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The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.
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Enfermedad Aguda , Enfermedad Crónica , Genes Bacterianos , Oxígeno , Infecciones por Pseudomonas , Pseudomonas aeruginosa , ARN Bacteriano , Animales , Humanos , Oxígeno/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Fibrosis Quística/microbiología , Heridas y Lesiones/microbiología , Ubiquinona/biosíntesis , Anaerobiosis , Genes Bacterianos/genética , Sepsis/complicaciones , Sepsis/microbiologíaRESUMEN
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
Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.
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Infecciones Bacterianas , Fibrosis Quística , Infecciones por Pseudomonas , Humanos , Ecosistema , Infecciones por Pseudomonas/microbiología , Transcriptoma , Células Epiteliales/microbiología , Medios de Cultivo/metabolismo , Fibrosis Quística/microbiología , Pseudomonas aeruginosa/genética , Esputo/microbiologíaRESUMEN
BACKGROUND: Pseudomonas aeruginosa is a multidrug-resistant pathogen causing recalcitrant pulmonary infections in people with cystic fibrosis (pwCF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have been developed that partially correct the defective chloride channel driving disease. Despite the many clinical benefits, studies in adults have demonstrated that while P. aeruginosa sputum load decreases, chronic infection persists. Here, we investigate how P. aeruginosa in pwCF may change in the altered lung environment after CFTR modulation. METHODS: P. aeruginosa strains (n = 105) were isolated from the sputum of 11 chronically colonized pwCF at baseline and up to 21 months posttreatment with elexacaftor-tezacaftor-ivacaftor or tezacaftor-ivacaftor. Phenotypic characterization and comparative genomics were performed. RESULTS: Clonal lineages of P. aeruginosa persisted after therapy, with no evidence of displacement by alternative strains. We identified commonly mutated genes among patient isolates that may be positively selected for in the CFTR-modulated lung. However, classic chronic P. aeruginosa phenotypes such as mucoid morphology were sustained, and isolates remained just as resistant to clinically relevant antibiotics. CONCLUSIONS: Despite the clinical benefits of CFTR modulators, clonal lineages of P. aeruginosa persist that may prove just as difficult to manage in the future, especially in pwCF with advanced lung disease.
RESUMEN
Cystic fibrosis-related diabetes (CFRD), the most common comorbidity in cystic fibrosis (CF), leads to increased mortality by accelerating the decline in lung function. Scnn1b-Tg transgenic mice overexpressing the epithelial sodium channel ß subunit exhibit spontaneous CF-like lung disease, including airway mucus obstruction and chronic inflammation. Here, we established a chronic CFRD-like model utilizing Scnn1b-Tg mice made diabetic by injection of streptozotocin. In Ussing chamber recordings of trachea, Scnn1b-Tg mice exhibited larger amiloride-sensitive currents and forskolin-activated currents, without a difference in ATP-activated currents compared to wildtype (WT) littermates. Both diabetic WT (WT-D) and diabetic Scnn1b-Tg (Scnn1b-Tg-D) mice on the same genetic background exhibited substantially elevated blood glucose at 8 weeks; glucose levels also were elevated in bronchoalveolar lavage fluid (BALF) Bulk lung RNA-seq data showed significant differences between WT-D and Scnn1b-Tg-D mice. Neutrophil counts in BALF were substantially increased in Scnn1b-Tg-D lungs compared to controls (Scnn1b-Tg-con) and compared to WT-D lungs. Lung histology data showed enhanced parenchymal destruction, alveolar wall thickening, and neutrophilic infiltration in Scnn1b-Tg-D mice compared to WT-D mice, consistent with development of a spontaneous lung infection. We intranasally administered Pseudomonas aeruginosa to induce lung infection in these mice for 24 hours, which led to severe lung leukocytic infiltration and an increase in pro-inflammatory cytokine levels in the BALF. In summary, we established a chronic CFRD-like lung mouse model using the Scnn1b-Tg mice. The model can be utilized for future studies toward understanding the mechanisms underlying the lung pathophysiology associated with CFRD and developing novel therapeutics.
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When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus. However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.
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Sistemas de Transporte de Aminoácidos , Fibrosis Quística , Infecciones por Pseudomonas , Infecciones Estafilocócicas , Staphylococcus aureus , Sistemas de Transporte de Aminoácidos/genética , Biopelículas , Fibrosis Quística/complicaciones , Glutamatos/genética , Glutamatos/metabolismo , Glutamatos/farmacología , Mutación , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/genética , Proteínas Bacterianas/genéticaRESUMEN
There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are 20 different O antigens composed of different repeat sugar structures conferring serogroup specificity, and 10 are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is 1 of the 10 serogroups commonly found in infection, but it has never been developed into a vaccine, due in part to the acid-labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing bacteria and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here, we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.
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Antígenos O , Infecciones por Pseudomonas , Ratones , Animales , Lipopolisacáridos , Pseudomonas aeruginosa , Serogrupo , Vacunas Bacterianas , Anticuerpos AntibacterianosRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic-resistant, nosocomial infections in immuno-compromised individuals and is a high priority for antimicrobial development. Key to pathogenicity in P. aeruginosa are biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that the activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by the substitution of an active site glutamate residue with tryptophan [PqsE(E182W)]. Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR attenuates P. aeruginosa infection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insights into drug discovery against P. aeruginosa with PqsE as the target.
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Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/química , Biopelículas , Dominio Catalítico , Humanos , Ratones , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/metabolismo , Percepción de QuorumRESUMEN
Antibiotic resistance (AMR) is an increasing public health crisis worldwide. This threatens our ability to adequately care for patients with infections due to multi-drug-resistant (MDR) pathogens. As such, there is an urgent need to develop new classes of antimicrobials that are not based on currently utilized antibiotic scaffolds. One promising avenue of antimicrobial research that deserves renewed examination involves the use of peptides. Although antimicrobial peptides (AMPs) have been studied for a number of years, innovations in peptide design and their applications are increasingly making this approach a viable alternative to traditional small-molecule antibiotics. This review will provide updates on two ways in which peptides are being explored as antibiotics. The first topic will focus on novel types of peptides and conjugation methods that are being exploited to act as antibiotics themselves. These direct-acting modified peptides could serve as potentially useful drugs while mitigating many of the known liabilities of AMPs. The second topic relates to the use of peptides as delivery vehicles for other active compounds with antimicrobial activity. Cell-penetrating peptides (CPPs) are peptides designed to carry compounds across cell membranes and are a promising method for delivering a variety of antimicrobial compounds. When conjugated to other compounds, CPPs have been shown to be effective at increasing the uptake of both small- and large-molecular-weight compounds. This includes conjugation to antisense molecules and traditional antibiotics, resulting in increased effectiveness of these antimicrobials. One particular approach utilizes CPPs conjugated to phosphorodiamidate morpholino oligomers (PMOs). PMOs are designed to target particular pathogens in a gene-specific way. They target mRNA and block protein translation. Peptide-conjugated PMOs (PPMOs) allow for efficient delivery into the Gram-negative cytoplasm, and recent updates to their in vitro and in vivo activity are reviewed. This includes recent data to suggest that PPMOs maintain activity in the setting of multi-drug-resistant (MDR) strains, an important finding as it relates to the further development of this therapeutic approach. Other topics include the ability to have activity in the biofilm setting, a finding that likely relates to the peptide portion of the conjugate. Finally, what is known and anticipated related to the development of resistance to these peptides will be discussed.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacosRESUMEN
The Pseudomonas aeruginosa type III secretion system (T3SS) is a complex molecular machine that delivers toxic proteins from the bacterial cytoplasm directly into host cells. This apparatus spans the inner and outer membrane and employs a needle-like structure that penetrates through the eucaryotic cell membrane into the host cell cytosol. The expression of the P. aeruginosa T3SS is highly regulated by environmental signals including low calcium and host cell contact. P. aeruginosa strains with mutations in T3SS genes are less pathogenic, suggesting that the T3SS is a virulence mechanism. Given that P. aeruginosa is naturally antibiotic resistant and multidrug resistant isolates are rapidly emerging, new antibiotics to target P. aeruginosa are needed. Furthermore, even if new antibiotics were to be developed, the timeline between when an antibiotic is released and resistance development is relatively short. Therefore, the concept of targeting virulence factors has garnered attention. So-called "antivirulence" approaches do not kill the microbe but instead focus on rendering it harmless and therefore unable to cause damage. Since these therapies target a particular system or pathway, the normal microbiome is unlikely to be affected and there is less concern about the spread to other microbes. Finally, and most importantly, since any antivirulence drug does not kill the microbe, there should be less selective pressure to develop resistance to these inhibitors. The P. aeruginosa T3SS has been well studied due to its importance for pathogenesis in numerous human and animal infections. Thus, many P. aeruginosa T3SS inhibitors have been described as potential antivirulence therapeutics, some of which have progressed to clinical trials.
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Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Humanos , Pseudomonas aeruginosa/genética , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Calcio/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/químicaRESUMEN
BACKGROUND: Microbiome sequencing has brought increasing attention to the polymicrobial context of chronic infections. However, clinical microbiology continues to focus on canonical human pathogens, which may overlook informative, but nonpathogenic, biomarkers. We address this disconnect in lung infections in people with cystic fibrosis (CF). METHODS: We collected health information (lung function, age, and body mass index [BMI]) and sputum samples from a cohort of 77 children and adults with CF. Samples were collected during a period of clinical stability and 16S rDNA sequenced for airway microbiome compositions. We use ElasticNet regularization to train linear models predicting lung function and extract the most informative features. RESULTS: Models trained on whole-microbiome quantitation outperformed models trained on pathogen quantitation alone, with or without the inclusion of patient metadata. Our most accurate models retained key pathogens as negative predictors (Pseudomonas, Achromobacter) along with established correlates of CF disease state (age, BMI, CF-related diabetes). In addition, our models selected nonpathogen taxa (Fusobacterium, Rothia) as positive predictors of lung health. CONCLUSIONS: These results support a reconsideration of clinical microbiology pipelines to ensure the provision of informative data to guide clinical practice.
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Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Microbiota , Adolescente , Adulto , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Humanos , Pulmón/microbiología , Pulmón/fisiología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Valor Predictivo de las Pruebas , ARN Ribosómico 16S/genética , Esputo/microbiología , Adulto JovenRESUMEN
Pseudomonas aeruginosa is a wide-spread γ-proteobacterium that produces the biosurfactant rhamnolipid that has a great commercial value due to excellent properties of low toxicity and high biodegradability. However, this bacterium is an opportunist pathogen that constitutes an important health hazard due to its production of virulence-associated traits and its high antibiotic resistance. Thus, it is highly desirable to have a non-virulent P. aeruginosa strain for rhamnolipid production. It has been reported that strain ATCC 9027 is avirulent in mouse models of infection, and it is still able to produce rhamnolipid. Thus, it has been proposed to be suitable for it industrial production, since it encodes a defective LasR quorum sensing (QS) transcriptional regulator that is the head of this regulatory network. However, the restoration of virulence factor production by overexpression of rhlR (the gene encoding a QS-transcriptional regulator which is under the transcriptional control of LasR) is not sufficient to restore its virulence in mice. It is desirable to obtain a deeper understanding of ATCC 9027 attenuated-virulence phenotype and to assess the safety of this strain to be used at an industrial scale. In this work we determined whether increasing the expression of the pore-forming toxin encoded by the exlBA operon in strain ATCC 9027 had an impact on its virulence using Galleria mellonella and mouse models of infections. We increased the expression of the exlBA operon by overexpressing from a plasmid its transcriptional activator Vfr or of the Vfr ligand cyclic AMP produced by CyaB. We found that in G. mellonella ATCC 9027/pUCP24-vfr and ATCC 9027/pUCP24-cyaB gained a virulent phenotype, but these strains remained avirulent in murine models of P. aeruginosa infection. These results reinforce the possibility of using ATCC 9027 for industrial biosurfactants production.
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Proteínas Bacterianas , Pseudomonas aeruginosa , Animales , Proteínas Bacterianas/genética , Ratones , Operón , Pseudomonas aeruginosa/genética , Percepción de Quorum , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa is a leading cause of life-threatening nosocomial infections. Many virulence factors produced by P. aeruginosa are controlled by the cell-to-cell communication process called quorum sensing (QS). QS depends on the synthesis, release, and groupwide response to extracellular signaling molecules called autoinducers. P. aeruginosa possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, that produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Previously, we discovered that RhlR regulates both RhlI-dependent and RhlI-independent regulons, and we proposed that an alternative ligand functions together with RhlR to control the target genes in the absence of RhlI. Here, we report the identification of an enzyme, PqsE, which is the alternative-ligand synthase. Using biofilm analyses, reporter assays, site-directed mutagenesis, protein biochemistry, and animal infection studies, we show that the PqsE-produced alternative ligand is the key autoinducer that promotes virulence gene expression. Thus, PqsE can be targeted for therapeutic intervention. Furthermore, this work shows that PqsE and RhlR function as a QS-autoinducer synthase-receptor pair that drives group behaviors in P. aeruginosa.
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Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/fisiología , Tioléster Hidrolasas/metabolismo , Proteínas Bacterianas/genética , Tioléster Hidrolasas/genéticaRESUMEN
Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon, leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing, since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22, rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA, resulting in uncontrolled AlgT and continuous production of alginate in addition to the expression of â¼300 additional genes. Here, we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.
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Alginatos/metabolismo , Proteínas Bacterianas/genética , Pseudomonas aeruginosa/genética , Factor sigma/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Operón/genética , Fenotipo , Proteolisis , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface. We also show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5-6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminal sequence to facilitate Lys-5 modification. Our findings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.
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Proteínas Bacterianas/metabolismo , Metiltransferasas/metabolismo , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Evolución Molecular , Metiltransferasas/química , Metiltransferasas/genética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality worldwide. To survive in both the environment and the host, P. aeruginosa must cope with redox stress. In P. aeruginosa, a primary mechanism for protection from redox stress is the antioxidant glutathione (GSH). GSH is a low-molecular-weight thiol-containing tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) that can function as a reversible reducing agent. GSH plays an important role in P. aeruginosa physiology and is known to modulate several cellular and social processes that are likely important during infection. However, the role of GSH biosynthesis during mammalian infection is not well understood. In this study, we created a P. aeruginosa mutant defective in GSH biosynthesis to examine how loss of GSH biosynthesis affects P. aeruginosa virulence. We found that GSH is critical for normal growth in vitro and provides protection against hydrogen peroxide, bleach, and ciprofloxacin. We also studied the role of P. aeruginosa GSH biosynthesis in four mouse infection models, including the surgical wound, abscess, burn wound, and acute pneumonia models. We discovered that the GSH biosynthesis mutant was slightly less virulent in the acute pneumonia infection model but was equally virulent in the three other models. This work provides new and complementary data regarding the role of GSH in P. aeruginosa during mammalian infection.
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Glutatión/biosíntesis , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Infecciones de los Tejidos Blandos/microbiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desinfectantes/farmacología , Farmacorresistencia Bacteriana , Interacciones Huésped-Patógeno , Humanos , Viabilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
The opportunistic bacterial pathogen Pseudomonas aeruginosa causes acute and chronic infections that are notoriously difficult to treat. In people with cystic fibrosis, P. aeruginosa can cause lifelong lung infections, and isolation of mucoid P. aeruginosa, resulting from the overproduction of alginate, is associated with chronic infection. The histone-like protein AlgP has previously been implicated in the control of alginate gene expression in mucoid strains, but this regulation is unclear. To explore AlgP in further detail, we deleted algP in mucoid strains and demonstrated that the deletion of algP did not result in a nonmucoid phenotype or a decrease in alginate production. We showed that the algP promoter is expressed by both the nonmucoid strain PAO1 and the isogenic mucoid strain PDO300, suggesting that there may be genes that are differentially regulated between these strains. In support of this, using RNA sequencing, we identified a small AlgP regulon that has no significant overlap between PAO1 and PDO300 and established that alginate genes were not differentially regulated by the deletion of algP. Of note, we found that deleting algP in PAO1 increased expression of the nitric oxide operon norCBD and the nitrous oxide reductase genes nosRZ and subsequently promoted growth of PAO1 under anaerobic conditions. Altogether, we have defined a narrow regulon of genes controlled by AlgP and provided evidence that alginate production is not greatly affected by AlgP, countering the long-standing premise in the field.
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Alginatos/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Pseudomonas aeruginosa/genética , Regulón , Factores de Transcripción/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Humanos , Óxido Nítrico/metabolismo , Operón , Regiones Promotoras Genéticas , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.
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Fibrosis Quística/metabolismo , PPAR gamma/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Respuesta de Proteína Desplegada , Células A549 , Arildialquilfosfatasa/metabolismo , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Estrés del Retículo Endoplásmico , Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Interleucina-8/metabolismo , Mitocondrias/metabolismo , PPAR gamma/agonistas , Pioglitazona , Infecciones por Pseudomonas/inmunología , Factor de Transcripción CHOP/metabolismo , beta-Defensinas/metabolismoRESUMEN
Quorum sensing (QS) is a bacterial cell-to-cell communication process that relies on the production, release, and response to extracellular signaling molecules called autoinducers. QS controls virulence and biofilm formation in the human pathogen Pseudomonas aeruginosa. P. aeruginosa possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, which produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Here, we use biofilm analyses, reporter assays, RNA-seq studies, and animal infection assays to show that RhlR directs both RhlI-dependent and RhlI-independent regulons. In the absence of RhlI, RhlR controls the expression of genes required for biofilm formation as well as genes encoding virulence factors. Consistent with these findings, ΔrhlR and ΔrhlI mutants have radically different biofilm phenotypes and the ΔrhlI mutant displays full virulence in animals whereas the ΔrhlR mutant is attenuated. The ΔrhlI mutant cell-free culture fluids contain an activity that stimulates RhlR-dependent gene expression. We propose a model in which RhlR responds to an alternative ligand, in addition to its canonical C4-homoserine lactone autoinducer. This alternate ligand promotes a RhlR-dependent transcriptional program in the absence of RhlI.
Asunto(s)
4-Butirolactona/análogos & derivados , Proteínas Bacterianas/metabolismo , Biopelículas , Regulación Bacteriana de la Expresión Génica , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/genética , Percepción de Quorum , Regulón , VirulenciaRESUMEN
Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P.â aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.