RESUMEN
Introduction: Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in Coxiella- infected cells remained unclear. Methods: We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth. Results: The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication. Discussion: Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.
Asunto(s)
Coxiella burnetii , Endosomas , Interacciones Huésped-Patógeno , Vacuolas , Proteínas de Unión al GTP rab , Coxiella burnetii/metabolismo , Coxiella burnetii/crecimiento & desarrollo , Coxiella burnetii/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Vacuolas/metabolismo , Vacuolas/microbiología , Células HeLa , Endosomas/metabolismo , Endosomas/microbiología , Fiebre Q/microbiología , Fiebre Q/metabolismoRESUMEN
Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.
Asunto(s)
Legionella pneumophila , Lisosomas , Macrófagos , Fagosomas , Proteínas de Unión al GTP rab , Proteínas de Unión a GTP rab7 , Legionella pneumophila/metabolismo , Legionella pneumophila/genética , Animales , Proteínas de Unión al GTP rab/metabolismo , Ratones , Fagosomas/metabolismo , Fagosomas/microbiología , Lisosomas/metabolismo , Lisosomas/microbiología , Macrófagos/microbiología , Macrófagos/metabolismo , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Sumoilación , Ratones Endogámicos C57BL , Endosomas/metabolismo , Endosomas/microbiologíaRESUMEN
Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.
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Células Epiteliales , Mastitis Bovina , Fagocitosis , Infecciones Estafilocócicas , Staphylococcus aureus , Proteínas de Unión al GTP rab , Animales , Bovinos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Staphylococcus aureus/fisiología , Femenino , Células Epiteliales/microbiología , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/microbiología , Mastitis Bovina/microbiología , Glándulas Mamarias Animales/microbiología , Endosomas/metabolismo , Endosomas/microbiología , Lisosomas/metabolismo , Lisosomas/microbiología , Línea Celular , Fagosomas/microbiologíaRESUMEN
Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.
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Endosomas , Membranas Intracelulares , Lisosomas , Macrófagos , Gránulos de Estrés , Humanos , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Endosomas/microbiología , Endosomas/patología , Membranas Intracelulares/metabolismo , Membranas Intracelulares/microbiología , Membranas Intracelulares/patología , Lisosomas/metabolismo , Lisosomas/microbiología , Lisosomas/patología , Mycobacterium tuberculosis/metabolismo , Gránulos de Estrés/metabolismo , Técnicas In Vitro , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patologíaRESUMEN
Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endosomas/enzimología , Flavoproteínas/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/enzimología , Macrófagos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Vacuolas/enzimología , Animales , Proteínas Bacterianas/genética , Células COS , Chlorocebus aethiops , Endocitosis , Endosomas/genética , Endosomas/microbiología , Evolución Molecular , Femenino , Flavoproteínas/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crecimiento & desarrollo , Enfermedad de los Legionarios/microbiología , Macrófagos/microbiología , Ratones , Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Células U937 , Vacuolas/genética , Vacuolas/microbiología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.
Asunto(s)
Factor 6 de Ribosilación del ADP/metabolismo , Brucella abortus/fisiología , Brucelosis/metabolismo , Brucelosis/microbiología , Interacciones Huésped-Patógeno , Vacuolas/microbiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Brucelosis/inmunología , Endosomas/metabolismo , Endosomas/microbiología , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Ratones , Modelos Biológicos , Unión Proteica , Transporte de Proteínas , Sistemas de Secreción Tipo IV , Red trans-GolgiRESUMEN
Large volumes of liquid and other materials from the extracellular environment are internalised by eukaryotic cells via an endocytic process called macropinocytosis. It is now recognised that this fundamental and evolutionarily conserved pathway is hijacked by numerous intracellular pathogens as an entry portal to the host cell interior. Yet, an increasing number of additional cellular functions of macropinosomes in pathologic processes have been reported beyond this role for fluid internalisation. It emerges that the identity of macropinosomes can vary hugely and change rapidly during their lifetime. A deeper understanding of this important multi-faceted compartment is based on novel methods for their investigation. These methods are either imaging-based for the tracking of macropinosome dynamics, or they provide the means to extract macropinosomes at high purity for comprehensive proteomic analyses. Here, we portray these new approaches for the investigation of macropinosomes. We document how these method developments have provided insights for a new understanding of the intracellular lifestyle of the bacterial pathogens Shigella and Salmonella. We suggest that a systematic complete characterisation of macropinosome subversion with these approaches during other infection processes and pathologies will be highly beneficial for our understanding of the underlying cellular and molecular processes.
Asunto(s)
Disentería Bacilar/microbiología , Endosomas/microbiología , Interacciones Huésped-Patógeno , Infecciones por Salmonella/microbiología , Salmonella/patogenicidad , Shigella/patogenicidad , HumanosRESUMEN
The antimicrobial peptide LL-37 inhibits the growth of the major human pathogen Mycobacterium tuberculosis (Mtb), but the mechanism of the peptide-pathogen interaction inside human macrophages remains unclear. Super-resolution imaging techniques provide a novel opportunity to visualize these interactions on a molecular level. Here, we adapt the super-resolution technique of stimulated emission depletion (STED) microscopy to study the uptake, intracellular localization and interaction of LL-37 with macrophages and virulent Mtb. We demonstrate that LL-37 is internalized by both uninfected and Mtb infected primary human macrophages. The peptide localizes in the membrane of early endosomes and lysosomes, the compartment in which mycobacteria reside. Functionally, LL-37 disrupts the cell wall of intra- and extracellular Mtb, resulting in the killing of the pathogen. In conclusion, we introduce STED microscopy as an innovative and informative tool for studying host-pathogen-peptide interactions, clearly extending the possibilities of conventional confocal microscopy.
Asunto(s)
Catelicidinas/metabolismo , Catelicidinas/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos , Pared Celular/microbiología , Células Cultivadas , Endosomas/microbiología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Lisosomas/microbiología , Macrófagos/microbiología , MicroscopíaRESUMEN
The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation of SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Endosomas/metabolismo , Endosomas/microbiología , Células HeLa , Humanos , Lisosomas/metabolismo , Lisosomas/microbiología , ARN Interferente PequeñoRESUMEN
Salmonella enterica is an important gastrointestinal and facultative intracellular pathogen. After invasion of host cells, it resides in a specialized, replication-permissive compartment, the Salmonella-containing vacuole (SCV). During maturation of the SCV, Salmonella remodels the host endosomal system to form a variety of membranous extensions from the SCV, one type designated Salmonella-induced filaments (SIFs). It was long unclear how Salmonella is able to sustain replication within the SCV, thought to be a nutrient-poor environment. Recent studies started to characterize the metabolic pathways used by intracellular Salmonella. Besides, new insights into the ultrastructure and biogenesis of SIFs and their essential role in nutrition were obtained lately. Here, we review the recent progress with focus on observations gained by various cellular models.
Asunto(s)
Metabolismo Energético/fisiología , Salmonella typhimurium/metabolismo , Vacuolas/microbiología , Proteínas Bacterianas/metabolismo , Microambiente Celular/fisiología , Endosomas/microbiología , Células Epiteliales/microbiología , Humanos , Macrófagos/microbiología , Salmonella typhimurium/crecimiento & desarrollo , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1-positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well-characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.
Asunto(s)
Endosomas/microbiología , Endosomas/fisiología , Escherichia coli Uropatógena/fisiología , Actinas/metabolismo , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Citoesqueleto/fisiología , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Femenino , Humanos , Ratones Endogámicos C57BL , Transducción de Señal , Vejiga Urinaria , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Evasión Inmune , Monocitos , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Endosomas/inmunología , Endosomas/microbiología , Endosomas/patología , Humanos , Monocitos/inmunología , Monocitos/microbiología , Monocitos/patología , Infecciones por Salmonella/patología , Proteínas de Unión al GTP rab/inmunología , Proteína de Unión al GTP rab3A/inmunologíaRESUMEN
AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.
Asunto(s)
Toxinas Bacterianas/metabolismo , Ciclofilina A/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Metaloproteasas/metabolismo , Peptidil-Prolil Isomerasa F/metabolismo , Photobacterium/metabolismo , Animales , Células Cultivadas , Endocitosis , Endosomas/metabolismo , Endosomas/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Masculino , Ratones Endogámicos C57BL , Photobacterium/patogenicidad , VirulenciaRESUMEN
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, ß1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/microbiología , Membrana Celular/patología , Endosomas/microbiología , Endosomas/patología , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/patología , Células HeLa , HumanosRESUMEN
Macrophages can internalize the invading pathogens by raft/caveolae and/or clathrin-dependent endocytosis and elicit an immune response against infection. However, the molecular mechanism for macrophage endocytosis remains elusive. Here we report that LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains) is required for caveolae-mediated endocytosis. Lapf-deficient macrophages have impaired capacity to endocytose and eliminate bacteria. Macrophage-specific Lapf-deficient mice are more susceptible to Escherichia coli (E. coli) infection with higher bacterial loads. Moreover, Lapf deficiency impairs TLR4 endocytosis, resulting in attenuated production of TLR-triggered proinflammatory cytokines. LAPF is localized to early endosomes and interacts with caveolin-1. Phosphorylation of LAPF by the tyrosine kinase Src is required for LAPF-Src-Caveolin complex formation and endocytosis and elimination of bacteria. Collectively, our work demonstrates that LAPF is critical for endocytosis of bacteria and induction of inflammatory responses, suggesting that LAPF and Src could be potential targets for the control of infectious diseases.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caveolina 1/metabolismo , Endocitosis/inmunología , Infecciones por Escherichia coli/inmunología , Macrófagos/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Caveolina 1/inmunología , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endosomas/inmunología , Endosomas/metabolismo , Endosomas/microbiología , Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Inmunidad Innata , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Noqueados , Cultivo Primario de Células , Familia-src Quinasas/inmunología , Familia-src Quinasas/metabolismoRESUMEN
Intracellular pathogens often exploit RAB functions to establish a safe haven in which to survive and proliferate. Ehrlichia chaffeensis, an obligatory intracellular bacterium, resides in specialized membrane-bound inclusions that have early endosome-like characteristics, e.g., resident RAB5 GTPase and RAB5 effectors, including VPS34 (the catalytic subunit of class III phosphatidylinositol 3-kinase), but the inclusions lack late endosomal or lysosomal markers. Within inclusions, Ehrlichia obtains host-derived nutrients by inducing RAB5-regulated autophagy using Ehrlichia translocated factor-1 deployed by its type IV secretion system. This manipulation of RAB5 by a bacterial molecule offers a simple strategy for Ehrlichia to avoid destruction in lysosomes and obtain nutrients, membrane components, and a homeostatic intra-host-cell environment in which to grow.
Asunto(s)
Muerte Celular Autofágica , Ehrlichia chaffeensis/fisiología , Ehrlichiosis/metabolismo , Interacciones Huésped-Parásitos/fisiología , Sistemas de Secreción Tipo IV/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Ehrlichiosis/patología , Endosomas/metabolismo , Endosomas/microbiología , Humanos , Lisosomas/metabolismo , Lisosomas/microbiologíaRESUMEN
Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin ß1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin ß1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin ß1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.
Asunto(s)
Células Epiteliales/microbiología , Mycoplasma hyopneumoniae/patogenicidad , Neumonía Porcina por Mycoplasma/microbiología , Animales , Membrana Celular/metabolismo , Membrana Celular/microbiología , Endosomas/metabolismo , Endosomas/microbiología , Células Epiteliales/metabolismo , Fibronectinas/metabolismo , Integrina beta1/metabolismo , Neumonía Porcina por Mycoplasma/metabolismo , PorcinosRESUMEN
Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.
Asunto(s)
Toxinas Bacterianas/metabolismo , Endocitosis , Células Epiteliales/microbiología , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Interacciones Huésped-Patógeno , Listeria monocytogenes/fisiología , Proteínas de la Membrana/análisis , Proteoma/análisis , Endosomas/metabolismo , Endosomas/microbiología , Células HeLa , Humanos , Listeria monocytogenes/metabolismo , Lisosomas/metabolismo , Lisosomas/microbiologíaRESUMEN
SipA is a major effector of Salmonella, which causes gastroenteritis and enteric fever. Caspase-3 cleaves SipA into two domains: the C-terminal domain regulates actin polymerization, whereas the function of the N terminus is unknown. We show that the cleaved SipA N terminus binds and recruits host Syntaxin8 (Syn8) to Salmonella-containing vacuoles (SCVs). The SipA N terminus contains a SNARE motif with a conserved arginine residue like mammalian R-SNAREs. SipAR204Q and SipA1-435R204Q do not bind Syn8, demonstrating that SipA mimics a cognate R-SNARE for Syn8. Consequently, Salmonella lacking SipA or that express the SipA1-435R204Q SNARE mutant are unable to recruit Syn8 to SCVs. Finally, we show that SipA mimicking an R-SNARE recruits Syn8, Syn13, and Syn7 to the SCV and promotes its fusion with early endosomes to potentially arrest its maturation. Our results reveal that SipA functionally substitutes endogenous SNAREs in order to hijack the host trafficking pathway and promote Salmonella survival.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endosomas/metabolismo , Interacciones Huésped-Patógeno , Fusión de Membrana , Proteínas de Microfilamentos/metabolismo , Proteínas Qa-SNARE/metabolismo , Salmonella/fisiología , Proteínas Bacterianas/genética , Endosomas/microbiología , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Proteínas Qa-SNARE/genéticaRESUMEN
Many pathogenic intracellular bacteria manipulate the host phago-endosomal system to establish and maintain a permissive niche. The fate and identity of these intracellular compartments is controlled by phosphoinositide lipids. By mechanisms that have remained undefined, a Francisella pathogenicity island-encoded secretion system allows phagosomal escape and replication of bacteria within host cell cytoplasm. Here we report the discovery that a substrate of this system, outside pathogenicity island A (OpiA), represents a family of wortmannin-resistant bacterial phosphatidylinositol (PI) 3-kinase enzymes with members found in a wide range of intracellular pathogens, including Rickettsia and Legionella spp. We show that OpiA acts on the Francisella-containing phagosome and promotes bacterial escape into the cytoplasm. Furthermore, we demonstrate that the phenotypic consequences of OpiA inactivation are mitigated by endosomal maturation arrest. Our findings suggest that Francisella, and likely other intracellular bacteria, override the finely tuned dynamics of phagosomal PI(3)P in order to promote intracellular survival and pathogenesis.