Alexander Buntru

Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms

ABSTRACT Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization of Neisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms.

Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectin-binding pathogens.

Staphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin α5β1, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin β1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominant-negative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1−/−) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1−/− cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membrane-microdomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.

Phosphatidylinositol 3′-Kinase Activity Is Critical for Initiating the Oxidative Burst and Bacterial Destruction during CEACAM3-mediated Phagocytosis

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing Gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3′-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.

Fingerprinting differential active site constraints of ATPases

The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different ATPases suitable tools are required. Here, we present the synthesis of six new ATP-based ATPase probes modified at three different positions of the nucleobase and the ribose, respectively. Subsequently, we studied the ATPases focal adhesion kinase FAK, the ubiquitin-activating protein UBA1 and the kinesin Eg5 as examples for ATPases that process ATP by different mechanisms. We find that for each of these enzymes at least one position in ATP can be modified without loss of acceptance by the enzyme. However, the positions at which modifications are tolerated significantly differ between the studied enzymes allowing fingerprints to be drawn for reactivity. The introduced ATP analogues may form the basis for the design of tailored probes with increased affinity and specificity for a specific ATPase of interest.

HemITAM signaling by CEACAM3, a human granulocyte receptor recognizing bacterial pathogens.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to the immunoglobulin superfamily and contribute to cell-cell adhesion and signal modulation in various tissues. In humans, several CEACAMs are targeted by pathogenic bacteria. One peculiar member of this family, CEACAM3, is exclusively expressed by human granulocytes and functions as an opsonin-independent phagocytic receptor for CEACAM-binding bacteria. Here, we will discuss CEACAM3-dependent processes by summarizing recent insight into the phosphotyrosine-based signaling complex formed upon CEACAM3 engagement. Compared to different well-studied phagocytic receptors, such as Fcγ receptors and Dectin-1, CEACAM3 appears as an example of a hemITAM-containing innate immune receptor, which promotes rapid internalization and intracellular destruction of a diverse group of CEACAM-binding bacteria. The particular efficiency of CEACAM3 arises from the direct coupling of upstream activators and downstream effectors of the small GTPase Rac by the cytoplasmic domain of CEACAM3, which co-ordinates actin cytoskeleton re-arrangements and bactericidal effector mechanisms of granulocytes.

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

BackgroundBacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells.ResultsLive-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2) domain of Hck (Hck-SH2) was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet)-Hck-SH2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet) and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact.ConclusionThese data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

BackgroundDisease manifestations of Staphylococcus aureus are connected to the fibronectin (Fn)-binding capacity of these Gram-positive pathogens. Fn deposition on the surface of S. aureus allows engagement of α5β1 integrins and triggers uptake by host cells. For several integrin- and actin-associated cytoplasmic proteins, including FAK, Src, N-WASP, tensin and cortactin, a functional role during bacterial invasion has been demonstrated. As reorganization of the actin cytoskeleton is critical for bacterial entry, we investigated whether vinculin, an essential protein linking integrins with the actin cytoskeleton, may contribute to the integrin-mediated internalization of S. aureus.ResultsComplementation of vinculin in vinculin -/- cells, vinculin overexpression, as well as shRNA-mediated vinculin knock-down in different eukaryotic cell types demonstrate, that vinculin does not have a functional role during the integrin-mediated uptake of S. aureus.ConclusionsOur results suggest that vinculin is insignificant for the integrin-mediated uptake of S. aureus despite the critical role of vinculin as a linker between integrins and F-actin.

The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin αMβ2, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

Grb14 Is a Negative Regulator of CEACAM3-mediated Phagocytosis of Pathogenic Bacteria

Background: CEACAM3-mediated phagocytosis is an innate mechanism to eliminate CEACAM-binding pathogenic bacteria. Results: Biochemical analyses and FRET-FLIM imaging reveal the direct binding of the adaptor protein Grb14 to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3, and this interaction limits phagocytosis. Conclusion: Grb14 is a negative regulator of CEACAM3-mediated phagocytosis. Significance: Deciphering CEACAM3 interaction partners illuminates the minimal set of cellular factors controlling phagocytosis. Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact.

Different Enzymatic Processing of γ-Phosphoramidate and γ-Phosphoester-Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.