These early FRET-based studies of bacterial exotoxins focused on understanding the intermolecular interactions between toxin subunits rather than between toxins and their host targets. Another timely and ambitious application of FRET has been to examine the cellular signaling pathways underpinning the interaction of bacterial pathogens with both phagocytic and non-phagocytic host cells.
outer membrane protein invasin acts as a high affinity ligand for cellular β1
-family integrins, transmembrane receptors involved in the formation of multi-protein structures termed focal adhesions that link the extracellular matrix to the intracellular cytoskeleton. Invasin-mediated clustering of integrins triggers host signaling on the cytoplasmic face of the plasma membrane. This requires the activation of the small GTPase Rac1, which subsequently binds downstream adaptors that promote cytoskeletal rearrangements and bacterial internalization. Ralph Isberg's laboratory demonstrated that Rac1 was activated at the site of internalization by normally non-phagocytic cells (Figure ), detecting the presence of activated CFP-Rac1 by its ability to bind to a YFP-labeled domain of its downstream adaptor PAK1 [4
]. Although Yersinia
is capable of entry into non-phagocytic cells, the bacterium paradoxically also utilizes a specialized (type III) secretion system to translocate effector proteins that prevent its uptake by phagocytic immune cells. Isberg and co-workers used FRET to show that the concerted action of two effectors, YopE, which suppresses Rac1 activation, and YopT, which alters Rac1 membrane localization, generated two spatially distinct Rac1 populations, an active pool in the nucleus and an inactive pool in the cytoplasm, leading to cellular paralysis. Thus, FRET, being uniquely suited to investigating the location and activation state of host molecules, was integral to uncovering this multifaceted manipulation of GTPase-dependent signaling [5
Figure 2 Yersinia pseudotuberculosis binding to host cells leads to local activation of Rac1 GTPase. COS1 cells, expressing mCFP-Rac1 and mYFP fused to the p21 binding domain of Pak1 (PBD) were incubated with an effector-deficient Y. pseudotuberculosis for 20 (more ...)
FRET can be combined with live cell imaging to reveal not only detailed interactions critical to manipulation of host cells by bacteria, as described above, but also the kinetics of those interactions, thereby establishing a specific sequence of events. Such a study resulted from a collaboration between the laboratories of Pascale Cossart and Joel Swanson, who combined FRET with live cell imaging to document the kinetics of signaling during Listeria
cell entry [6
]. Listeria monocytogenes
employs outer membrane proteins termed internalins to hijack Rac1-dependent receptor-mediated endocytosis. Internalin B (InlB) binds the hepatocyte growth factor receptor (HGFR/c-Met) to stimulate actin reorganization. They used FRET to investigate the activation of two host factors critical for the entry process, Rac1 and phosphoinositide (PI)-3-kinase, which generates the bioactive signaling lipids phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3]. Rac1 activation was monitored similarly to that described above. To monitor activation of PI-3-kinase, the investigators co-expressed YFP and CFP derivatives of the pleckstrin-homology (PH) domain of the serine/threonine kinase Akt, which specifically interacts with these phosphoinositides. Bacterial attachment resulted in a localized FRET signal, as PI(3,4)P2 and PI(3,4,5)P3 were sufficiently concentrated at the plasma membrane to permit a FRET signal between the bound Akt PH domains. Their kinetic analysis of living cells showed that activation of PI3-kinase and concomitant generation of 3'-phosphoinositides at bacterial entry sites occurs upstream of Rac1 activation, which in turn is critical for F-actin assembly. Thus, FRET enabled both the spatial and temporal mapping of lipid- and protein-based signaling at the plasma membrane.
Most recently, in their study in BMC Biology
], Hauck and co-workers combined live fluorescence microscopy and FRET techniques to study events triggered by the association of Neisseria gonorrhoeae
surface (Opa) proteins with the mammalian transmembrane receptor CEACAM3. Uptake of CEACAM3-bound bacteria depends on an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence within the cytoplasmic domain of the receptor, which is rapidly phosphorylated upon ligand binding. This is engaged by multiple host signaling proteins that contain a Src-homology 2 (SH2) domain, including the Src-family kinase (SFK) Hck. Using acceptor photobleaching FRET, as well as other approaches, Hauck and colleagues demonstrated that Hck and CEACAM3 transiently but directly interact specifically at sites of bacterial attachment [7
]. Although the interaction of these two mammalian signaling proteins was predicted from earlier biochemical studies, this investigation confirmed the prediction and also revealed the dynamic nature of the association in living cells.