Alteration of immunoproteome profile of Echinococcus granulosus hydatid fluid with progression of cystic echinococcosis

Abstract

Background: Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed.

Methods: We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages.

Results: We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis.

Conclusions: Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Figure 1

Electrophoretic profile of E. granulosus HF collected from sheep livers. HF (200 μg proteins) harvested from single fertile cyst of CE1 (A) and CE2 (B) stages were electrofocused on IPG strips (pH 6–11) and resolved by 15% SDS-PAGE. The gels were stained with CBB. The protein spots were subjected to in-gel trypsin digestion and processed with MALDI-TOF-MS. Proteins identified are marked by Arabic numbers (1–78) and are summarized in Table 1 and Additional file 2. Representative ultrasonographic images of CE1 and CE2 cysts are shown. M _r, molecular weights in kDa; pI, isoelectric point.

Figure 2

Global immunoproteome characteristics of CE2 HF. (A) Immunoproteome analysis of HF against pooled sera of different CE stages. The patients were classified by typical US findings [4]. CE2 HF was separated by 2-DE, transferred to nitrocellulose membranes, and probed with pooled CE1-CE5 sera (n = 3 per group) as described in the Methods section. Immunoreactions of EgAg5 (purple), EgAgB1 subunit (blue), EgAgB1 (red), EgAgB subunit 2 (khaki), EgAgB2 (brown), and EgAgB4 (green) are indicated. All signals were detected by ECL after 2 min exposure. Spots at ca. 24 kDa could not be identified on 2-DE gel, but showed immunoreactivity (box, left panel). Each corresponding band from CE1 and CE2 HF was independently analyzed by nano-LC-ESI-MS/MS. (B) Identification of proteins within box in A by PMF. (C) Immunoreactivity of each spot in Figure 1A (left panel) was compared to the volumes of the corresponding protein spots. The open bars indicate the relative volumes (left Y-axis) and normalized protein volumes (right Y-axis) in the CBB stained gels. The solid bars indicate the relative volumes of the immunoblot.

Figure 3

Changes of immunoproteome profile according to CE progression. Blots containing CE2 HF were probed with individual CE1-CE5 sera as described in the Methods section. Markings are the same as in Figure 2A. Panels A-E show representative immunoblot images from respective disease stages (CE1-CE5). M _r, molecular weights in kDa; pI, isoelectric point.

Figure 4

Cross-reactivity with other pathological conditions and normal controls. Blots containing CE2 HF were reacted with individual/pooled (n = 10 per group) serum of AE (A), neurocysticercosis (B), sparganosis (C), primary hepatocellular carcinoma (D), and normal controls (E). EgAgB1 shows cross-reactivity with AE patient sera (boxes in panel A). Markings are the same as in Figure 2A. AE, alveolar echinococcosis; NC, neurocysticercosis; HCC, primary hepatocellular carcinoma. M _r, molecular weights in kDa; pI, isoelectric point.