Internalization of fibronectin-binding S. aureus into mammalian cells via integrins and membrane microdomains

Abstract

Staphylococcus aureus is one of the leading nosocomial pathogens in industrialized countries. It can cause diseases from mild to severe infections by expressing a wide range of virulence factors, including superantigens, toxins and several of adhesins. The fibronectin-binding proteins (FnBPs) play an essential role for the tight adherence of the bacteria to their host cells. The adhesion of the bacteria via FnBPs to integrin α5β1 leads to the recruitment of a focal adhesion like complex and the rearrangement of the actin cytoskeleton resulting in the internalization of S. aureus by non-professional phagocytes. In this study we analyzed, how the bacterial protein FnBP turns the cell adhesion receptor integrin α5β1 into an endocytotic molecule. Therefore, we addressed the structure-function relationship of FnBPs and functionally characterized cellular components, which are involved in the internalization of S. aureus in vitro. Interestingly an accumulation of GM1-enriched membrane microdomains to cell attached S. aureus and also to FnBP-A coupled beads was observed. By using a panel of genetically deficient mouse fibroblasts, we were able to demonstrate that besides integrin β1, none of the characterized integrin/actin-associated proteins was required for MM recruitment. Furthermore, we could determine a crucial role for membrane microdomains in bacterial uptake as disruption of microdomains leads to a dramatic decrease of internalized bacteria. Thereupon we investigated microdomain associated proteins concerning their role in pathogenic uptake and surprisingly we observed enhanced pathogenic internalization in caveolin1 knockout cells suggesting an inhibitory role for caveolin1. Caveolin1 is a major component of a special subtype of microdomains, caveolae, that are highly immobile fractions within the plasma membrane. We could demonstrate that membrane mobility directly correlates with bacterial uptake. By enhancing membrane mobility using different temperatures or reagents, numbers of internalized bacteria increased. Our data foster the assumption that numbers of internalized bacteria increase during the infection process caused by increased body temperature and therefore enhanced membrane mobility. After recruitment of membrane microdomains also a focal adhesion like complex is recruited to cell attached S. aureus. Several proteins have been shown to be important for the uptake process, such as FAK, Src-PTKs and cortactin. Interestingly, vinculin-deficient mouse embryonic fibroblasts showed an increased bacterial uptake. Using a vinculin mutant, which cannot bind to PIP2 anymore we could almost restore numbers of internalized bacteria comparable to vinculin knockout cells. We conclude that through the mutation of PIP2 binding or loss of vinculin at all, the level of PIP2 in cell increases resulting in enhanced bacterial internalization. PIP2 plays not only a role in migration, proliferation and in numerous other signal transduction pathways but also activates several proteins that are known to be involved in the uptake process of S. aureus. Previous in vivo and in vitro studies revealed that S. aureus FnBP alone is necessary and sufficient to drive bacterial uptake into mammalian cells. Recently, we sequenced FnBP from S. aureus Cowan strain and expressed different FnBP-A domains as recombinant fusion proteins. Now, we had a perfect tool, to analyze these domains regarding to their ability of Fn binding, internalization in host cells and adherence to host cells. Furthermore we had now the opportunity to compare the FnBP B-domain of S. aureus Cowan and NCTC 8325 strain. Our results lead to the assumption that in contrast to the B domain of NCTC 8325 strain, the B domain of Cowan strain showed enhanced internalization, adherence and Fn binding, which directly correlates with enhanced invasiveness. Furthermore we could demonstrate that binding of FnBP-A domains to Fn results in a conformational change from a "compact" to an "extended" state exposing ligand binding sites. This is a highly interesting observation as an activation of Fn by virulence factors was not detected until now. In summary the data obtained in this study provide an advanced view on the internalization process of pathogenic S. aureus into mammalian host cells but also for the general process of integrin internalization.