Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells

PLoS One. 2014 Oct 27;9(10):e110047. doi: 10.1371/journal.pone.0110047. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 6
  • ADP-Ribosylation Factors / metabolism*
  • Adhesins, Bacterial / metabolism*
  • Cell Line
  • Epithelial Cells / metabolism*
  • HSP90 Heat-Shock Proteins / metabolism*
  • Humans
  • Intracellular Space
  • Neisseria meningitidis / physiology
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Binding
  • Protein Transport
  • Proteolysis
  • Recombinant Proteins
  • Temperature
  • rab GTP-Binding Proteins / metabolism*

Substances

  • ADP-Ribosylation Factor 6
  • Adhesins, Bacterial
  • HSP90 Heat-Shock Proteins
  • NadA protein, Neisseria meningitidis
  • Phosphoinositide-3 Kinase Inhibitors
  • Recombinant Proteins
  • rab11 protein
  • ADP-Ribosylation Factors
  • ARF6 protein, human
  • rab GTP-Binding Proteins

Grant support

This work is entirely funded by Novartis Vaccines; no external entities have contributed to cover project expenses. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.