RESUMO
During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase-1, which in turn triggers macrophage pyroptosis and IL-1ß/IL-18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore-forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase-11 activation. Surprisingly, previous studies indicated that a T3SS-induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS-negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore-forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll-like receptors, and thus enhanced the expression of inflammatory proteins including pro-IL-1ß and TNF-α. However, mature-IL-1ß and IL-18 were undetectable in wild-type mice, suggesting that ExlA failed to effectively activate caspase-1. Nevertheless, caspase-1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA-induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome-dependent process.
Assuntos
Inflamassomos/imunologia , Leucocidinas/toxicidade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Animais , Citocinas/biossíntese , Inflamassomos/metabolismo , Inflamação , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Infiltração de Neutrófilos , Fragmentos de Peptídeos/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/metabolismo , Sistemas de Secreção Tipo III , VirulênciaRESUMO
Pore-forming toxins are potent virulence factors secreted by a large array of bacteria. Here, we deciphered the action of ExlA from Pseudomonas aeruginosa and ShlA from Serratia marcescens on host cell-cell junctions. ExlA and ShlA are two members of a unique family of pore-forming toxins secreted by a two-component secretion system. Bacteria secreting either toxin induced an ExlA- or ShlA-dependent rapid cleavage of E-cadherin and VE-cadherin in epithelial and endothelial cells, respectively. Cadherin proteolysis was executed by ADAM10, a host cell transmembrane metalloprotease. ADAM10 activation is controlled in the host cell by cytosolic Ca2+ concentration. We show that Ca2+ influx, induced by ExlA or ShlA pore formation in the plasma membrane, triggered ADAM10 activation, thereby leading to cadherin cleavage. Our data suggest that ADAM10 is not a cellular receptor for ExlA and ShlA, further confirming that ADAM10 activation occurred via Ca2+ signalling. In conclusion, ExlA- and ShlA-secreting bacteria subvert a regulation mechanism of ADAM10 to activate cadherin shedding, inducing intercellular junction rupture, cell rounding and loss of tissue barrier integrity.
Assuntos
Proteína ADAM10/metabolismo , Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Proteínas Hemolisinas/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Western Blotting , Cálcio/metabolismo , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Pseudomonas aeruginosa/patogenicidade , Serratia marcescens/patogenicidade , Virulência/fisiologia , Fatores de Virulência/metabolismoRESUMO
To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.
Assuntos
Células Epiteliais/metabolismo , Infecções por Pseudomonas/virologia , Pseudomonas aeruginosa/patogenicidade , Animais , Divisão Celular/fisiologia , Linhagem Celular , Senescência Celular/fisiologia , Cães , Humanos , Imuno-Histoquímica , Junções Intercelulares/metabolismo , Células Madin Darby de Rim Canino , Microscopia Confocal , TransfecçãoRESUMO
We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/genética , Cichorium intybus/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Infection of the vascular system by Pseudomonas aeruginosa (Pa) occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS) toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS) effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-types were sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE)-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349). VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E)-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells.
Assuntos
Antígenos CD/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Caderinas/metabolismo , Metaloendopeptidases/metabolismo , Infecções por Pseudomonas/metabolismo , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Most clinical strains of Pseudomonas aeruginosa, a leading agent of nosocomial infections, are multiresistant to antibiotherapy. Because of the paucity of new available antibiotics, the investigation of strategies aimed at limiting the action of its major virulence factors has gained much interest. The type 3 secretion system of P. aeruginosa and its effectors are known to be major determinants of toxicity and are required for bacterial dissemination in the host. Bacterial transmigration across the vascular wall is considered to be an important step in the infectious process. Using human endothelial primary cells, we demonstrate that forskolin (FSK), a drug inducing cyclic AMP (cAMP) elevation in eukaryotic cells, strikingly reduced the cell retraction provoked by two type 3 toxins, ExoS and ExoT, found in the majority of clinical strains. Conversely, cytotoxicity of a strain carrying the type 3 effector ExoU was unaffected by FSK. In addition, FSK altered the capacity of two ExoS/ExoT strains to transmigrate across cell monolayers. In agreement with these findings, other drugs and a cytokine inducing the increase of cAMP intracellular levels have also protected cells from retraction. cAMP is an activator of both protein kinase A and EPAC, a GTPase exchange factor of Rap1. Using activators or inhibitors of either pathway, we show that the beneficial effect of FSK is exerted by the activation of the EPAC/Rap1 axis, suggesting that its protective effect is mediated by reinforcing cell-cell and cell-substrate adhesion.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Colforsina/metabolismo , Células Endoteliais/microbiologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Pseudomonas aeruginosa/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Ativadores de Enzimas/metabolismo , Humanos , Complexo ShelterinaRESUMO
Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica , Pseudomonas aeruginosa/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/microbiologia , Humanos , Lipoproteínas/genética , Masculino , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Análise de Sobrevida , Fatores de Virulência/genéticaRESUMO
The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.
Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Toxinas Bacterianas , Dobramento de Proteína , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E(2), or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Caderinas/metabolismo , Proteínas de Membrana/metabolismo , Pré-Eclâmpsia/metabolismo , Proteína ADAM10 , Células Endoteliais/química , Feminino , Humanos , Hidrólise , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/urina , Placentação , Gravidez , Protocaderinas , Trofoblastos/química , Regulação para CimaRESUMO
Fibrillin-1, the major component of extracellular microfibrils that associate with insoluble elastin in elastic fibres, is mainly synthesized during development and postnatal growth and is believed to guide elastogenesis. Mutations in the fibrillin-1 gene cause Marfan syndrome, a multisystem disorder characterized by aortic aneurysms and dissections. The recent finding that early deficiency of elastin modifies vascular ageing has raised the possibility that fibrillin-1 deficiency could also contribute to late-onset pathology of vascular remodelling. To address this question, we examined cardiovascular function in 3-week-old, 6-month-old, and 24-month-old mice that are heterozygous for a hypomorphic structural mutation of fibrillin-1 (Fbn1{+/mgΔ} mice). Our results indicate that Fbn1{+/mgΔ} mice, particularly those that are 24 months old, are slightly more hypotensive than wild-type littermates. Additionally, aneurysm and aortic insufficiency were more frequently observed in ageing Fbn1{+/mgΔ}$ mice than in the wild-type counterparts. We also noted substantial fragmentation and decreased number of elastic lamellae in the aortic wall of Fbn1{+/mgΔ} mice, which were correlated with an increase in aortic stiffness, a decrease in vasoreactivity, altered expression of elastic fibre-related genes, including fibrillin-1 and elastin, and a decrease in the relative ratio between tissue elastin and collagen. Collectively, our findings suggest that the heterozygous mgΔ mutation accelerates some aspects of vascular ageing and eventually leads to aortic manifestations resembling those of Marfan syndrome. Importantly, our data also indicate that vascular abnormalities in Fbn1{+/mgΔ} mice are opposite to those induced by elastin haploinsufficiency during ageing that affect blood pressure, vascular dimensions, and number of elastic lamellae.
Assuntos
Envelhecimento/patologia , Síndrome de Marfan/genética , Proteínas dos Microfilamentos/deficiência , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Aorta/diagnóstico por imagem , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Fibrilina-1 , Fibrilinas , Regulação da Expressão Gênica/fisiologia , Hemodinâmica , Masculino , Síndrome de Marfan/patologia , Síndrome de Marfan/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Estresse Mecânico , UltrassonografiaRESUMO
Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.
RESUMO
Endothelial cells express two different classical cadherins, vascular endothelial (VE) cadherin and neural (N) cadherin, having distinct functions in the vascular system. VE-cadherin is specific to endothelial adherens junctions and is strictly necessary for vascular morphogenesis. On the contrary, N-cadherin shows diffuse localization on the cell surface and interacts with mural cells for vessel stabilization. In this study, we sought to clarify the cellular mechanisms leading to the distinct cellular locations and functions of the two cadherins in the endothelium. VE-cadherin has been shown to be responsible for the junctional exclusion of N-cadherin. Using several endothelial models, we demonstrate that this property is dependent on VE-cadherin binding to p120 catenin (p120(ctn)). Moreover, although in the absence of VE-cadherin N-cadherin can localize to cell contacts, angiogenesis remains impaired, demonstrating that endothelial junction formation is not sufficient for normal vessel development. Interestingly, we show that VE-cadherin, but not N-cadherin, is partially associated with cholesterol-enriched microdomains. Lipid raft-associated-VE-cadherin is characterized by a very high level of p120(ctn) association, and this association is necessary for VE-cadherin recruitment into lipid rafts. Altogether, our results indicate a critical role for p120(ctn) in regulating the membrane distribution of endothelial cadherins with functional consequences in terms of cadherin stabilization and intracellular signaling.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Cateninas/fisiologia , Endotélio Vascular/metabolismo , Animais , Western Blotting , Adesão Celular , Membrana Celular/metabolismo , Células Cultivadas , Imunofluorescência , Humanos , Imunoprecipitação , Junções Intercelulares , Microdomínios da Membrana , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Veias Umbilicais , delta CateninaRESUMO
ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Membrana Celular/química , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , GMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Infecções por Pseudomonas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Pseudomonas aeruginosa/patogenicidade , Animais , Membrana Celular/patologia , Infecção Hospitalar/microbiologia , Drosophila melanogaster/genética , Genoma Bacteriano/genética , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo III/metabolismoRESUMO
The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.
RESUMO
Protocadherins are transmembrane proteins exhibiting homophilic adhesive activities through their extracellular domain. Protocadherin 12 (Pcdh12) is expressed in angiogenic endothelial cells, mesangial cells of kidney glomeruli, and glycogen cells of the mouse placenta. To get insight into the role of this protein in vivo, we analyzed PCDH12-deficient mice and investigated their placental phenotype. The mice were alive and fertile; however, placental and embryonic sizes were reduced compared with wild-type mice. We observed defects in placental layer segregation and a decreased vascularization of the labyrinth associated with a reduction in cell density in this layer. To understand the molecular events responsible for the phenotypic alterations observed in Pcdh12(-/-) placentas, we analyzed the expression profile of embryonic day 12.5 mutant placentas compared with wild-type placentas, using pangenomic chips: 2,289 genes exhibited statistically significant changes in expressed levels due to loss of PCDH12. Functional grouping of modified genes was obtained by GoMiner software. Gene clusters that contained most of the differentially expressed genes were those involved in tissue morphogenesis and development, angiogenesis, cell-matrix adhesion and migration, immune response, and chromatin remodeling. Our data show that loss of PCDH12 leads to morphological alterations of the placenta and to notable changes in its gene expression profile. Specific genes emerging from the microarray screen support the biological modifications observed in PCDH12-deficient placentas.
Assuntos
Caderinas/deficiência , Perfilação da Expressão Gênica , Morfogênese , Placenta/embriologia , Placenta/metabolismo , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Adesão Celular , Movimento Celular , Decídua/citologia , Decídua/metabolismo , Feminino , Glicogênio/metabolismo , Camundongos , Tamanho do Órgão , Placenta/citologia , Gravidez , Protocaderinas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bacterial pore-forming toxins induce a rapid and massive increase in cytosolic Ca2+ concentration due to the formation of pores in the plasma membrane and/or activation of Ca2+-channels. As Ca2+ is an essential messenger in cellular signaling, a sustained increase in Ca2+ concentration has dramatic consequences on cellular behavior, eventually leading to cell death. However, host cells have adapted mechanisms to protect against Ca2+ intoxication, such as Ca2+ efflux and membrane repair. The final outcome depends upon the nature and concentration of the toxin and on the cell type. This review highlights the repercussions of Ca2+ overload on the induction of cell death, repair mechanisms, cellular adhesive properties, and the inflammatory response.
Assuntos
Toxinas Bacterianas/toxicidade , Cálcio/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacosRESUMO
Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.
Assuntos
Fenômenos Fisiológicos Bacterianos , Células Eucarióticas/microbiologia , Células Eucarióticas/patologia , Imagem Molecular/métodos , Animais , Células Endoteliais , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Células NIH 3T3RESUMO
Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.
Assuntos
Células Epiteliais Alveolares/microbiologia , Bacteriemia/microbiologia , Toxinas Bacterianas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , FagocitoseRESUMO
Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. IMPORTANCE: The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.