Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.     

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.     

Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.     

Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells

Although human gingival epithelium prevents intrusions by periodontal bacteria, Porphyromonas gingivalis, the most well‐known periodontal pathogen, is able to invade gingival epithelial cells and pass through the epithelial barrier into deeper tissues. We previously reported that intracellular P. gingivalis exits from gingival epithelial cells via a recycling pathway. However, the underlying molecular process remains unknown. In the present study, we found that the pathogen localized in early endosomes recruits VAMP2 and Rab4A. VAMP2 was found to be specifically localized in early endosomes, although its localization remained unclear in mammalian cells. A single transmembrane domain of VAMP2 was found to be necessary and sufficient for localizing in early endosomes containing P. gingivalis in gingival epithelial cells. VAMP2 forms a complex with EXOC2/Sec5 and EXOC3/Sec6, whereas Rab4A mediates dissociation of the EXOC complex followed by recruitment of RUFY1/Rabip4, Rab4A effector, and Rab14. Depletion of VAMP2 or Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells. It is suggested that these novel dynamics allow P. gingivalis to exploit fast recycling pathways promoting further bacterial penetration of gingival tissues.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

The recycling endosome and bacterial pathogens

Bacterial pathogens have developed a wide range of strategies to survive within human cells. A number of pathogens multiply in a vacuolar compartment, whereas others can rupture the vacuole and replicate in the host cytosol. A common theme among many bacterial pathogens is the use of specialised secretion systems to deliver effector proteins into the host cell. These effectors can manipulate the host's membrane trafficking pathways to remodel the vacuole into a replication‐permissive niche and prevent degradation. As master regulators of eukaryotic membrane traffic, Rab GTPases are principal targets of bacterial effectors. This review highlights the manipulation of Rab GTPases that regulate host recycling endocytosis by several bacterial pathogens, including Chlamydia pneumoniae, Chlamydia trachomatis, Shigella flexneri, Salmonella enterica serovar Typhimurium, Uropathogenic Escherichia coli, and Legionella pneumophila. Recycling endocytosis plays key roles in a variety of cellular aspects such as nutrient uptake, immunity, cell division, migration, and adhesion. Though much remains to be understood about the molecular basis and the biological relevance of bacterial pathogens exploiting Rab GTPases, current knowledge supports the notion that endocytic recycling Rab GTPases are differentially targeted to avoid degradation and support bacterial replication. Thus, future studies of the interactions between bacterial pathogens and host endocytic recycling pathways are poised to deepen our understanding of bacterial survival strategies.     

TLR sorting by Rab11 endosomes maintains intestinal epithelial‐microbial homeostasis

Compartmentalization of Toll‐like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell‐intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild‐type controls, germ‐free Rab11a‐deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host‐microbial homeostasis at least partially via sorting TLRs.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Rabs and the exocyst in ciliogenesis, tubulogenesis and beyond

The exocyst, an octameric protein complex mediating vesicle tethering at the plasma membrane for exocytosis, is a downstream effector of the Rab proteins Rab8 and Rab11, which are key regulators of membrane trafficking from the trans-Golgi network and recycling endosome to the plasma membrane. Rab11 and Rab8 coordinate their actions via Rabin8, the guanine nucleotide exchange factor of Rab8. A cascade of protein-protein interactions involving the Rabs and the exocyst complex couples the generation of secretory vesicles at donor compartments to their docking and fusion at the plasma membrane. Here, we discuss recent work implicating Rab proteins and the exocyst in primary ciliogenesis and epithelial lumenogenesis. In addition, we discuss early work in the budding yeast Saccharomyces cerevisiae, which provided the initial insight into the molecular mechanisms of polarized exocytosis.     

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.     

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α₂-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (k_ex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (k_en). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing k_ex 30–40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.     

The exocyst is required for melanin exocytosis from melanocytes and transfer to keratinocytes

Skin pigmentation involves the production of the pigment melanin by melanocytes, in melanosomes and subsequent transfer to keratinocytes. Within keratinocytes, melanin polarizes to the apical perinuclear region to form a protective cap, shielding the DNA from ultraviolet radiation‐induced damage. Previously, we found evidence to support the exocytosis by melanocytes of the melanin core, termed melanocore, followed by endo/phagocytosis by keratinocytes as a main form of transfer, with Rab11b playing a key role in the process. Here, we report the requirement for the exocyst tethering complex in melanocore exocytosis and transfer to keratinocytes. We observed that the silencing of the exocyst subunits Sec8 or Exo70 impairs melanocore exocytosis from melanocytes, without affecting melanin synthesis. Moreover, we confirmed by immunoprecipitation that Rab11b interacts with Sec8 in melanocytes. Furthermore, we found that the silencing of Sec8 or Exo70 in melanocytes impairs melanin transfer to keratinocytes. These results support our model as melanocore exocytosis from melanocytes is essential for melanin transfer to keratinocytes and skin pigmentation and suggest that the role of Rab11b in melanocore exocytosis is mediated by the exocyst.     

Host microtubule plus‐end binding protein CLASP1 influences sequential steps in the Trypanosoma cruzi infection process

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin‐dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus‐end tracking proteins (+TIPs) in the co‐ordination of T. cruzi trypomastigote internalization and post‐entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium‐regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1‐depleted cells. These post‐entry phenotypes correlate with a generalized impairment of minus‐end directed transport of lysosomes in CLASP1 knock‐down cells and mimic the effects ofdynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1‐dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite‐elicited signalling, host microtubule plus‐end tracking proteins and dynein‐based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.     

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane

The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.     

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP‐binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra‐lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32–39, 2009. © 2008 Wiley‐Liss, Inc.     

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.     

The RAB5‐GEF Function of RIN1 Regulates Multiple Steps During Listeria monocytogenes Infection

Listeria monocytogenes is a food‐borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome‐mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5‐directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1‐mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.