Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells.
Bottom Line: 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.
Affiliation: Novartis Vaccines, Siena, Italy.
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.
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Mentions: The functional role of external Hsp90 in rNadA internalization was further explored by means of a synthetic membrane-impermeable inhibitor, the FITC-GA, that specifically target the extracellular population of the chaperone. To verify that FITC-GA did not enter the cells we used the cellular AKT levels as a marker for the functionality of the intracellular chaperone . Hsp90 inhibitors (e.g. 17-AAG) promote the proteasomal degradation of this intracellular kinase. Cells were incubated with medium containing 2.5 and 10 µM of either 17-AAG or FITC-GA for 1, 4, 24 and 48 hrs. At the end of each period, cell lysates were prepared and equal amount of proteins were analyzed by electrophoresis and Western blot to reveal the amount of intracellular AKT. As shown in figure 8A, the levels of AKT did not change until 4 hrs of treatment with the inhibitors (figure 8A, lanes 2–7). At 24 and 48 hrs the intracellular kinase was degraded in cells incubated in presence of 17-AAG at both concentrations (figure 8A, lanes 9–10, 13–14) while its amount was not modified in FITC-GA treated cells (figure 8A, lanes 11–12, 15–16). This result strongly suggested that the Hsp90 inhibitor FITC-GA do not exert any activity on the intracellular population of the chaperone.