Antignani, Vincenzo (2010) Protein translocation motifs: a common trait between plant and human pathogens. [Tesi di dottorato] (Unpublished)

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Some oomycetes and fungal effectors carry RXLR-dEER domains. These domains are necessary for the host cell entry and without any pathogen-encoded machinery. Using this information and bioinformatic based analyses, we identified potential cell entry motifs in the N-terminal regions of 13 secreted proteins from plant pathogens (Alternaria brassiciola, Pythium ultimum. Albugo candida), human pathogens (Aspergillus fumigatus, Cryptococcus neoformans, Coccidioides immitus), beneficial (Trichoderma atroviride) and saprophytic (Aspergillus flavus) microorganisms. Filter binding assays was used to test whether the N-terminal domains of the effector proteins bound phosphoinositides. Twelve out of thirteen of the fusions proteins bind PI3P, and more weakly to PI4P and/or PI5P. The capability to enter the host cell was confirmed by a soybean root uptake or an epithelial cell uptake assays. Mutations in the functional RXLR-like motifs of effectors resulted in a loss of binding. Alanine substitutions in the fungal RxLR motifs also abolished the activity of the N-terminal domain-GFP fusion protein, preventing the protein accumulation in human epithelial cells. To test the hypothesis that the fungal effectors bound external PI3P in order to enter human cells, we incubated the effector-GFP fusions with a mammalian cell culture in the presence of a molar excess of each of the PI3P- and PI4P-binding biosensor proteins. We used a highly specific biosensors for PI3P and PI4P by fusing the PH domains of the human proteins PEPP1 to mCherry. The PI3P-binding proteins completely abolished entry by the six effector-GFP fusions into the epithelial cells. Protein accumulation was strongly inhibited in each case by competing PI3P-binding proteins (which entered the cells instead). To determine if the effector-GFP fusions bound to the same sites on the cells as the PI3P-binding proteins, equimolar concentrations of the biosensors and effectors were incubated with A549 cells at 2°C. The effector fusions colocalized with each biosensor in a punctate pattern on the surface of the cells. At 37°C, the biosensor and the effector fusions were colocalized inside the cells within endosome-like structures. The rule of one of these proteins in the virulence was evaluated by generating a knocked-out mutant of A. fumigatus unable to produce the Af2 effector. Conidia from the wild type and from the mutants were compared with respect to their capability to activate phagocytosis in mammalian epithelial cells and for their intracellular survival.

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