Functional Analysis of Different Streptococcal Invasion Mechanisms and Consequences for Intracellular Survival

The human pathogen S. pyogenes subverts the host immune defense by several strategies including invasion of host cells. The major adhesion and invasion factor SfbI consists of a N-terminal aromatic amino acid rich domain (AroD), a central proline rich domain (ProD) and C-terminal fibronectin (Fn)-binding repeats (FnBR). While the FnBRs and their interaction with Fn to initiate bacterial uptake are well characterized, no information were available for the function of the AroD and the ProD. SfbI triggers integrin-clustering and caveolae-aggregation on the host cell surface subsequently leading to bacterial internalization within large membrane invaginations. SfbI-expressing streptococci avoid the classical intracellular trafficking route which normally leads to fusion of the bacterial-containing compartments with lysosomes and would result in bacterial killing. In the present study it was observed that the AroD as well as the ProD affect the SfbI-mediated uptake process; besides the already described entry a morphological different internalization mechanism which is attended by the formation of membrane protrusions was detectable. Analysis of co-incubation assays from recombinant protein-constructs, latex beads coated with recombinant protein or S. gordonii heterologous expressing distinct SfbI-constructs with host cells revealed that distinct AroD types or lack of the ProD avoid typical SfbI-mediated uptake: integrin-clustering and caveolae-aggregation were prevented; rather cytoskeleton rearrangements and signaling events of classical phagocytic processes were induced. Moreover, bacterial survival is affected by the intracellular trafficking route. AroD might modulate the SfbI protein conformation with consequences for Fn-binding capacity and ProD characteristic. ProD is supposed to modify amount and composition of host cell integrins and, moreover, to prevent the remodulation of the actin cytoskeleton by directly inhibiting Arp2/3 complex dependent actin-branching.