The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5ß1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

Controling the cytoskeleton during CEACAM3-mediated phagocytosis.

Phagocytosis, an innate defense mechanism of multicellular animals, is initiated by specialized surface receptors. A phagocytic receptor expressed by human polymorphonuclear granulocytes, the major professional phagocytes in our body, is one of the fastest evolving human proteins implying a special role in human biology. This receptor, CEACAM3, is a member of the CarcinoEmbryonic Antigen-related Cell Adhesion Molecule (CEACAM) family and dedicated to the immediate recognition and rapid internalization of human-restricted pathogens. In this focused contribution, we will review the special adaptations of this protein, which co-evolves with different species of mucosa-colonizing bacteria. While the extracellular Immunoglobulin-variable (IgV)-like domain recognizes various bacterial adhesins, an Immunoreceptor Tyrosine-based Activation Motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 constitutes the central signaling hub to trigger actin rearrangements needed for efficient phagocytosis. A major emphasis of this review will be placed on recent findings, which have revealed the multi-level control of this powerful phagocytic device. As tyrosine phosphorylation and small GTPase activity are central for CEACAM3-mediated phagocytosis, the counterregulation of CEACAM3 activity involves the receptor-type protein tyrosine phosphatase J (PTPRJ) as well as the Rac-GTP scavenging protein Cyri-B. Interference with such negative regulatory circuits has revealed that CEACAM3-mediated phagocytosis can be strongly enhanced. In principle, the knowledge gained by studying CEACAM3 can be applied to other phagocytic systems and opens the door to treatments, which boost the phagocytic capacity of professional phagocytes.