The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.     

Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection

Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin dynamics at bacterial internalization sites.     

The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

The carboxyl-terminal SH3 domain of the mammalian adaptor CrkII promotes internalization of Listeria monocytogenes through activation of host phosphoinositide 3-kinase

The intracellular bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to its host receptor, the Met Receptor Tyrosine Kinase. InlB-induced activation of Met stimulates host signal transduction pathways that culminate in cell surface changes driving pathogen engulfment. One mammalian protein with the potential to couple Met to downstream signalling is the adaptor CrkII. CrkII contains an unusual carboxyl-terminal SH3 domain (SH3C) that promotes entry of Listeria. However, binding partners or downstream effectors of SH3C remain unknown. Here, we use RNA interference and overexpression studies to demonstrate that SH3C affects bacterial uptake, at least in part, through stimulation of host phosphatidylinositide (PI) 3-kinase. Experiments with latex beads coated with InlB protein indicated that one potential role of SH3C and PI 3 kinase is to promote changes in the F-actin cytoskeleton necessary for particle engulfment. Taken together, our results indicate that the CrkII SH3C domain engages a cellular ligand that regulates PI 3 kinase activity and host cell surface rearrangements.     

Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to meningitis or abortion. Listeria provokes its internalization ('entry') into mammalian cells that are normally non-phagocytic, such as intestinal epithelial cells and hepatocytes. Entry provides access to a nutrient-rich cytosol and allows translocation across anatomical barriers. Here I discuss the two major internalization pathways used by Listeria. These pathways are initiated by binding of the bacterial surface proteins InlA or InlB to their respective host receptors, E-cadherin or Met. InlA mediates traversal of the intestinal barrier, whereas InlB promotes infection of the liver. At the cellular level, both InlA- and InlB-dependent entry require host signalling that promotes cytoskeletal rearrangements and pathogen engulfment. However, many of the specific signalling proteins in the two entry routes differ. InlA-mediated uptake uses components of adherens junctions that are coupled to F-actin and myosin, whereas InlB-dependent entry involves cytosolic adaptors that bridge Met to regulators of F-actin, including phosphoinositide 3-kinase and activators of the Arp2/3 complex. Unexpectedly, entry directed by InlB also involves endocytic components. Future work on InlA and InlB will lead to a better understanding of virulence, and may also provide novel insights into the normal biological functions of E-cadherin and Met.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes

Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin post-translational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrin-independent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.     

gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.     

Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta

Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.     

Involvement of CD44v6 in InlB-dependent Listeria invasion

Listeria monocytogenes , a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.     

Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB-phagosomes

Internalization of Listeria monocytogenes into non-phagocytic cells is mediated by the interactions between the two bacterial invasion proteins InlA (internalin) and InlB and their cellular surface receptors E-cadherin and c-Met. To get an insight into all the cellular components necessary for uptake and early intracellular life, we undertook a global proteomic characterization of the early listerial phagosome in the human epithelial cell line LoVo. First, we proceeded to an immunocytochemical characterization of intracellular marker recruitment to phagosomes containing latex beads coated with InlA or InlB. E-cadherin and c-Met were, as expected, rapidly recruited to the phagosomal formation site. Phagosomes subsequently acquired the early endosomal antigen 1 (EEA1) and the lysosomal-associated membrane protein 1 (LAMP1), while presenting a more delayed enrichment of the lysosomal hydrolase cathepsin D. Early phagosomes devoid of lysosomal, endoplasmic reticulum and Golgi enzymatic activities could then be isolated by subcellular fractionation of LoVo cells. Two-dimensional gel electrophoresis (2DPAGE) revealed differences between the protein profiles of InlA- or InlB-phagosomes and those of early/late endosomes or lysosomes of naive LoVo cells. One major protein specifically recruited on the InlB-phagosomes was identified by mass spectrometry as MSF, a previously reported member of the septin family of GTPases. MSF forms filaments that co-localize with the actin cytoskeleton in resting cells and it is recruited to the entry site of InlB-coated beads. These results suggest that MSF is a putative effector of the InlB-mediated internalization of L. monocytogenes into host cells.     

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.     

Phosphatidylinositol 4-kinase IIIβ is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.     

Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells

The bacterial pathogen Listeria monocytogenes uses the surface protein InlB to invade a variety of cell types. The interaction of InlB with the hepatocyte growth-factor receptor, Met, is crucial for infection to occur. Remarkably, the ubiquitin ligase Cbl is rapidly recruited to InlB-activated Met. Recent studies have shown that ligand-dependent endocytosis of Met and other receptor tyrosine kinases is triggered by monoubiquitination of the receptor, a process that is mediated by Cbl. Here, we show that purified InlB induces the Cbl-dependent monoubiquitination and endocytosis of Met. We then demonstrate that the bacterium exploits the ubiquitin-dependent endocytosis machinery to invade mammalian cells. First, we show that L. monocytogenes colocalizes with Met, EEA1, Cbl, clathrin and dynamin during entry. Then, we assess the role of different proteins of the endocytic machinery during L. monocytogenes infection. Over-expression or down-regulation of Cbl, respectively, increases or decreases bacterial invasion. Furthermore, RNA interference-mediated knock-down of major components of the endocytic machinery (for example, clathrin, dynamin, eps15, Grb2, CIN85, CD2AP, cortactin and Hrs), inhibit bacterial entry, establishing that the endocytic machinery is key to the bacterial internalization process.     

Phosphatidylinositol 3-kinases in carcinoembryonic antigen-related cellular adhesion molecule-mediated internalization of Neisseria gonorrhoeae

Neisseria gonorrhoeae can be internalized by mammalian cells through interactions between bacterial opacity-associated (Opa) adhesins and members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. We examined the role of phosphatidylinositol 3-kinases (PI3Ks) in gonococcal invasion of epithelial cell lines expressing either CEACAM1 or CEACAM3. CEACAM3-mediated internalization, but not that mediated by CEACAM1, was accompanied by localized and transient accumulation of the class I PI3K product phosphatidylinositol 3,4,5-trisphosphate at sites of bacterial engulfment. Inhibition of phosphatidylinositol 3-kinases reduced CEACAM3-mediated uptake but, paradoxically, led to an increase in intracellular survival of bacteria internalized via either CEACAM1 or CEACAM3, suggesting additional roles for PI3K products. Consistent with this finding, the class III PI3K product phosphatidylinositol 3-phosphate accumulated and persisted in the membrane of gonococcal phagosomes after internalization. Inhibition of PI3K blocked phagosomal acquisition of the late endosomal marker lysosome-associated membrane protein 2 and reduced phagosomal acidification. Inhibiting phagosomal acidification with concanamycin A also increased survival of intracellular gonococci. These results suggest two modes of action of phosphatidylinositol 3-kinases during internalization of gonococci: synthesis of phosphatidylinositol 3,4,5-trisphosphate is important for CEACAM3-mediated uptake, while phosphatidylinositol 3-phosphate is needed for phagosomal maturation and acidification, which are required for optimal bacterial killing.     

InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin

Internalization of Listeria monocytogenes into non-phagocytic cells is tightly controlled by host cell actin dynamics and cell membrane alterations. However, knowledge about the impact of phosphatidylcholine cleavage driven by host cell phospholipase D (PLD) on Listeria internalization into epithelial cells is limited. Here, we report that L. monocytogenes activates PLD in Vero cells during the internalization. With immunostaining it was shown that both PLD1 and PLD2 surrounded partially or completely the phagocytic cup of most L. monocytogenes. Either up- or down-regulation of PLD expression (activity) diminished Listeria internalization. Both PLD1 and PLD2 in Vero cells were required for efficient Listeria internalization, and could substitute for each other in the regulation of Listeria internalization. Further, exogenous InlB activated host cell PLD1 and PLD2 via the Met receptor, and restored host PLD activation by InlB-deficient L. monocytogenes. InlB-induced PLD activation and Listeria internalization were tightly controlled by phospho-cycling of cofilin. PLD1, but not PLD2, was involved in cofilin-mediated PLD activation and Listeria internalization. These data indicate that cofilin-dependent PLD activation induced by InlB may represent a novel regulation mechanism for efficient Listeria internalization into epithelial cells.