The overall goal of my PhD thesis is the understanding of the molecular basis exploited by NadA to contribute in the complex pathophysiology of N. meningitidis. Knowledge of the NadA function is not as accurate as its immunological potential. From the early reports, NadA was shown to target human epithelial cells promoting adhesion. Later on it was shown to bind human monocytes/macrophages and dendritic cells. Until now, the host receptors have remained elusive. Our group demonstrated that the direct interaction of NadA with the external molecular chaperone Hsp90 is a crucial step to direct NadA-driven epithelial intracellular trafficking through the recycling pathway. During the first part of my PhD, I participated and finalized a project focused on the understanding of the mechanism exploited by NadA to enter human epithelial cells, thus a lane to traverse the epithelial barrier. Altogether, our experiments show that the rNadA is able to enter into Chang epithelial cells through a PI3K-dependent endocytosis process regulated by ARF6. Its cellular co-localization with Rab11 suggest that the adhesin enter a recycling pathway; the interaction with the extracellular pool of Hsp90 is crucial to enter this pathway since treatments with Hsp90 inhibitors lead to intracellular accumulation of rNadA. Finally, HDX-MS analysis performed with rNadA in complex with Hsp90 allowed us to identify the NadA Head domain residues crucial for the interaction. As logical pursuing of the studies described above, I started a project aimed to identify NadA receptors. I approached this target by performing an unbiased large-scale human protein microarray screening. With this method a discrete number of putative NadA interactors were identified and the effective binding was confirmed for 4 of them with different techniques. One of the identified proteins (Lox-1) is a receptor predominantly express on endothelial cells while the other three are mainly expressed on cells of myeloid origin and better characterized on monocytes/macrophages. Intriguingly none of the candidates are expressed on epithelial cells suggesting a broad range of biological function extending to several cell types. However, to date the identity of the NadA receptor on epithelial cells is still missing. Although spending part of my time on Lox-1 recognition by NadA (see below), in the second part of my PhD I focused my attention to the characterization of the NadA binding to monocytes in order to elucidate the contribution of this N. meningitidis antigen in this step of bacteria pathogenesis. As anticipated, the protein microarray identified three different monocytic receptors involved in the NadA binding: Siglec 5, Siglec 14 and FcγR2A Here we report evidence of a novel interaction with a N. meningitidis surface protein suggesting a model in which meningococcus sialic acid acts as tethering factor whereas NadA establish a stronger adhesive interaction. The putative interactions were further corroborated by cellular binding assays and by biophysical and functional studies with the soluble recombinant proteins. Recently, we suggested an additional role for NadA at the host peripheral microvasculature and at the blood brain barrier through the identification of the interaction with Lox-1. In this work we confirm the interaction with biophysical and functional studies. In spite of the strong biochemical evidence of the NadA/Lox-1 binding, the biological outcome of this interaction has not been approached. In order to fill this gap, I spent three months of my PhD at X. Nassif laboratory in Paris. Overall, the results I obtained confirm the master role of type IV pili for the adhesion to endothelial cells but also highlight a role for NadA in this process. All considered, NadA is able to interact with all three cell types involved in the bacterium cycle of infection suggesting unknown and multifaceted roles for this protein in the complex multi-step mechanism of MenB pathogenesis

Benucci, B. (2016). Role of Neisseria meningitidis Adhesin A (NadA) in bacteria pathogenesis.

Role of Neisseria meningitidis Adhesin A (NadA) in bacteria pathogenesis

BENUCCI, BARBARA
2016-01-01

Abstract

The overall goal of my PhD thesis is the understanding of the molecular basis exploited by NadA to contribute in the complex pathophysiology of N. meningitidis. Knowledge of the NadA function is not as accurate as its immunological potential. From the early reports, NadA was shown to target human epithelial cells promoting adhesion. Later on it was shown to bind human monocytes/macrophages and dendritic cells. Until now, the host receptors have remained elusive. Our group demonstrated that the direct interaction of NadA with the external molecular chaperone Hsp90 is a crucial step to direct NadA-driven epithelial intracellular trafficking through the recycling pathway. During the first part of my PhD, I participated and finalized a project focused on the understanding of the mechanism exploited by NadA to enter human epithelial cells, thus a lane to traverse the epithelial barrier. Altogether, our experiments show that the rNadA is able to enter into Chang epithelial cells through a PI3K-dependent endocytosis process regulated by ARF6. Its cellular co-localization with Rab11 suggest that the adhesin enter a recycling pathway; the interaction with the extracellular pool of Hsp90 is crucial to enter this pathway since treatments with Hsp90 inhibitors lead to intracellular accumulation of rNadA. Finally, HDX-MS analysis performed with rNadA in complex with Hsp90 allowed us to identify the NadA Head domain residues crucial for the interaction. As logical pursuing of the studies described above, I started a project aimed to identify NadA receptors. I approached this target by performing an unbiased large-scale human protein microarray screening. With this method a discrete number of putative NadA interactors were identified and the effective binding was confirmed for 4 of them with different techniques. One of the identified proteins (Lox-1) is a receptor predominantly express on endothelial cells while the other three are mainly expressed on cells of myeloid origin and better characterized on monocytes/macrophages. Intriguingly none of the candidates are expressed on epithelial cells suggesting a broad range of biological function extending to several cell types. However, to date the identity of the NadA receptor on epithelial cells is still missing. Although spending part of my time on Lox-1 recognition by NadA (see below), in the second part of my PhD I focused my attention to the characterization of the NadA binding to monocytes in order to elucidate the contribution of this N. meningitidis antigen in this step of bacteria pathogenesis. As anticipated, the protein microarray identified three different monocytic receptors involved in the NadA binding: Siglec 5, Siglec 14 and FcγR2A Here we report evidence of a novel interaction with a N. meningitidis surface protein suggesting a model in which meningococcus sialic acid acts as tethering factor whereas NadA establish a stronger adhesive interaction. The putative interactions were further corroborated by cellular binding assays and by biophysical and functional studies with the soluble recombinant proteins. Recently, we suggested an additional role for NadA at the host peripheral microvasculature and at the blood brain barrier through the identification of the interaction with Lox-1. In this work we confirm the interaction with biophysical and functional studies. In spite of the strong biochemical evidence of the NadA/Lox-1 binding, the biological outcome of this interaction has not been approached. In order to fill this gap, I spent three months of my PhD at X. Nassif laboratory in Paris. Overall, the results I obtained confirm the master role of type IV pili for the adhesion to endothelial cells but also highlight a role for NadA in this process. All considered, NadA is able to interact with all three cell types involved in the bacterium cycle of infection suggesting unknown and multifaceted roles for this protein in the complex multi-step mechanism of MenB pathogenesis
2016
Benucci, B. (2016). Role of Neisseria meningitidis Adhesin A (NadA) in bacteria pathogenesis.
Benucci, Barbara
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1003521
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