Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

The clathrin endocytic pathway in viral infection.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.     

Alternative infectious entry pathways for dengue virus serotypes into mammalian cells

The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-β-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells.     

Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

Porphyromonas gingivalis fimbriae binds to neoglycoproteins: evidence for a lectin-like interaction

Porphyromonas gingivalis is a likely major pathogen in adult periodontitis. Fimbriae in particular have been suggested as playing an important role in facilitating the initial interaction between the bacteria and the host and triggers host responses. Murakami et al. [Biochem. Biophys. Res. Commun. 192 (1993) 826] have shown that fimbriae of P. gingivalis strongly induced TNF-alpha gene expression in macrophages and expression of TNF-alpha was inhibited by N-acetyl-D-galactosamine, but not inhibited by other sugars. Studies by Sojar et al. [FEBS Lett. 422 (1998) 205] suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction of P. gingivalis fimbriae and human lactoferrin. In the present study, purified fimbriae from P. gingivalis and neoglycoproteins were used to assess lectin-like interaction of fimbriae. In dot blot and overlay assays, iodinated purified P. gingivalis fimbriae as well as biotinylated purified P. gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy-L-fucose) and by lesser extent to albumin-N-acetyl-D-galactosamine (albumin-p-aminophenyl-N-acetyl-beta-D-galactosaminide). However, fimbriae failed to bind carbohydrate free bovine serum albumin, which was used in preparation of the neoglycoproteins. These results suggests that P. gingivalis fimbriae bind to glycoconjugates through lectin-like interaction with carbohydrate. This protein-carbohydrate interactions may be important for triggering events in these cells, which mediate the host response of this pathogen.     

Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle

Porphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.     

Pathways of clathrin-independent endocytosis

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.     

The role of palmitoylation in directing dopamine D1 receptor internalization through selective endocytic routes

We previously determined that D1 receptors can endocytose through caveolae, a subset of lipid rafts, in addition to internalization via a clathrin-dependent pathway. In this report, we investigated the potential role that palmitoylation might have on directing D1 receptor internalization through either a clathrin or caveolar-dependent route. Through whole cell binding analysis and sucrose gradient fractionation studies, we demonstrated that although palmitoylation of the D1 receptor was not required for agonist-independent localization to caveolae, agonist induced internalization kinetics of a de-palmitoylated D1 receptor were accelerated ∼8-fold in comparison to wild-type D1 receptor and were very similar to that observed for clathrin-dependent D1 receptor internalization. Additionally, inhibition of the clathrin mediated pathway led to significant attenuation in the extent of agonist induced internalization of the de-palmitoylated D1 receptor, suggesting the de-palmitoylated D1 receptor was directed to a clathrin-dependent internalization pathway. Taken together, these data suggest that palmitoylation may be involved in directing agonist-dependent D1 receptor internalization through selective endocytic routes.     

The RprY response regulator of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with chronic adult periodontitis. Its ecological niche is the gingival crevice, where the organism adapts to the challenges of the infectious process such as host defence and bacterial products. Bacterial responses to environmental changes are partly regulated by two-component signal transduction systems. Several intact systems were annotated in the genome of P. gingivalis, as well as an orphan regulator encoding a homologue of RprY, a response regulator from Bacteroides fragilis. With the goal of defining the environmental cues that activate RprY in P. gingivalis, we used several strategies to identify its regulon. Results from gene expression and DNA-protein binding assays identified target genes that were either involved in transport functions or associated with oxidative stress, and indicated that RprY can act as an activator and a repressor. RprY positively activated the primary sodium pump, NADH : ubiquinone oxidoreductase (NQR), and RprY protein also interacted with the promoter regions of nqrA genes from B. fragilis and Vibrio cholerae. Given that gingival bleeding and infiltration of host defence cells are symptoms of periodontal infection, iron products released from blood and reactive oxygen species from polymorphonuclear leucocytes may be potential inducers of the RprY regulon.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.     

Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae

Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.     

Auxilin depletion causes self-assembly of clathrin into membraneless cages in vivo

Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an ∼50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an ∼1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2

Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two major alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1] [2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3] [4] [5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1] [5]. SJ170, which differs from SJ145 by the presence of a carboxy-terminal extension, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was previously shown to bind Eps15 [6], a protein involved in receptor-mediated endocytosis. Here, we show that the same domain also binds clathrin and the clathrin adaptor AP-2. These interactions occur both in vitro and in vivo and are direct. Binding of AP-2 is mediated by the ear domain of its alpha-adaptin subunit and binding of clathrin by the amino-terminal domain of its heavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-length SJ170 or its unique carboxy-terminal region caused mislocalization of Eps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transferrin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the regulation of clathrin coat dynamics.     

Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process

Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.     

Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of beta(2)-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.     

Characterization of Human Astrovirus Cell Entry

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.