Autophagosomes form at ER-mitochondria contact sites.

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate--the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER-mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER-mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER-mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER-mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER-mitochondria contact site is important in autophagosome formation.

Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway.

Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well-known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11- and RalA-positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell-to-cell spreading.

Endocytic recycling in yeast is regulated by putative phospholipid translocases and the Ypt31p/32p-Rcy1p pathway.

Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3Delta crf1Delta background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1Delta mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p-Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.

Entry of Porphyromonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment.

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

Porphyromonas gingivalis outer membrane vesicles enter human epithelial cells via an endocytic pathway and are sorted to lysosomal compartments.

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis.

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae.

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast.

Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.

Cell entry and exit by periodontal pathogen via recycling pathway.

In the oral cavity, gingival epithelial cell (GEC) layers function as an innate host defense system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well-known periodontal pathogen, can enter GECs and pass through the epithelial barrier into deeper tissues. An intracellular location is considered advantageous for bacteria to escape from immune surveillance by the host as well as antibiotic pressure, leading to intracellular persistence, multiplication and dissemination to adjacent tissues. P. gingivalis are invaginated by gingival epithelial cells via the endocytic pathway, and some intracellular bacteria are sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms are sorted to Rab11- and RalA-positive recycling endosomes, followed by bacterial exit from the cells. Exited bacteria can re-enter fresh cells. However, dominant negative forms and RNAi-knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturb the exit of P. gingivalis. These are the first known results to show that the endocytic recycling pathway mediates bacterial exit from infected cells to neighboring cells and may provide important information regarding the exit mechanisms of various invasive pathogens.

Cellular machinery to fuse antimicrobial autophagosome with lysosome.

Autophagy is an intracellular bulk degradation/recycling system that turns over cellular constituents and also functions to degrade intracellular foreign microbial invaders by a process termed xenophagy (antimicrobial autophagy). We previously showed that intracellular group A Streptococcus (GAS) organisms are captured by xenophagosomes, then degraded following fusion with lysosomes. Very recently, we analyzed the molecular mechanism underlying xenophagosome/lysosome fusion and found that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) were involved. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbed autophagic fusion with lysosomes, and cellular bactericidal efficiency was significantly diminished. Furthermore, knockdown of those SNAREs inhibited the fusion of canonical autophagosomes with lysosomes. In addition, important findings showed that Vti1b is derived from autophagic compartments, whereas VAMP8 originates from lysosomes. Together, these results strongly suggest that SNARE proteins Vti1b and VAMP8 mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

Outer membrane vesicles function as offensive weapons in host-parasite interactions.

Outer membrane vesicles (OMVs), ubiquitously shed from Gram-negative bacteria, contain various virulence factors such as toxins, proteases, adhesins, and lipopolysaccharide, which are utilized to establish a colonization niche, modulate host defense and response, and impair host cell function. Thus, OMVs can be considered as a type of bacterial offensive weapon. This review discusses the entry mechanism of OMVs into host cells as well as their etiological roles in host-parasite interactions.

Mediatory molecules that fuse autophagosomes and lysosomes.

Autophagy functions to degrade intracellular foreign microbial invaders by a process that is termed xenophagy (antimicrobial autophagy). Xenophagosomes undergo a stepwise maturation process culminating in a fusion event with lysosomes, after which the cargoes are degraded. Recent investigations by our laboratory demonstrate that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are involved in the fusion between xenophagosomes and lysosomes. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbs the colocalization of LC3 with LAMP-1. We also find that the invasive efficiency of group A Streptococcus into cells is not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency is significantly diminished, indicating that xenophagy is functionally impaired. In addition, knockdown of these SNAREs inhibits the fusion of canonical autophagosomes with lysosomes. Together, these findings indicate that VAMP8 and Vti1b mediate fusion with lysosomes in both antimicrobial and canonical autophagy.

Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes.

Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7, VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluorescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.