Giardia secretome highlights secreted tenascins as a key component of pathogenesis

Abstract

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood.

Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites.

Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.

Figure 1:

Neighbour joining tree showing clustering of (A) cathepsin B and (B) tenascin gene families. Genes were retrieved by gene name search on GiardiaDB. Gene sequences were downloaded and aligned using ClustalW generated with the MEGA 6 software package. Maximum composite likelihood method was used, with 2000 bootstrap replicates. Bootstrap values greater than 50% are shown above the branches. ♦Proteins confirmed to be secreted using our proteomic analysis.

Figure 2:

The effect of co-culture with Giardia or Giardia supernatants on the electrophysiological properties of CaCo-2 monolayers. (A) Transepithelial electrical resistance in CaCo-2 monolayers following seeding on permeable supports. Data show an increase in TEER as the monolayer develops. Confluence occurred around day 6. Giardia were added on day 6 after the confluent monolayer formed and co-cultured with the Caco-2 monolayer for 24 hours. TEER was measured after 24 hours and compared with TEER in monolayers that had not been exposed to Giardia (n = 6). (B) A representative short circuit current (Isc) against time recording from single monolayers of CaCo-2 cells in an Ussing chamber. The trace shows the activation of CFTR chloride channels (basolateral application of 10 μM of forskolin) and calcium-activated chloride channels (basolateral application of 100 μM of UTP). Specificity of activation is confirmed by inhibition of Isc by the specific CFTR channel blocker, GlyH101; and specific calcium-activated chloride channel blocker, DIDS. The effect on Isc of 24-hour co-incubation of CaCo-2 monolayers with Giardia or with Giardia supernatant (1:1000 dilution) is also shown. (C) Effect of 24-hour co-incubation of CaCo-2 monolayers with different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3). (D) Effect of supernatant co-incubation from different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3) from Caco-2 monolayers. The results were analysed by the Student t test and expressed as mean values ± standard error mean (SEM). Significant difference expressed as *P < 0.05, **P < 0.01 compared with control.

Figure 3:

Proposed novel mechanism of pathogenicity for Giardia involving PNPO, extracellular nuclease, GCATB, tenascin. PNPO () renders the intestinal environment more favourable to trophozoite's growth. Once a new Giardia colony is established, trophozoites release extracellular nuclease (), GCATB (), and tenascin (). Extracellular nuclease may contribute to reducing the viscosity of the intestinal outer mucus layer, while GCATB may degrade mucins and disrupt intracellular junction. Finally, tenascins may maintain intestinal cells apart by attaching to the EGF receptors present at the surface of intestinal cells that could, over time, lead to the apoptosis of these isolated intestinal cells.