scholarly journals Small molecules with antibiofilm, antivirulence and antibiotic synergy activities againstPseudomonas aeruginosa

2016 ◽  
Author(s):  
Erik van Tilburg Bernardes ◽  
Laetitia Charron-Mazenod ◽  
David Reading ◽  
Shauna L. Reckseidler-Zenteno ◽  
Shawn Lewenza
Keyword(s):  
Cystic Fibrosis ◽  
Extracellular Matrix ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Small Molecules ◽  
Biofilm Formation ◽  
Chronic Infections ◽  
Term Survival ◽  
Lung Infections

AbstractBiofilm formation is a universal bacterial strategy for long-term survival in nature and during infections. Biofilms are dense microbial communities enmeshed within a polymeric extracellular matrix that protects bacteria from antibiotic exposure and the immune system and thus contribute to chronic infections.Pseudomonas aeruginosais an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in Cystic Fibrosis (CF) patients. The extracellular matrix ofP. aeruginosabiofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and non-mucoid isolates ofP. aeruginosaproduces the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation and immune evasion. Given the central importance of the Pel and Psl EPS in biofilm structure, they are attractive targets for novel anti-infective compounds. In this study we used a high throughput gene expression screen to identify compounds that repress expression ofpelandpslgenes as measured by transcriptionalluxfusions. Testing of thepel/pslrepressors demonstrated an antibiofilm activity against microplate and flow chamber biofilms formed by wild type and hyperbiofilm forming strains. To determine the potential role of EPS in virulence, mutants inpel/pslwere shown to have reduced virulence in the feeding behavior and slow killing virulence assays inCaenorhabditis elegans. The antibiofilm molecules also reducedP. aeruginosaPAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds were synergistic in killingP. aeruginosabiofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronicP. aeruginosainfections.Author summaryBacteria use the strategy of growing as a biofilm to promote long-term survival and therefore to cause chronic infections. One of the best examples isPseudomonas aeruginosaand the chronic lung infections in individuals with Cystic Fibrosis (CF). Biofilms are generally a dense community of bacteria enmeshed in an extracellular matrix that protects bacteria from numerous environmental stresses, including antibiotics and the immune system. In this study we developed an approach to identifyP. aeruginosabiofilm inhibitors by repressing the production of the matrix exopolysaccharide (EPS) polymers. Bacteria treated with compounds and then fed to the nematode also had showed reduced virulence by promoting nematode survival. To tackle the problem of biofilm tolerance of antibiotics, the compounds identified here also had the beneficial property of increasing the biofilm sensitivity to different classes of antibiotics. The compounds disarm bacteria but they do not kill or limit growth like antibiotics. We provide further support that disarmingP. aeruginosamay be a critical anti-infective strategy that limits the development of antibiotic resistance, and provides a new way for treating chronic infections.

scholarly journals Psl Produced by Mucoid Pseudomonas aeruginosa Contributes to the Establishment of Biofilms and Immune Evasion

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Christopher J. Jones ◽  
Daniel J. Wozniak
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Immune Evasion ◽  
Biofilm Formation ◽  
Primary Concern ◽  
Pulmonary Infections ◽  
Chronic Infections ◽  
Key Factor ◽  
Current Therapies ◽  
Lung Infections

ABSTRACT Despite years of research and clinical advances, chronic pulmonary infections with mucoid Pseudomonas aeruginosa remain the primary concern for cystic fibrosis patients. Much of the research on these strains has focused on the contributions of the polysaccharide alginate; however, it is becoming evident that the neutral polysaccharide Psl also contributes to biofilm formation and the maintenance of chronic infections. Here, we demonstrate that Psl produced by mucoid strains has significant roles in biofilm structure and evasion of immune effectors. Though mucoid strains produce less Psl than nonmucoid strains, the Psl that is produced is functional, since it mediates adhesion to human airway cells and epithelial cell death. Additionally, Psl protects mucoid bacteria from opsonization and killing by complement components in human serum. Psl production by mucoid strains stimulates a proinflammatory response in the murine lung, leading to reduced colonization. To determine the relevance of these data to clinical infections, we tested Psl production and biofilm formation of a panel of mucoid clinical isolates. We demonstrated three classes of mucoid isolates, those that produce Psl and form robust biofilms, those that did not produce Psl and have a poor biofilm phenotype, and exopolysaccharide (EPS) redundant strains. Collectively, these experimental results demonstrate that Psl contributes to the biofilm formation and immune evasion of many mucoid strains. This is a novel role for Psl in the establishment and maintenance of chronic pulmonary infections by mucoid strains. IMPORTANCE Cystic fibrosis patients are engaged in an ongoing battle against chronic lung infections by the bacterium Pseudomonas aeruginosa. One key factor contributing to the maintenance of chronic infections is the conversion to a mucoid phenotype, where the bacteria produce copious amounts of the polysaccharide alginate. Once the bacteria become mucoid, existing treatments are poorly effective. We proposed that mucoid bacteria produce an additional polysaccharide, Psl, which is important for their establishment and maintenance of chronic infections. This work demonstrates that Psl enhances attachment of mucoid bacteria to lung surfaces and leads to inflammation and damage in the lung. Additionally, we find that 50% of mucoid bacteria isolated from patients with chronic infections rely on Psl for the structure of their biofilm communities, suggesting that treatments against Psl should be investigated to enhance the success of current therapies. IMPORTANCE Cystic fibrosis patients are engaged in an ongoing battle against chronic lung infections by the bacterium Pseudomonas aeruginosa. One key factor contributing to the maintenance of chronic infections is the conversion to a mucoid phenotype, where the bacteria produce copious amounts of the polysaccharide alginate. Once the bacteria become mucoid, existing treatments are poorly effective. We proposed that mucoid bacteria produce an additional polysaccharide, Psl, which is important for their establishment and maintenance of chronic infections. This work demonstrates that Psl enhances attachment of mucoid bacteria to lung surfaces and leads to inflammation and damage in the lung. Additionally, we find that 50% of mucoid bacteria isolated from patients with chronic infections rely on Psl for the structure of their biofilm communities, suggesting that treatments against Psl should be investigated to enhance the success of current therapies.

scholarly journals Metabotypes of Pseudomonas aeruginosa Correlate with Antibiotic Resistance, Virulence and Clinical Outcome in Cystic Fibrosis Chronic Infections

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 63
Author(s):  
Oriane Moyne ◽  
Florence Castelli ◽  
Dominique J. Bicout ◽  
Julien Boccard ◽  
Boubou Camara ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Multiscale Analysis ◽  
Resistant Bacteria ◽  
Chronic Infections ◽  
Antibiotic Resistant ◽  
Causes Of Mortality ◽  
Lung Infections ◽  
Clinical Measurements

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a “metabotypes”, expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections

scholarly journals Heterogenous response to R-pyocin killing in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

2020 ◽  
Author(s):  
Madeline Mei ◽  
Jacob Thomas ◽  
Stephen P. Diggle
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Genotypic Diversity ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Bacterial Populations ◽  
Heterogeneous Populations ◽  
Lung Infections ◽  
The Impact ◽  
Lps Binding

AbstractBacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources and (ii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.ImportanceR-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection, however little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics, as well as evolve phenotypic and genotypic diversity over time; thus it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is likely heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

scholarly journals Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections

2014 ◽  
Vol 9 (6) ◽  
pp. 1391-1398 ◽  
Author(s):  
Chloe E James ◽  
Emily V Davies ◽  
Joanne L Fothergill ◽  
Martin J Walshaw ◽  
Colin M Beale ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lytic Activity ◽  
Lung Infections

scholarly journals Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
Yuqing Long ◽  
Weixin Fu ◽  
Su Wang ◽  
Xuan Deng ◽  
Yongxin Jin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Bacterial Resistance ◽  
Global Gene Expression ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type ◽  
Regulatory Pathways ◽  
Opportunistic Pathogenic

ABSTRACT Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa. In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa. The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

scholarly journals Overproduction of the AlgT Sigma Factor Is Lethal to Mucoid Pseudomonas aeruginosa

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Ashley R. Cross ◽  
Vishnu Raghuram ◽  
Zihuan Wang ◽  
Debayan Dey ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Continuous Production ◽  
Common Mutation ◽  
Wild Type ◽  
Chronic Infections ◽  
Truncated Form ◽  
Content Type ◽  
Lung Infections

ABSTRACT 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. IMPORTANCE Pseudomonas 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 ∼300 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.

scholarly journals Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

2020 ◽  
Author(s):  
Wai Leong ◽  
Carla Lutz ◽  
Jonathan Williams ◽  
Yan Hong Poh ◽  
Benny Yeo Ken Yee ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Late Stage ◽  
Early Stage ◽  
Acanthamoeba Castellanii ◽  
Opportunistic Pathogen ◽  
Human Neutrophils ◽  
Phenotypic Traits ◽  
Chronic Infections

AbstractThe opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types.Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

scholarly journals Overproduction of the AlgT sigma factor is lethal to mucoid Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Ashley R. Cross ◽  
Vishnu Raghuram ◽  
Zihuan Wang ◽  
Debayan Dey ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Continuous Production ◽  
Bacterial Pathogen ◽  
Common Mutation ◽  
Wild Type ◽  
Chronic Infections ◽  
Truncated Form ◽  
Lung Infections

ABSTRACTPseudomonas 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. Our findings support a model where 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 ~300 additional genes including algT itself. 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.

scholarly journals Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

2022 ◽  
Author(s):  
Wai Leong ◽  
Wee Han Poh ◽  
Jonathan Williams ◽  
Carla Lutz ◽  
M. Mozammel Hoque ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Late Stage ◽  
Early Stage ◽  
Opportunistic Pathogen ◽  
Phenotypic Traits ◽  
Nucleotide Polymorphisms ◽  
Chronic Infections ◽  
The Impact

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

scholarly journals Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

2020 ◽  
Vol 203 (2) ◽  
pp. e00300-20
Author(s):  
Katie V. Farrant ◽  
Livia Spiga ◽  
Jane C. Davies ◽  
Huw D. Williams
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Immune System ◽  
Hypochlorous Acid ◽  
Secretion System ◽  
Content Type ◽  
Type 3 Secretion System ◽  
Lung Infections ◽  
Type 3

ABSTRACTPseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

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