Proteomic analysis inGiardia duodenalisyields insights into strain virulence and antigenic variation

Abstract

Giardia duodenalis is a protozoan parasite of the small intestine in vertebrates, including humans. Assemblage A of G. duodenalis is one of the two discrete subtypes that infects humans, and is considered a zoonotic assemblage. Two G. duodenalis Assemblage A strains BRIS/95/HEPU/2041 and BRIS/83/HEPU/106, constituting virulent and control strains respectively, were analyzed in one of the first comparative shotgun proteomic studies performed in this parasite. Protein extracts were prepared using a multiplatform approach with both an in-gel and in-solution sample preparation to enable us to assess the complementarity for future Giardia proteomic studies. Protein analysis revealed that BRIS/95/HEPU/2041 possessed a wider and more varied repertoire of variant surface proteins (VSPs), which are hypothesized to be involved in host adaptation, immune evasion, and virulence. A total of 35 VSPs were identified, with three common to both strains, six unique to BRIS/82/HEPU/106, and twenty-six unique to BRIS/95/HEPU/2041. Additionally, up to 25.6% of all differentially expressed proteins in BRIS/95/HEPU/2041 belonged to the VSP family, a trend not seen in the control BRIS/83/HEPU/106. Greater antigen variation in BRIS/95/HEPU/2041 may explain aspects of virulence phenotypes in G. duodenalis, with a highly diverse population capable of evading host immune responses.

The generation gap: Proteome changes and strain variation during encystation in Giardia duodenalis

“…In total, there were 2 large functional clusters that varied between strains and life cycle stages; 'VSP/Furin-like/EGFlike' and 'ankyrin-repeat'. Both these clusters feature proteins from the Giardia variable genome already implicated in inter-and intra-assemblage variation [23,49,51]. These data reinforce the hypothesis that the diversity seen in such families at the genome level translates to crucial differences at the level of the expressed proteome between strains at both life cycle stages.…”

“…In a previous study, we examined strain variation between H-106 and B-2041 trophozoites from axenic culture, and found that surface antigen proteins from the VSP family varied in both number and subpopulation distribution between strains [23]. In this study we show that other proteins that we previously identified as differentially expressed between H-106 and B-2041 trophozoites are also differentially expressed in the corresponding cysts.…”

“…The functional analysis of differentially expressed proteins in Section 3.3 highlighted that H-106 and B-2041 are overall remarkably similar metabolically and structurally in axenic culture, and that proteomic variation in cysts appears to be generated from the variable genome, as we have seen previously in trophozoites [23]. Of the 15 functional clusters identified, six were common between strains in the same life cycle stage (Fig.…”

“…In G. duodenalis post-transcriptional regulations means only a single VSP is expressed on the entirety of the parasite surface [54], and in axenic culture the absence of immune selection allows multiple VSP variants in the population to proliferate, which are detected in proteomics [6,23,51]. Though microRNAs (miRNAs) derived from small nucleolar RNAs (snoRNAS) play a role in VSP-switching [55] it is not confirmed experimentally if exogenous signals from environment, media or host affect the proliferation and survival of some VSP variants.…”

“…For each sample 250 g of protein was extracted from SDS sample buffer using chloroform-methanol [19], with the protein pellet washed 2-3 times with methanol. In-solution digestion was performed using the filter aided sample preparation (FASP) method [20,21], modified to solubilise protein in 2,2,2-trifluoroethanol (TFE) [22] as previously described [23]. Each extract was reconstituted to 60 L with 2% formic acid, 2% TFE and stored at −20 • C until analysis by LC-MS/MS.…”