Pathogenic bacteria tightly interact with their host, often exploiting adhesin-mediated engagement of eukaryotic surface receptors to trigger intracellular signaling events [1
]. As bacteria-induced responses are of critical importance during the initiation and progression of the infection, signaling processes in the host cell are usually studied in molecular detail. Both biochemical and genetic approaches have shed light on protein-protein interactions and signaling connections that occur in infected eukaryotic cells. However, widely used biochemical approaches to investigate protein-protein interactions such as glutathione S-transferase (GST)-pull-down assays or coimmunoprecipitation from cell lysates have two major drawbacks: firstly, it is always possible that the two associated proteins are not directly interacting, but rather are linked by a third protein; secondly, biochemical approaches disrupt the cellular context and therefore lack spatial resolution. Similarly, genetic methods such as yeast two-hybrid screens, although applicable even in a high-throughput format, do not provide any information on where these processes occur under physiological conditions at the subcellular level.
By contrast, the introduction of green fluorescent protein (GFP) from Aequorea victoria, has greatly facilitated the microscopic investigation of proteins in living cells. Though the use of GFP and its spectral variants allows the observation of colocalization of multiple proteins in real time, the resolution of light microscopes (about 250 nm) is too low to prove a direct interaction of two colocalized putative binding partners. Therefore, there is a need for methods that combine the power of biochemical studies to pinpoint molecular interactions with the ability to study the subcellular context as provided by fluorescence microscopy.
Within the last few years, the phenomenon of fluorescence resonance energy transfer (FRET), first described by Förster in 1948, has garnered increasing interest as a method to address protein-protein interactions in the context of the cell [2
]. During FRET, energy is transferred from a donor fluorophore in its excited state in a non-radiative way by dipole-dipole interactions to an acceptor molecule [5
]. The efficiency of fluorescence resonance energy transfer is defined by: E = 1/[1 + (r/R0
]. Apparently, the efficiency of energy transfer depends on the sixth power of the distance 'r' separating the donor and the acceptor molecule. Therefore, FRET only takes place to a significant extent if molecules are spaced within a few nm (about 1 to 10 nm) [6
]. The additional parameter 'R0
', called the Förster radius, is defined as the distance where efficiency of energy transfer from donor to acceptor is 50%. R0
is FRET-pair specific and is influenced by the spectral overlap of donor emission and acceptor excitation, the quantum yield of the donor, the absorption coefficient of the acceptor and the relative orientation of donor and acceptor. As a consequence, FRET is only likely to occur if two proteins labeled with a donor and an appropriate acceptor molecule are in direct contact.
To exploit this methodology in the study of pathogen-induced host cell signaling and to provide a general framework on how to approach FRET analysis in the context of receptor-initiated signaling cascades, we have used the example of carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-mediated contact with the Gram-negative pathogen Neisseria gonorrhoeae
. Over the last few years, our group and others have demonstrated that CEACAM3, a granulocyte-expressed member of this receptor family, functions as an opsonin-independent phagocytic receptor [7
]. CEACAM3 recognizes colony opacity associated (Opa) proteins of N. gonorrhoeae
(Ngo) as well as additional outer membrane adhesins of other Gram-negative bacteria and, upon binding of bacteria, initiates an intracellular signaling cascade [9
Efficient uptake of CEACAM3-bound bacteria depends on an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic part of the receptor, which is phosphorylated within minutes of receptor engagement [7
]. Biochemical analyses have demonstrated that Src homology 2 (SH2) domains of several signaling molecules, including the protein tyrosine kinases (PTKs) c-Src and Hck, are able to bind to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 [10
]. As well as the SH2 domain of Src PTKs, the SH2 domains of phosphatidylinositol-3 kinase, phospholipase Cγ, and Syk have also been found to colocalize with the receptor upon bacterial binding [11
]. However, it is unclear if this colocalization is indeed due to direct interaction of the respective signaling molecule with the phosphorylated receptor, or if there are additional molecules involved.
In the present investigation, we took advantage of a novel FRET pair of fluorophores, enhanced cyan fluorescent protein (CyPet) and enhanced yellow fluorescent protein (YPet), which have been recently developed by random mutagenesis and which display enhanced FRET efficiency compared to the often used cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) pair [13
]. Tagging the cytoplasmic domain of CEACAM3 with CyPet and coexpressing a YPet-SH2 domain fusion protein (Figure ), we observed FRET in cell lysates and by flow cytometry in intact cells expressing phosphorylated CEACAM3-CyPet together with YPet-SH2 Hck, but not with YPet-SH2 SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). Importantly, FRET measurements in infected cells using acceptor bleaching revealed the direct interaction between CEACAM3 and Hck exclusively at sites of bacterial contact with host cell. Together, these data highlight the use of FRET approaches to visualize cellular signaling in response to bacterial host cell contact and provide conclusive evidence for a direct interaction between CEACAM3 and Hck upon N. gonorrhoea
Figure 1 Overview of the used constructs. (a) Wild type carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3 WT) comprising an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in its cytoplasmic domain (CT) or CEACAM3 ΔCT (more ...)