title: Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells.
abstract: Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Bozza
authors.affiliation: Unit of Genomic Approaches to Membrane Traffic, Fondazione Mario Negri Sud, S. Maria Imbaro (CH), Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Capitani
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Montanari
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Benucci
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Biancucci
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Nardi-Dei
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Caproni
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Barrile
authors.affiliation: Unit of Genomic Approaches to Membrane Traffic, Fondazione Mario Negri Sud, S. Maria Imbaro (CH), Italy.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Picciani
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Savino
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Aricò
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Pizza
authors.affiliation: Institute of Protein Biochemistry, CNR, Naples, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Luini
authors.affiliation: Unit of Genomic Approaches to Membrane Traffic, Fondazione Mario Negri Sud, S. Maria Imbaro (CH), Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Sallese
authors.affiliation: Novartis Vaccines, Siena, Italy; Department of Biology, University of Naples "Federico II", Naples, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Merola
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D018829:Adhesins, Bacterial / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space; D009345:Neisseria meningitidis / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis; D011994:Recombinant Proteins; D013696:Temperature; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D018829:Adhesins, Bacterial; D018841:HSP90 Heat-Shock Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C501982:NadA protein, Neisseria meningitidis; C000715348:ARF6 protein, human