RESUMEN
Individual Atg8 (autophagy related 8) paralogs, comprising MAP1LC3A/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE16, play a crucial role in canonical macroautophagy/autophagy. However, their functions remain unclear owing to functional redundancy. In a previous study, we reported that intracellular Streptococcus pneumoniae triggers hierarchical autophagy in response to bacterial infection. This process commences with the induction of conjugation of Atg8 paralogs (Atg8s) to single membranes (CASM), followed by CASM shedding and subsequent induction of xenophagy. In our recent study, we performed functional analysis of Atg8s during pneumococci-induced hierarchical autophagy. Our findings suggest that LC3A and GABARAPL1 are crucial for CASM induction, whereas GABARAPL2 and GABARAP play sequential roles in CASM shedding and subsequent induction of xenophagy, respectively.Abbreviation: Atg8: autophagy related 8; Atg8s: Atg8 paralogs; CASM: conjugation of Atg8s to single membranes; mpi: minutes post-infection; mpi: minutes post-infection; PcAV: pneumococci-containing autophagic vesicles; PcLV: LC3-associated phagosome (LAPosome)-like vacuole; PcV: pneumococci-containing vesicles; Sp: S. pneumoniae.
RESUMEN
Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia , Streptococcus pneumoniae , Animales , Ratones , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Macroautofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/metabolismo , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/metabolismoRESUMEN
Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.
Asunto(s)
Neuraminidasa , Infecciones Neumocócicas , Humanos , Neuraminidasa/metabolismo , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Background. Streptococcus pneumoniae is a major causative bacteria of pneumonia and invasive pneumococcal disease (IPD); however, the mechanisms underlying its severity and invasion remain to be defined. Pneumococcal colonies exhibit opaque and transparent opacity phase variations, which have been associated with invasive infections and nasal colonization, respectively, in animal studies. This study evaluated the relationship between the opacity of pneumococcal colonies and the clinical presentation of pneumococcal pneumonia.Methods. This retrospective study included adult patients hospitalized with pneumococcal pneumonia between 2012 and 2019 at four tertiary medical institutions. Pneumococcal strains from lower respiratory tract specimens were determined for their serotypes and microscopic colony opacity, and the association between the opacity phase and the severity of pneumonia was evaluated. Serotypes 3 and 37 with mucoid colony phenotypes were excluded from the study because their colony morphologies were clearly different.Results. A total of 92 patients were included. Most patients were older adults (median age: 72 years) and males (67â%), and 59â% had community-acquired pneumonia. Of the 92 patients, 41 (45â%), 12 (13â%), and 39 (42â%) patients had opaque, transparent, and mixed variants in their pneumococcal colony, respectively. The opaque and non-opaque pneumococcal variants had no statistically significant difference in patient backgrounds. Although the pneumonia severity index score did not differ between the opaque and non-opaque groups, the rate of bacteremia was significantly higher in the opaque group than in the non-opaque group. Serotype distribution was similar between the groups.Conclusions. Opaque pneumococcal variants may cause pneumonia and invasive diseases in humans. This study could help elucidate IPD, and opacity assessment may serve as a predictor for IPD.
Asunto(s)
Infecciones Neumocócicas , Neumonía Neumocócica , Animales , Masculino , Humanos , Anciano , Streptococcus pneumoniae , Variación de la Fase , Estudios RetrospectivosRESUMEN
Streptococcus pneumoniae is a major, encapsulated Gram-positive pathogen that causes diseases including community-acquired pneumonia, meningitis, and sepsis. This pathogen colonizes the nasopharyngeal epithelia asymptomatically but can often migrate to sterile tissues and cause life-threatening invasive infections (invasive pneumococcal disease). Although multivalent pneumococcal polysaccharides and conjugate vaccines are available and effective, they also have major shortcomings with respect to the emergence of vaccine-resistant serotypes. Therefore, alternative therapeutic approaches are needed, and the molecular analysis of host-pathogen interactions and their applications to pharmaceutical development and clinical practice has recently received increased attention. In this review, we introduce pneumococcal surface virulence factors involved in pathogenicity and highlight recent advances in our understanding of host autophagy recognition mechanisms against intracellular S. pneumoniae and pneumococcal evasion from autophagy.
Asunto(s)
Infecciones Neumocócicas , Streptococcus pneumoniae , Humanos , Streptococcus pneumoniae/genética , Macroautofagia , Infecciones Neumocócicas/microbiología , Factores de Virulencia , Virulencia , Vacunas NeumococicasRESUMEN
We report 2 adult cases of invasive disease in Japan caused by Streptococcus oralis that expressed the serotype 3 pneumococcal capsule and formed mucoid colonies. Whole-genome sequencing revealed that the identical serotype 3 pneumococcal capsule locus and hyl fragment were recombined into the genomes of 2 distinct S. oralis strains.
Asunto(s)
Infecciones Neumocócicas , Adulto , Humanos , Japón , Vacunas Neumococicas , Serogrupo , Streptococcus oralis/genética , Streptococcus pneumoniae/genéticaRESUMEN
Although the involvement of macroautophagy/autophagy in hepatitis B virus (HBV) infection has become clearer recently, whether selective autophagy plays an important role in suppressing HBV remains uncertain. We recently found that LGALS9 (galectin 9) is an interferon (IFN)-inducible protein involved in the suppression of HBV replication. Expression of LGALS9 in HBV-infected cells causes the formation of cytoplasmic puncta that degrade the HBV core protein (HBc) in conjunction with RSAD2/viperin, another IFN-inducible protein. LGALS9 binds to HBc via RSAD2 and promotes the autoubiquitination of RNF13 (ring finger protein 13) to recruit SQSTM1/p62, resulting in the formation of LC3-positive autophagosomes that degrade HBc. Both LGALS9 and RSAD2 are encoded by IFN-stimulated genes that act synergistically to induce HBc proteolysis in HBV-infected hepatocytes in an IFN-dependent manner. These results reveal a crosstalk mechanism between the innate immune system and selective autophagy during viral infection.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Autofagia , Hepatocitos , Humanos , Sistema Inmunológico , Macroautofagia , Replicación ViralRESUMEN
Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.
Asunto(s)
Galectinas/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Macroautofagia/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Galectinas/genética , Galectinas/metabolismo , Expresión Génica , Células HEK293 , Células Hep G2 , Hepatitis B , Virus de la Hepatitis B/metabolismo , Humanos , Proteolisis , Proteína Sequestosoma-1/genéticaRESUMEN
STREPTOCOCCUS PNEUMONIAE: is an opportunistic bacterial pathogen that can promote severe infection by overcoming the epithelial and blood-brain barrier. Pneumococcal cell-surface virulence factors, including cell wall-anchored choline-binding proteins (Cbps) play pivotal roles in promoting invasive disease. We reported previously that intracellular pneumococci were detected by hierarchical macroautophagic/autophagic processes that ultimately lead to bacterial elimination. However, whether intracellular pneumococci can evade autophagy by deploying Cbps remains unclear. In this study, we explore the biological functions of Cbps and reveal their roles in manipulating the autophagic process. Specifically, we found that CbpC-activated autophagy takes place via its interactions with ATG14 (autophagy related 14) and SQSTM1/p62 (sequestosome1). Importantly, CbpC dampens host autophagy by promoting ATG14 degradation via the ATG14-CbpC-SQSTM1/p62 axis. CbpC-induced reductions in ATG14 levels result in impaired ATG14-STX17 complex formation. In pneumococcal-infected cells, ATG14 levels are dramatically reduced in a CbpC-dependent manner that results in suppression of autophagy-mediated degradation and enhanced bacterial survival. Taken together, our results reveal a novel mechanism via which pneumococci can manipulate host autophagy responses, in this case, by employing CbpC as a trap to promote ATG14 depletion. Our findings highlight a novel and sophisticated tactic used by S. pneumoniae that serves to promote intracellular survival.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Bacterianas/metabolismo , Proteolisis , Streptococcus pneumoniae/fisiología , Células HeLa , Humanos , Macroautofagia , Viabilidad MicrobianaRESUMEN
Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC. Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Proteínas Relacionadas con la Autofagia , Autofagia , Proteínas Bacterianas/metabolismo , Streptococcus pneumoniae , Animales , Proteínas Relacionadas con la Autofagia/genética , Línea Celular , Humanos , Proteínas de la Membrana , Ratones , Streptococcus pneumoniae/genéticaRESUMEN
Multiple autophagic processes are triggered in response to bacterial infection as the host attempts to eliminate intracellular invaders. However, it is still unclear how the mechanisms contributing to canonical macroautophagy/autophagy, including xenophagy, coordinate with the more recently described features that are characteristic of noncanonical autophagy. Recently, we revealed that infection with Streptococcus pneumoniae can trigger the formation of RB1CC1/FIP200-independent LC3-associated phagosome-like vacuoles (PcLVs) that contain the pneumococci at an early stage of infection. We also found that interactions of SQSTM1/p62 with the ATG16L1 WD domain are essential for PcLV formation. Intriguingly, PcLVs were required for the subsequent generation of bactericidal autophagic vacuoles (PcAVs). Furthermore, we also identified LC3-delocalized SQSTM1-positive PcLVs as intracellular intermediates that link PcLVs and PcAVs. These findings reveal a novel multi-step mechanism that contributes to xenophagy of the critical S. pneumoniae respiratory pathogen.
Asunto(s)
Autofagia , Streptococcus pneumoniae , Autofagosomas , Proteínas Bacterianas , FagosomasRESUMEN
In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases.
Asunto(s)
Autofagia , Vesículas Citoplasmáticas/metabolismo , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/fisiología , Animales , Biomarcadores , Línea Celular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Humanos , Ratones , Modelos Biológicos , Infecciones Neumocócicas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.
Asunto(s)
Autofagia , Células Epiteliales/inmunología , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Poliubiquitina/metabolismo , Streptococcus pneumoniae/inmunología , Estreptolisinas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Células Epiteliales/microbiología , Humanos , UbiquitinaciónRESUMEN
The evolutionarily conserved processes of endosome-lysosome maturation and macroautophagy are established mechanisms that limit survival of intracellular bacteria. Similarly, another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report that an intracellular vacuolar pathogen, Legionella dumoffii, is specifically targeted by LAP over classical endocytic maturation and macroautophagy pathways. Upon infection, the majority of L. dumoffii resides in ER-like vacuoles and replicate within this niche, which involves inhibition of classical endosomal maturation. The establishment of the replicative niche requires the bacterial Dot/Icm type IV secretion system (T4SS). Intriguingly, the remaining subset of L. dumoffii transiently acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently undamaged single membrane, and fail to associate with the molecules implicated in selective autophagy, such as ubiquitin or adaptors. The process requires toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an intracellular pathogen, the survival of which in infected cells is limited predominantly by LAP. The results suggest that L. dumoffii is a valuable model organism for examining the mechanistic details of LAP, particularly induced by bacterial infection.
Asunto(s)
Sistemas de Secreción Bacterianos , Legionella/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fagocitosis , Vacuolas/metabolismo , Animales , Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Biomarcadores/metabolismo , Diglicéridos/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Células HEK293 , Células HeLa , Humanos , Espacio Intracelular/microbiología , Legionella/ultraestructura , Legionelosis/enzimología , Legionelosis/patología , Ratones , Viabilidad Microbiana , NADPH Oxidasas/metabolismo , Células RAW 264.7 , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Ubiquitina/metabolismo , Vacuolas/ultraestructuraRESUMEN
Genital mycoplasmas, including Ureaplasma spp., are among the smallest human pathogenic bacteria and are associated with preterm birth. Electron microscopic observation of U. parvum showed that these prokaryotes have a regular, spherical shape with a mean diameter of 146 nm. U. parvum was internalized into HeLa cells by clathrin-mediated endocytosis and survived for at least 14 days around the perinuclear region. Intracellular U. parvum reached endosomes in HeLa cells labeled with EEA1, Rab7, and LAMP-1 within 1 to 3 hr. After 3 hr of infection, U. parvum induced the cytosolic accumulation of galectin-3 and was subsequently entrapped by the autophagy marker LC3. However, when using atg7-/- MEF cells, autophagy was inadequate for the complete elimination of U. parvum in HeLa cells. U. parvum also colocalized with the recycling endosome marker Rab11. Furthermore, the exosomes purified from infected HeLa cell culture medium included U. parvum. In these purified exosomes ureaplasma lipoprotein multiple banded antigen, host cellular annexin A2, CD9, and CD63 were detected. This research has successfully shown that Ureaplasma spp. utilize the host cellular membrane compartments possibly to evade the host immune system.
Asunto(s)
Autofagosomas/inmunología , Autofagia , Células Epiteliales/inmunología , Interacciones Huésped-Patógeno , Ureaplasma/inmunología , Autofagosomas/microbiología , Autofagosomas/ultraestructura , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Células HeLa , Humanos , Evasión Inmune , Microscopía Electrónica , Ureaplasma/ultraestructuraRESUMEN
Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.
Asunto(s)
Autofagia , Burkholderia pseudomallei/fisiología , Espacio Intracelular/microbiología , Viabilidad Microbiana , Neutrófilos/microbiología , Sistemas de Secreción Bacterianos , Biomarcadores/metabolismo , Burkholderia pseudomallei/ultraestructura , Gránulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Lisosomas/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Neutrófilos/ultraestructura , Fagosomas/metabolismo , Fagosomas/ultraestructuraRESUMEN
Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.
Asunto(s)
Choque Séptico/microbiología , Infecciones Estreptocócicas/diagnóstico , Streptococcus agalactiae/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/farmacología , Femenino , Humanos , Japón/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Serogrupo , Streptococcus agalactiae/efectos de los fármacosRESUMEN
When nucleotide-binding oligomerization domain-like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN(+/-) mice were more responsive to inflammasome activation than those from GLMN(+/+) mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Inflamasomas/metabolismo , Macrófagos/metabolismo , Proteínas Musculares/metabolismo , Shigella flexneri/metabolismo , Animales , Antígenos Bacterianos/genética , Apoptosis , Proteínas Bacterianas/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Femenino , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Células Jurkat , Macrófagos/microbiología , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Fluorescente , Proteínas Musculares/genética , Unión Proteica , Shigella flexneri/genética , Shigella flexneri/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Persistent colonization of the gastric mucosa by Helicobacter pylori (Hp) elicits chronic inflammation and aberrant epithelial cell proliferation, which increases the risk of gastric cancer. Here we examine the ability of microRNAs to modulate gastric cell proliferation in response to persistent Hp infection and find that epigenetic silencing of miR-210 plays a key role in gastric disease progression. Importantly, DNA methylation of the miR-210 gene is increased in Hp-positive human gastric biopsies as compared with Hp-negative controls. Moreover, silencing of miR-210 in gastric epithelial cells promotes proliferation. We identify STMN1 and DIMT1 as miR-210 target genes and demonstrate that inhibition of miR-210 expression augments cell proliferation by activating STMN1 and DIMT1. Together, our results highlight inflammation-induced epigenetic silencing of miR-210 as a mechanism of induction of chronic gastric diseases, including cancer, during Hp infection.
Asunto(s)
Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Infecciones por Helicobacter/genética , Metiltransferasas/metabolismo , MicroARNs/metabolismo , Estatmina/metabolismo , Neoplasias Gástricas/genética , Animales , Ciclo Celular/genética , Proliferación Celular , Enfermedad Crónica , Metilación de ADN , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Perfilación de la Expresión Génica , Gerbillinae , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Helicobacter pylori/fisiología , Humanos , Masculino , Metiltransferasas/genética , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Estatmina/genética , Neoplasias Gástricas/complicaciones , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.