RESUMEN
The bacterium Pseudomonas aeruginosa is an exceptionally resilient opportunistic pathogen, presenting formidable challenges for treatment due to its proclivity for developing drug resistance. To address this predicament, we have devised a self-assembled supramolecular antibiotic known as dHTSN1@pHPplus, which can circumvent the drug resistance mechanism of Pseudomonas aeruginosa and effectively combat Pseudomonas aeruginosa infection by impeding the secretion of key virulence factors through the inhibition of the type III secretion system while simultaneously mobilizing immune cells to eradicate Pseudomonas aeruginosa. Furthermore, dHTSN1@pHPplus was ingeniously engineered with infection-targeting capabilities, enabling it to selectively concentrate precisely at the site of infection. As anticipated, the administration of dHTSN1@pHPplus exhibited a remarkable therapeutic efficacy in combating dual resistance to Meropenem and imipenem in a mouse model of P. aeruginosa lung infection. The results obtained from metagenomic detection further confirmed these findings, demonstrating a significant reduction in the proportion of Pseudomonas aeruginosa compared to untreated mice with Pseudomonas aeruginosa-infected lungs. Additionally, no notable acute toxicity was observed in the acute toxicity experiments. The present study concludes that the remarkable efficacy of dHTSN1@pHPplus in treating drug-resistant P. aeruginosa infection confirms its immense potential as a groundbreaking antibiotic agent for combating drug-resistant P. aeruginosa.
Asunto(s)
Antibacterianos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Factores de Virulencia , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Animales , Antibacterianos/farmacología , Antibacterianos/química , Ratones , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Inmunidad Adaptativa/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Humanos , Farmacorresistencia Bacteriana/efectos de los fármacos , Ratones Endogámicos BALB C , FemeninoRESUMEN
Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.
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Fibrosis Quística , Modelos Animales de Enfermedad , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Animales , Infecciones por Pseudomonas/microbiología , Ratones , Humanos , Infecciones del Sistema Respiratorio/microbiología , Interacciones Huésped-Patógeno , Esputo/microbiologíaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in compromised hosts. P. aeruginosa infections are difficult to treat because of the inherent ability of the bacteria to develop antibiotic resistance, secrete a variety of virulence factors, and form biofilms. The secreted aminopeptidase (PaAP) is an emerging virulence factor, key in providing essential low molecular weight nutrients and a cardinal modulator of biofilm development. PaAP is therefore a new potential target for therapy of P. aeruginosa infections. The present review summarizes the current knowledge of PaAP, with special emphasis on its biochemical and enzymatic properties, activation mechanism, biological roles, regulation, and structure. Recently developed specific inhibitors and their potential as adjuncts in the treatment of P. aeruginosa infections are also described.
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Aminopeptidasas , Pseudomonas aeruginosa , Factores de Virulencia , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/metabolismo , Aminopeptidasas/metabolismo , Humanos , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , AnimalesRESUMEN
Pseudomonas aeruginosa (P. aeruginosa), a common opportunistic pathogen, is highly prone to chronic infection and is almost impossible to eradicate, especially attributed to virulence factors and adaptive mutations. In the present study, pseudomonas effector candidate 1 (Pec 1), a novel virulence factor of P. aeruginosa, was investigated, which inhibited bacterial clearance by the host and aggravated lung injury. Further, it demonstrated that Pec 1 inhibited miR-155 via suppressing integrin ß3 expression, thereby activating PI3K-AKT-mTOR and inhibiting autophagy in macrophages. Additionally, the identification of Pec 1 in sputum was related to the bacterial load and assisted in rapid diagnosis of P. aeruginosa infection. This finding underlined the importance of Pec 1 in the pathogenesis of P. aeruginosa infection and indicated that Pec 1 could be a vital independent virulence factor during chronic infection with P. aeruginosa, providing new insights in rapid diagnosis, therapeutic targets, and vaccine antigens of P. aeruginosa infection.
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Autofagia , Macrófagos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Factores de Virulencia , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Macrófagos/microbiología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/inmunología , Animales , Ratones , Humanos , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ratones Endogámicos C57BL , Interacciones Huésped-Patógeno , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Infections, such as that by the multiresistant opportunistic bacterial pathogen Pseudomonas aeruginosa, may pose a serious health risk, especially on vulnerable patient populations. The nematode Caenorhabditis elegans provides a simple organismal model to investigate both pathogenic mechanisms and the emerging role of innate immunity in host protection. Here, we review the virulence and infection strategies of P. aeruginosa and host defenses of C. elegans. We summarize the recognition mechanisms of patterns of pathogenesis, including novel pathogen-associated molecular patterns and surveillance immunity of translation, mitochondria, and lysosome-related organelles. We also review the regulation of antimicrobial and behavioral defenses by the worm's neuroendocrine system. We focus on how discoveries in this rich field align with well-characterized evolutionary conserved protective pathways, as well as on potential crossovers to human pathogenesis and innate immune responses.
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Caenorhabditis elegans , Interacciones Huésped-Patógeno , Inmunidad Innata , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/inmunología , Pseudomonas aeruginosa/patogenicidad , Interacciones Huésped-Patógeno/inmunología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/inmunología , Humanos , Modelos Animales de Enfermedad , VirulenciaRESUMEN
Pseudomonas aeruginosa is one of the most common nosocomial pathogens and part of the top emergent species associated with antimicrobial resistance that has become one of the greatest threat to public health in the twenty-first century. This bacterium is provided with a wide set of virulence factors that contribute to pathogenesis in acute and chronic infections. This review aims to summarize the impact of multidrug resistance on the virulence and fitness of P. aeruginosa. Although it is generally assumed that acquisition of resistant determinants is associated with a fitness cost, several studies support that resistance mutations may not be associated with a decrease in virulence and/or that certain compensatory mutations may allow multidrug resistance strains to recover their initial fitness. We discuss the interplay between resistance profiles and virulence from a microbiological perspective but also the clinical consequences in outcomes and the economic impact.
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Farmacorresistencia Bacteriana Múltiple , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Farmacorresistencia Bacteriana Múltiple/genética , Virulencia , Humanos , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Factores de Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.
Asunto(s)
Antibacterianos , Fibrosis Quística , Fenotipo , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Masculino , Infecciones por Pseudomonas/microbiología , Antibacterianos/farmacología , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Esputo/microbiología , Factores de Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: As antimicrobial resistance (AMR) has become a global health crisis, new strategies against AMR infection are urgently needed. Quorum sensing (QS), responsible for bacterial communication and pathogenicity, is among the targets for anti-virulence drugs that thrive as one of the promising treatments against AMR infection. METHODS: We identified a natural compound, Harmine, through virtual screening based on three QS receptors of Pseudomonas aeruginosa (P. aeruginosa) and explored the effect of Harmine on QS-controlled and pathogenicity-related phenotypes including pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14. The protective effect of Harmine on Caenorhabditis elegans (C. elegans) and mice infection models was determined and the synergistic effect of Harmine combined with common antibiotics was explored. The underlaying mechanism of Harmine's QS inhibitory effect was illustrated by molecular docking analysis, transcriptomic analysis, and target verification assay. RESULTS: In vitro results suggested that Harmine possessed QS inhibitory effects on pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14, and in vivo results displayed Harmine's protective effect on C. elegans and mice infection models. Intriguingly, Harmine increased susceptibility of both PA14 and clinical isolates of P. aeruginosa to polymyxin B and kanamycin when used in combination. Moreover, Harmine down-regulated a series of QS controlled genes associated with pathogenicity and the underlying mechanism may have involved competitively antagonizing autoinducers' receptors LasR, RhlR, and PqsR. CONCLUSIONS: This study shed light on the anti-virulence potential of Harmine against QS targets, suggesting the possible use of Harmine and its derivates as anti-virulence compounds.
Asunto(s)
Antibacterianos , Biopelículas , Caenorhabditis elegans , Harmina , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Percepción de Quorum , Percepción de Quorum/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/genética , Animales , Harmina/farmacología , Caenorhabditis elegans/microbiología , Ratones , Virulencia/efectos de los fármacos , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Simulación del Acoplamiento Molecular , Pruebas de Sensibilidad Microbiana , Piocianina , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana/efectos de los fármacos , FemeninoRESUMEN
Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3â%) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69â%) and cefepime (43.8â%). Four multidrug-resistant (MDR) isolates (25â%) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.
Asunto(s)
Antibacterianos , Pie Diabético , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/patogenicidad , Pie Diabético/microbiología , Túnez/epidemiología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/epidemiología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Estudios Prospectivos , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Virulencia/genética , Tipificación de Secuencias Multilocus , Adulto , Factores de Virulencia/genética , Farmacorresistencia Bacteriana Múltiple/genética , Anciano de 80 o más Años , PrevalenciaRESUMEN
The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.
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Fibrosis Quística , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Fibrosis Quística/microbiología , Evolución Molecular , Transferencia de Gen Horizontal , Adaptación al Huésped , Especificidad del Huésped , Macrófagos/microbiología , Macrófagos/inmunología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Infecciones por Pseudomonas/microbiología , Interacciones Huésped-PatógenoRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that poses a significant threat to individuals suffering from cystic fibrosis (CF). The pathogen is highly prevalent in CF individuals and is responsible for chronic infection, resulting in severe tissue damage and poor patient outcome. Prolonged antibiotic administration has led to the emergence of multidrug resistance in P. aeruginosa. In this direction, antivirulence strategies achieving targeted inhibition of bacterial virulence pathways, including quorum sensing, efflux pumps, lectins, and iron chelators, have been explored against CF isolates of P. aeruginosa. Hence, this review article presents a bird's eye view on the pulmonary infections involving P. aeruginosa in CF patients by laying emphasis on factors contributing to bacterial colonization, persistence, and disease progression along with the current line of therapeutics against P. aeruginosa in CF. We further collate scientific literature and discusses various antivirulence strategies that have been tested against P. aeruginosa isolates from CF patients.
Asunto(s)
Antibacterianos , Fibrosis Quística , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Percepción de Quorum , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Fibrosis Quística/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Humanos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Percepción de Quorum/efectos de los fármacos , Virulencia/efectos de los fármacos , Factores de Virulencia , Farmacorresistencia Bacteriana Múltiple , AnimalesRESUMEN
Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.
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Células Caliciformes , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Mucosa Respiratoria , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Células Caliciformes/microbiología , Células Caliciformes/metabolismo , Humanos , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/citología , Infecciones por Pseudomonas/microbiología , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Organoides/microbiología , Traslocación BacterianaRESUMEN
Bacteriophages have evolved diverse strategies to overcome host defence mechanisms and to redirect host metabolism to ensure successful propagation. Here we identify a phage protein named Dap1 from Pseudomonas aeruginosa phage PaoP5 that both modulates bacterial host behaviour and contributes to phage fitness. We show that expression of Dap1 in P. aeruginosa reduces bacterial motility and promotes biofilm formation through interference with DipA, a c-di-GMP phosphodiesterase, which causes an increase in c-di-GMP levels that trigger phenotypic changes. Results also show that deletion of dap1 in PaoP5 significantly reduces genome packaging. In this case, Dap1 directly binds to phage HNH endonuclease, prohibiting host Lon-mediated HNH degradation and promoting phage genome packaging. Moreover, PaoP5Δdap1 fails to rescue P. aeruginosa-infected mice, implying the significance of dap1 in phage therapy. Overall, these results highlight remarkable dual functionality in a phage protein, enabling the modulation of host behaviours and ensuring phage fitness.
Asunto(s)
Terapia de Fagos , Infecciones por Pseudomonas , Fagos Pseudomonas , Pseudomonas aeruginosa , Proteínas Virales , Pseudomonas aeruginosa/virología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/genética , Animales , Ratones , Fagos Pseudomonas/genética , Fagos Pseudomonas/fisiología , Infecciones por Pseudomonas/terapia , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/inmunología , Virulencia , Proteínas Virales/genética , Proteínas Virales/metabolismo , Biopelículas/crecimiento & desarrollo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Femenino , Bacteriófagos/fisiología , Bacteriófagos/genéticaRESUMEN
Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.
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Drosophila melanogaster , Inmunidad Innata , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Drosophila melanogaster/microbiología , Drosophila melanogaster/inmunología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Virulencia , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide and has emerged as a serious public health threat, due in large part to its multiple virulence factors and remarkable resistance capabilities. Stk1, a eukaryotic-type Ser/Thr protein kinase, has been shown in our previous work to be involved in the regulation of several signalling pathways and biological processes. Here, we demonstrate that deletion of stk1 leads to alterations in several virulence- and resistance-related physiological functions, including reduced pyocyanin and pyoverdine production, attenuated twitching motility, and enhanced biofilm production, extracellular polysaccharide secretion, and antibiotic resistance. Moreover, we identified AlgR, an important transcriptional regulator, as a substrate for Stk1, with its phosphorylation at the Ser143 site catalysed by Stk1. Intriguingly, both the deletion of stk1 and the mutation of Ser143 of AlgR to Ala result in similar changes in the above-mentioned physiological functions. Furthermore, assays of algR expression in these strains suggest that changes in the phosphorylation state of AlgR, rather than its expression level, underlie changes in these physiological functions. These findings uncover Stk1-mediated phosphorylation of AlgR as an important mechanism for regulating virulence and resistance in P. aeruginosa.
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Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Proteínas Serina-Treonina Quinasas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/enzimología , Fosforilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Biopelículas/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Farmacorresistencia Bacteriana/genética , Infecciones por Pseudomonas/microbiología , TransactivadoresRESUMEN
It is becoming increasingly apparent that commensal skin bacteria have an important role in wound healing and infection progression. However, the precise mechanisms underpinning many of these probiotic interactions remain to be fully uncovered. In this work, we demonstrate that the common skin commensal Cutibacterium acnes can limit the pathogenicity of the prevalent wound pathogen Pseudomonas aeruginosa in vivo. We show that this impact on pathogenicity is independent of any effect on growth, but occurs through a significant downregulation of the Type Three Secretion System (T3SS), the primary toxin secretion system utilised by P. aeruginosa in eukaryotic infection. We also show a downregulation in glucose acquisition systems, a known regulator of the T3SS, suggesting that glucose availability in a wound can influence infection progression. C. acnes is well known as a glucose fermenting organism, and we demonstrate that topically supplementing a wound with glucose reverses the probiotic effects of C. acnes. This suggests that introducing carbon source competition within the wound microenvironment may be an effective way to prevent or limit wound infection.
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Glucosa , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Glucosa/metabolismo , Animales , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , Propionibacterium acnes/crecimiento & desarrollo , Propionibacterium acnes/fisiología , Propionibacterium acnes/metabolismo , Infección de Heridas/microbiología , Ratones , Infecciones por Pseudomonas/microbiología , Piel/microbiología , Carbono/metabolismo , Cicatrización de Heridas , Antibiosis , Progresión de la Enfermedad , HumanosRESUMEN
Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in homeostasis and protection against heavy metal toxicity and oxidative stress. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a bacterial MT known as PmtA. Utilizing genetically modified P. aeruginosa PAO1 strains (a human clinical wound isolate), we show that inducing pmtA increases levels of pyocyanin and biofilm compared to other PAO1 isogenic strains, supporting previous results that pmtA is important for pyocyanin and biofilm production. We also show that overexpression of pmtA in vitro provides protection for cells exposed to oxidants, which is a characteristic of inflammation, indicating a role for PmtA as an antioxidant in inflammation. We found that a pmtA clean deletion mutant is phagocytized faster than other PAO1 isogenic strains in THP-1 human macrophage cells, indicating that PmtA provides protection from the phagocytic attack. Interestingly, we observed that monoclonal anti-PmtA antibody binds to PmtA, which is accessible on the surface of PAO1 strains using both flow cytometry and enzyme-linked immunosorbent assay techniques. Finally, we investigated intracellular persistence of these PAO1 strains within THP-1 macrophages cells and found that the phagocytic endurance of PAO1 strains is affected by pmtA expression. These data show for the first time that a bacterial MT (pmtA) can play a role in the phagocytic process and can be found on the outer surface of PAO1. Our results suggest that PmtA plays a role both in protection from oxidative stress and in the resistance to the host's innate immune response, identifying PmtA as a potential therapeutic target in P. aeruginosa infection. IMPORTANCE: The pathogen Pseudomonas aeruginosa is a highly problematic multidrug-resistant (MDR) pathogen with complex virulence networks. MDR P. aeruginosa infections have been associated with increased clinical visits, very poor healthcare outcomes, and these infections are ranked as critical on priority lists of both the Centers for Disease Control and Prevention and the World Health Organization. Known P. aeruginosa virulence factors have been extensively studied and are implicated in counteracting host defenses, causing direct damage to the host tissues, and increased microbial competitiveness. Targeting virulence factors has emerged as a new line of defense in the battle against MDR P. aeruginosa strains. Bacterial metallothionein is a newly recognized virulence factor that enables evasion of the host immune response. The studies described here identify mechanisms in which bacterial metallothionein (PmtA) plays a part in P. aeruginosa pathogenicity and identifies PmtA as a potential therapeutic target.
Asunto(s)
Proteínas Bacterianas , Biopelículas , Macrófagos , Metalotioneína , Estrés Oxidativo , Fagocitosis , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/metabolismo , Humanos , Metalotioneína/genética , Metalotioneína/metabolismo , Macrófagos/microbiología , Macrófagos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Células THP-1 , Piocianina/metabolismoRESUMEN
Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses. IMPORTANCE: The notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa. Since the cupA pilus is an important adhesin of P. aeruginosa, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.
Asunto(s)
Macrófagos , Fagocitosis , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Macrófagos/microbiología , Macrófagos/inmunología , Ratones , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/inmunología , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Humanos , Células RAW 264.7RESUMEN
The nematode Caenorhabditis elegans is a powerful model organism for studying the molecular and cellular mechanisms of innate immunity governed by the intestine. Here, we present a protocol to perform C. elegans survival assays to infection by the bacterial pathogen Pseudomonas aeruginosa PA14. Specifically, we describe steps for preparing C. elegans strains and PA14 bacteria for survival assays. This protocol will assist researchers to study genes involved in intestinal innate immunity and gut defense against pathogen infection. For complete details on the use and execution of this protocol, please refer to Liu et al.1 and Zheng et al.2.
Asunto(s)
Caenorhabditis elegans , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/inmunología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Inmunidad InnataRESUMEN
Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology. Our findings show that ChiC exhibits activity against both insoluble (α- and ß-chitin) and soluble chitooligosaccharides. Enzyme kinetics toward (GlcNAc)4 revealed a kcat of 6.50 s-1 and a KM of 1.38 mM, the latter remarkably high for a canonical chitinase. In our label-free proteomics investigation, ChiC was among the most abundant proteins in the Pel biofilm, suggesting a potential contribution to PA biofilm formation. Using an intratracheal challenge model of PA pneumonia, the chiC::ISphoA/hah transposon insertion mutant paradoxically showed slightly increased virulence compared to the wild-type parent strain. Our results indicate that ChiC is a genuine chitinase that contributes to a PA pathoadaptive pathway.IMPORTANCEIn addition to performing chitin degradation, chitinases from the glycoside hydrolase 18 family have been found to play important roles during pathogenic bacterial infection. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing pneumonia in immunocompromised individuals. Despite not being able to grow on chitin, the bacterium produces a chitinase (ChiC) with hitherto unknown function. This study describes an in-depth characterization of ChiC, focusing on its potential contribution to the bacterium's disease-causing ability. We demonstrate that ChiC can degrade both polymeric chitin and chitooligosaccharides, and proteomic analysis of Pseudomonas aeruginosa biofilm revealed an abundance of ChiC, hinting at a potential role in biofilm formation. Surprisingly, a mutant strain incapable of ChiC production showed higher virulence than the wild-type strain. While ChiC appears to be a genuine chitinase, further investigation is required to fully elucidate its contribution to Pseudomonas aeruginosa virulence, an important task given the evident health risk posed by this bacterium.