RESUMO
The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.
Assuntos
ADP Ribose Transferases/metabolismo , Permeabilidade da Membrana Celular , Exotoxinas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Morte Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Mutação , Pseudomonas aeruginosa/efeitos dos fármacos , Sistemas de Secreção Tipo III/metabolismo , Vacúolos/metabolismoRESUMO
Effector proteins translocated into host cells by the Pseudomonas aeruginosa type III secretion system (T3SS) are critical for phagocytic avoidance and systemic spread of the microorganism. The T3SS genes are present in virtually all P. aeruginosa strains. When examined in environmental isolates and clinical specimens, expression of the T3SS genes is the rule. Isolates from the airways of cystic fibrosis (CF) patients are one exception, and these isolates usually carry mutations that disable T3SS gene expression. In this study, we describe two P. aeruginosa isolates, one pigmented brown and one green, from a keratitis-ichthyosis-deafness (KID) syndrome patient with a chronic cutaneous ankle wound. Similar to most isolates from CF, both of the KID isolates were defective for T3SS gene expression. Providing the primary activator of T3SS transcription (exsA) in trans restored T3SS function. Since the exsA sequences were identical to that of a reference strain with active T3SS gene expression, we examined the cAMP-Vfr system, a critical regulator of T3SS gene expression. Vfr is a cAMP-dependent transcription factor that activates exsA expression. Whereas T3SS activity was corrected in the brown isolate by restoring cAMP synthesis, the same was not observed for the green isolate. These findings suggest that distinct mechanisms resulted in loss of T3SS gene expression in the KID isolates. The mutations responsible for the T3SS defects were not clearly evident by comparison of the whole-genome sequences to a reference strain. Our findings suggest that loss of T3SS gene expression may be a trait common to both CF and non-CF chronic infections. IMPORTANCE A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation. In this study, we report loss of T3SS gene expression in non-CF chronic isolates. This finding suggests that loss of the T3SS may be a common and important trait that contributes to persistence and may open avenues to explore the significance further using non-CF chronic infection models.
Assuntos
Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Tornozelo , Traumatismos do Tornozelo , Proteínas de Bactérias/genética , Proteína Receptora de AMP Cíclico , Fibrose Cística , Regulação Bacteriana da Expressão Gênica , Humanos , Infecção Persistente , Regiões Promotoras Genéticas , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Infecção dos Ferimentos/genéticaAssuntos
Fibrose Cística/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III/genética , Proteínas de Bactérias/genética , Fibrose Cística/complicações , Fibrose Cística/fisiopatologia , Progressão da Doença , Evolução Molecular , Humanos , Masculino , Mutação de Sentido Incorreto , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa/genética , Proteínas Repressoras/genética , Análise de Sequência de DNA , Transativadores/genética , Virulência/genética , Adulto JovemRESUMO
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing small noncoding RNAs (sRNAs) and/or facilitating base pairing between sRNAs and mRNA targets. A previous study identified sRNA 1061, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. In this study, we screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression. Further characterization revealed that sRNA 179 inhibits the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small noncoding RNAs that sequester RsmA from target mRNAs. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA, leading to reduced expression of the T3SS and cAMP-Vfr regulons.IMPORTANCE Control of gene expression by small noncoding RNA (sRNA) is well documented but underappreciated. Deep sequencing of mRNA preparations from Pseudomonas aeruginosa suggests that >500 sRNAs are generated. Few of those sRNAs have defined roles in gene expression. To address that knowledge gap, we constructed an sRNA expression library and identified sRNA 179 as a regulator of the type III secretion system (T3SS) and the cAMP-Vfr regulons. The T3SS- and cAMP-Vfr-controlled genes are critical virulence factors. Increased understanding of the signals and regulatory mechanisms that control these important factors will enhance our understanding of disease progression and reveal potential approaches for therapeutic intervention.
Assuntos
Proteínas de Bactérias/genética , Proteína Receptora de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fator Proteico 1 do Hospedeiro/genética , Pseudomonas aeruginosa/genética , Pequeno RNA não Traduzido/genética , Sistemas de Secreção Tipo III/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/patogenicidade , RNA Bacteriano/genética , Regulon , Transcrição Gênica , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/genéticaRESUMO
Type III secretion systems (T3SS) are widely distributed in Gram-negative microorganisms and critical for host-pathogen and host-symbiont interactions with plants and animals. Central features of the T3SS are a highly conserved set of secretion and translocation genes and contact dependence wherein host-pathogen interactions trigger effector protein delivery and serve as an inducing signal for T3SS gene expression. In addition to these conserved features, there are pathogen-specific properties that include a unique repertoire of effector genes and mechanisms to control T3SS gene expression. The Pseudomonas aeruginosa T3SS serves as a model system to understand transcriptional and posttranscriptional mechanisms involved in the control of T3SS gene expression. The central regulatory feature is a partner-switching system that controls the DNA-binding activity of ExsA, the primary regulator of T3SS gene expression. Superimposed upon the partner-switching mechanism are cyclic AMP and cyclic di-GMP signaling systems, two-component systems, global regulators, and RNA-binding proteins that have positive and negative effects on ExsA transcription and/or synthesis. In the present review, we discuss advances in our understanding of how these regulatory systems orchestrate the activation of T3SS gene expression in the context of acute infections and repression of the T3SS as P. aeruginosa adapts to and colonizes the cystic fibrosis airways.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Transativadores/genética , Sistemas de Secreção Tipo III/genética , Animais , Humanos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/patogenicidade , Sistemas do Segundo Mensageiro , Transdução de Sinais , Transcrição GênicaRESUMO
Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis.IMPORTANCEP. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of the P. aeruginosa strains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103 fleQ mutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células Epiteliais/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Transativadores/metabolismo , Sistemas de Secreção Tipo III/metabolismo , ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Humanos , Estabilidade Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Transativadores/genética , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genéticaRESUMO
With the rise of multidrug resistance, Pseudomonas aeruginosa infections require alternative therapeutics. The injectisome (iT3SS) and flagellar (fT3SS) type III secretion systems are 2 virulence factors associated with poor clinical outcomes. iT3SS translocates toxins, rod, needle, or regulator proteins, and flagellin into the host cell cytoplasm and causes cytotoxicity and NLRC4-dependent inflammasome activation, which induces interleukin 1ß (IL-1ß) release and reduces interleukin 17 (IL-17) production and bacterial clearance. fT3SS ensures bacterial motility, attachment to the host cells, and triggers inflammation. INP1855 is an iT3SS inhibitor identified by in vitro screening, using Yersinia pseudotuberculosis Using a mouse model of P. aeruginosa pulmonary infection, we show that INP1855 improves survival after infection with an iT3SS-positive strain, reduces bacterial pathogenicity and dissemination and IL-1ß secretion, and increases IL-17 secretion. INP1855 also modified the cytokine balance in mice infected with an iT3SS-negative, fT3SS-positive strain. In vitro, INP1855 impaired iT3SS and fT3SS functionality, as evidenced by a reduction in secretory activity and flagellar motility and an increase in adenosine triphosphate levels. As a result, INP1855 decreased cytotoxicity mediated by toxins and by inflammasome activation induced by both laboratory strains and clinical isolates. We conclude that INP1855 acts by dual inhibition of iT3SS and fT3SS and represents a promising therapeutic approach.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Inflamassomos/metabolismo , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting ten CF lung pairs and studying â¼12,000 regional isolates, we were able to investigate whether clonally related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely diseased lung regions and found evolved differences in bacterial nutritional requirements, host defense and antibiotic resistance, and virulence due to hyperactivity of the type 3 secretion system. These findings suggest that bacterial intermixing is limited in CF lungs and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.
Assuntos
Fibrose Cística/complicações , Variação Genética , Pulmão/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Humanos , Dados de Sequência Molecular , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNARESUMO
UNLABELLED: The opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa. IMPORTANCE: The inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.
Assuntos
Flagelina/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Flagelina/genética , Humanos , Inflamassomos/fisiologia , Interleucina-1beta/metabolismo , Macrófagos/fisiologia , Camundongos , Mutação , Plasmídeos , Transporte ProteicoRESUMO
Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants.
Assuntos
Sistemas de Secreção Bacterianos , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana Transportadoras/genética , Polissacarídeos Bacterianos/metabolismo , Transdução de SinaisRESUMO
ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS) and a member of the AraC/XylS protein family. Each of the 10 ExsA-dependent promoter regions that define the T3SS regulon has two adjacent binding sites for monomeric ExsA. Whereas the promoter-proximal sites (binding site 1) contain highly conserved GnC and TGnnA sequences that are separated by â¼10 bp, the promoter-distal sites (binding site 2) share no obvious sequence similarity to each other or to the binding site 1 consensus. In the present study, we used footprinting with Fe-BABE (a protein-labeling reagent that can be conjugated to cysteine residues) to demonstrate that the two ExsA monomers bind to the P(exsC), P(exsD), P(exoT), and P(pcrG) promoters in a head-to-tail orientation. The footprinting data further indicate that the conserved GnC and TGnnA sequences constitute binding site 1. When bound to site 1, the first helix-turn-helix (HTH) motif of ExsA interacts with the conserved GnC sequence, and the second HTH interacts at or near the TGnnA sequences. Genetic data using the P(exoT) promoter indicate that residues L198 and T199 in the first HTH motif of ExsA contact the guanine in the GnC sequence and that residue K202, also in the first HTH motif, contacts the cytosine. Likewise, evidence is presented that residues Q248, Y250, T252, and R257 located in the second HTH motif contribute to the recognition of the TGnnA sequence. These combined data define interactions of ExsA with site 1 on the P(exoT) promoter and provide insight into the nature of the interactions involved in recognition of binding site 2.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Pseudomonas aeruginosa/metabolismo , Transativadores/metabolismo , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Proteínas Ativadoras de GTPase/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genéticaRESUMO
Many Gram-negative bacteria utilize a type III secretion system (T3SS) to deliver protein effectors to target host cells. Transcriptional control of T3SS gene expression is generally coupled to secretion through the release of a regulatory protein. T3SS gene expression in Pseudomonas aeruginosa is regulated by extracellular secretion of ExsE. ExsE is a small 81 residue protein that appears to lack a stable structural core as indicated by previous studies. In this study, we employed various NMR methods to characterize the structure of ExsE alone and when bound to its secretion chaperone ExsC. We found that ExsE is largely unfolded throughout the polypeptide chain, belonging to a class of proteins that are intrinsically disordered. The unfolded, extended conformation of ExsE may expedite efficient secretion through the narrow path of the T3SS secretion channel to activate gene expression in a timely manner. We also found that the structurally flexible ExsE samples through conformations with localized structurally ordered regions. Importantly, these transiently ordered elements are related to the secondary structures involved in binding ExsC based on a prior crystal structure of the ExsC-ExsE complex. These findings support the notion that preexisting structured elements facilitate binding of intrinsically disordered proteins to their targets.
Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Chaperonas Moleculares/química , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Desdobramento de ProteínaRESUMO
Diabetes mellitus predisposes the host to bacterial infections. Moreover, hyperglycemia has been shown to be an independent risk factor for respiratory infections. The luminal surface of airway epithelia is covered by a thin layer of airway surface liquid (ASL) and is normally sterile despite constant exposure to bacteria. The balance between bacterial growth and killing in the airway determines the outcome of exposure to inhaled or aspirated bacteria: infection or sterility. We hypothesized that restriction of carbon sources--including glucose--in the ASL is required for sterility of the lungs. We found that airway epithelia deplete glucose from the ASL via a novel mechanism involving polarized expression of GLUT-1 and GLUT-10, intracellular glucose phosphorylation, and low relative paracellular glucose permeability in well-differentiated cultures of human airway epithelia and in segments of airway epithelia excised from human tracheas. Moreover, we found that increased glucose concentration in the ASL augments growth of P. aeruginosa in vitro and in the lungs of hyperglycemic ob/ob and db/db mice in vivo. In contrast, hyperglycemia had no effect on intrapulmonary bacterial growth of a P. aeruginosa mutant that is unable to utilize glucose as a carbon source. Our data suggest that depletion of glucose in the airway epithelial surface is a novel mechanism for innate immunity. This mechanism is important for sterility of the airways and has implications in hyperglycemia and conditions that result in disruption of the epithelial barrier in the lung.
Assuntos
Glucose/deficiência , Imunidade Inata , Mucosa Respiratória/microbiologia , Animais , Líquidos Corporais , Células Cultivadas , Células Epiteliais/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Camundongos , Pseudomonas aeruginosa , Mucosa Respiratória/metabolismo , Sistema Respiratório/microbiologia , EsterilizaçãoRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that causes acute, invasive infections in immunocompromised individuals and chronic, persistent respiratory infections in individuals with cystic fibrosis (CF). The differential progression of acute or chronic infections involves the production of distinct sets of virulence factors. P. aeruginosa strains isolated from patients with acute respiratory infection are generally nonencapsulated and express a variety of invasive virulence factors, including flagella, the type III secretion system (T3SS), type IV pili (TFP), and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to the production of an alginate capsule. The mucoid phenotype results from loss-of-function mutations in mucA, which encodes an anti-sigma factor that normally prevents alginate synthesis. Here, we report that the cyclic AMP/Vfr-dependent signaling (CVS) pathway is defective in mucA mutants and that the defect occurs at the level of vfr expression. The CVS pathway regulates the expression of multiple invasive virulence factors, including T3SS, exotoxin A, protease IV, and TFP. We further demonstrate that mucA-dependent CVS inhibition involves the alternative sigma factor AlgU (AlgT) and the response regulator AlgR but does not depend on alginate production. Our findings show that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in acute and persistent infections. These results suggest that mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa in the CF lung.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/genética , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutação , Pseudomonas aeruginosa/genética , Regulon , Fator sigma/genética , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Fatores de VirulênciaRESUMO
Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína Receptora de AMP Cíclico/genética , DNA Bacteriano , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/genética , Fatores de Virulência/genéticaRESUMO
Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/genética , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/genética , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Western Blotting , Cromatografia em Gel , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Exotoxinas/genética , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/genética , Homologia de Sequência de Aminoácidos , Fatores de Virulência/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes.
Assuntos
Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Pseudomonas aeruginosa/fisiologia , Proteínas Repressoras/metabolismo , Transcrição Gênica , Fatores de Virulência/biossíntese , Antiporters/genética , Proteínas de Bactérias/genética , Linhagem Celular , Células Epiteliais/microbiologia , Técnicas de Inativação de Genes , HumanosRESUMO
Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.
Assuntos
Apoptose/fisiologia , Células de Kupffer/metabolismo , Células de Kupffer/microbiologia , Pseudomonas aeruginosa/patogenicidade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Substituição de Aminoácidos/genética , Apoptose/genética , Bacteriemia/metabolismo , Bacteriemia/microbiologia , Bacteriemia/patologia , Inibidores de Caspase , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células Clonais , Humanos , Células de Kupffer/citologia , Células de Kupffer/enzimologia , Fígado/citologia , Fígado/microbiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Transfecção , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Chronic Pseudomonas aeruginosa infection, as occurs in cystic fibrosis, is associated with decreased surfactant phospholipid levels. To investigate mechanisms, we measured synthesis of dipalmitoylphosphatidylcholine (DPPC), the major surfactant phospholipid. Mice received an agarose bead slurry alone, or were infected with beads containing a clinical mucoid isolate of P. aeruginosa. Bacterial infection after 3 days resulted in a approximately 50% reduction in surfactant DPPC content versus control. These changes in surfactant were associated with co-ordinate reductions in mRNAs and immunoreactive levels for CTP: phosphocholine cytidylyltransferase (CCTalpha), the rate-regulatory enzyme required for DPPC synthesis. P. aeruginosa infection of murine lung epithelia decreased CCTalpha gene transcription without altering mRNA stability and by a mechanism other than release of a soluble extracellular inhibitor. Promoter deletional analysis revealed that P. aeruginosa activates a negative response element from -1019 to -799 bp of the CCTalpha proximal 5'-flanking region. Exposure of cells to a P. aeruginosa mutant strain producing alginate reduced CCTalpha promoter activity, whereas these effects were not observed in strains defective in alginate synthesis. Murine type II cells isolated from P. aeruginosa-infected CCTalpha promoter-beta-galactosidase transgenic mice exhibited significantly reduced CCT and beta-galactosidase enzyme activities versus control. Thus, a mucoid P. aeruginosa strain reduces mRNA synthesis of a key biosynthetic enzyme thereby decreasing levels of surfactant.
Assuntos
Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Alginatos/metabolismo , Animais , Células Cultivadas , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Epitélio/metabolismo , Epitélio/microbiologia , Expressão Gênica , Immunoblotting , Pulmão/metabolismo , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Regiões Promotoras Genéticas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Type III secretion systems (T3SS) function by translocating effector proteins into eukaryotic host cells and are important for the virulence of many Gram-negative bacterial pathogens. Although the secretion and translocation machineries are highly conserved between different species, each pathogen translocates a unique set of effectors that subvert normal host cell physiology to promote pathogenesis. The uniqueness of each pathogen is further reflected in the diversity of mechanisms used to regulate T3SS gene expression. Pseudomonas aeruginosa utilizes a complex set of signalling pathways to modulate T3SS expression in response to extracellular and intracellular cues. Whereas some pathways are dedicated solely to regulating the T3SS, others co-ordinately regulate expression of the T3SS with multiple virulence functions on a global scale. Emerging regulatory themes include coupling of T3SS transcription with type III secretory activity, global regulatory control through modulation of cAMP biosynthesis, repression by a variety of stresses, involvement of multiple two component regulatory systems, and an inverse relationship between T3SS expression and multicellular behaviour. Factors controlling activation of T3SS expression likely contribute to the environmental survival of the organism and to the pathogenesis of acute P. aeruginosa infections. Conversely, active repression of the T3SS might contribute to the persistence of chronic infections.