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.
Highlights
Neisseria meningitidis is a Gram-negative diplococcus that causes severe invasive disease and represents one of the most devastating bacterial infections
Non permeabilized Chang cells were incubated with recombinant NadA (rNadA) and, after washing, the bound fraction was revealed by FACS analysis with anti-Neisseria meningitidis adhesin A (NadA) antibody
Even at 4uC the amount of cell-bound rNadA was measurably higher than the background Setting incubation temperature at 37uC, we analyzed the kinetics of rNadA binding to Chang cells
Summary
Neisseria meningitidis (meningococcus) is a Gram-negative diplococcus that causes severe invasive disease and represents one of the most devastating bacterial infections. Fatal if not treated on time, meningococcus invasion appears to be more an undesirable event of a usually commensal bacterium, probably due to a combination of host susceptibility and strain specific propensity to invasiveness [1,2]. The pathophysiology of the bacterium N. meningitidis is a process that requires several steps: penetration of the epithelial or mucosal barrier, reaching and surviving the bloodstream, crossing the blood-brain barrier, and eventually causing meningitis through extracellular proliferation [3]. A recent report shows that bacterial capsule and type 4 pili are important for epithelial cell transcytosis [9] but host and pathogen players involved in this process are far from being defined
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