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Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.

Entamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (β-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica.

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.

The extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5., (© 2012 Blackwell Publishing Ltd.)