship to lipid metabolism and fatty acid absorption: lysophospholipase and fatty acid binding protein Sm-14. The lysophospholipase is that previously identified with two splice variants [9]. This enzyme may control the cytolytic actions of lysophospholipids that have been identified in the tegument [10, 11], but may also be produced in the gut as a consequence of membrane remodeling associated with both the secretion of gastrodermal products and absorption of luminal contents. Sm14 has been localized to both the basal lamina of the tegument and gut epithelium [12]. It is a key scavenger protein for fatty acids given the inability of the parasite to synthesize sterols and lipids de novo [13]. Finally, immunophilin-binding protein (FKB12) has been implicated in schistosome-immune evasion and appears to regulate signal transduction via interaction with the TGF-beta family type I receptor (SmT-R-I, SmRK-1) and calcinerin [14]. This protein has not been previously identified in the gastrodermis, but is present in the tegument of both sexes, suggesting that the proteome analyzed may also contain some proteins originating from the tegument. Although gut proteases functioning in host protein digestion were not abundant enough in the regurgitant to be detected at this level of proteomic analysis, their presence has been confirmed by biochemical analysis. Cathepsin B was previously identified as the major gut protease using active site probes [15]. Cathepsin L was also identified by probe analysis and cathepsin D by direct solution assays using a fluorescent substrate [5]. Cathepsin C-like activity was histochemically localized to the gastrodermis and gonads [16], and leucine aminopeptidase predominantly to the gastrodermis by antibody [17]. Given the network of proteases that operate in the gut to digest host plasma proteins [5, 15], it is not surprising that a protease inhibitor, cystatin, is also found as a major protein in the regurgitant [18]. Cystatins are tight binding inhibitors of the papain family of cysteine proteases [19] and may inhibit the gut cysteine proteases cathepsins B and L to regulate their activities. Also intriguing was the identification of the catalytically inactive form of the asparaginyl endopeptidase SmAE (legumain or Sm32 [20, 21]) in which the active site cysteine is replaced by an asparagine. Apart from this substitution, the other residues constituting enzyme’s binding pocket are intact and it is therefore possible that inactive SmAE might compete with the active enzyme for substrates, thus providing post-translational regulation. Catalytically active SmAE is a key enzyme in physiology of the schistosome gut as it synergizes with other proteases to degrade host proteins [5]. It may also serve to process and activate gut-associated protease zymogens [22] as has been demonstrated in vitro for pro-cathepsin B1 [8].