scholarly journals Alginate-regulating genes are identified in the clinical cystic fibrosis isolate ofPseudomonas aeruginosaPA2192

2018 ◽  
Author(s):  
Brett Colbert ◽  
Hansi Kumari ◽  
Ana Piñon ◽  
Lior Frey ◽  
Sundar Pandey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Sigma Factor ◽  
Bacterial Colonization ◽  
Genetic Disorder ◽  
Clinical Strain ◽  
Coding Region ◽  
Alginate Production ◽  
Gene Coding ◽  
Cystic Fibrosis Isolate

ABSTRACTCystic fibrosis (CF) is a genetic disorder that leads to a buildup of mucus in the lungs ideal for bacterial colonization. WhenPseudomonas aeruginosaenters the CF lung, it undergoes a conversion from nonmucoid to mucoid; colonization by a mucoid strain ofP. aeruginosagreatly increases mortality. The mucoid phenotype is due to the production of alginate. The regulator of alginate production is the AlgT/U sigma factor. The observed phenotypic conversion is due to a mutation in themucAgene coding for an anti-sigma factor, MucA, which sequesters AlgT/U. This mucoid phenotype is unstable when the strains are removed from the lung as they acquire second-site mutations. Thisin vitroreversion phenomenon is utilized to identify novel genes regulating alginate production. Previously, second-site mutations were mapped toalgT/U, algO,andmucP, demonstrating their role in alginate regulation. Most of these studies were performed using a non-CF isolate. It was hypothesized that second site mutations in a clinical strain would be mapped to the same genes. In this study, a clinical, hyper-mucoidP. aeruginosastrain PA2192 was used to study the reversion phenomenon. This study found that PA2192 has a novelmucAmutation which was named themmucA180allele. Twelve colonies were sub-cultured for two weeks without aeration at room temperature in order to obtain nonmucoidsuppressors ofalginateproduction(sap). Only 41sapmutants were stable for more than 48 hours — a reversion frequency of 3.9% as compared to ~90% in laboratory strains showing that PA2192 has a stable mucoid phenotype. This phenotype was restored in 28 of the 41sapmutants when complemented with plasmids harboringalgT/U. Four of thesapmutants are complemented byalgO. Sequence analyses of thealgT/Umutants have found no mutations in the coding region or promoter leading to the hypothesis that there is another, as yet unidentified mechanism of alginate regulation in this clinical strain.

scholarly journals Most of Staphylococcus aureus and Pseudomonas aeruginosa co-infecting isolates coexist, a condition that may impact clinical outcomes in Cystic Fibrosis patients

2020 ◽  
Author(s):  
Paul Briaud ◽  
Sylvère Bastien ◽  
Laura Camus ◽  
Marie Boyadjian ◽  
Philippe Reix ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Multivariate Analysis ◽  
Natural History ◽  
Clinical Outcomes ◽  
Bacterial Colonization ◽  
Childhood And Adolescence ◽  
History Of

AbstractStaphylococcus aureus (SA) is the major colonizer of the lung of cystic fibrosis (CF) patient during childhood and adolescence. As patient aged, the prevalence of SA decreases and Pseudomonas aeruginosa (PA) becomes the major pathogen infecting adult lungs. Nonetheless, SA remains significant and patients harbouring both SA and PA are frequently found in worldwide cohort. Impact of coinfection remains controversial. Furthermore, co-infecting isolates may compete or coexist. The aim of this study was to analyse if co-infection and coexistence of SA and PA could lead to worse clinical outcomes. The clinical and bacteriological data of 212 Lyon CF patients were collected retrospectively, and patients were ranked into three groups, SA only (n=112), PA only (n=48) or SA plus PA (n=52). In addition, SA and PA isolates from co-infecting patients were tested in vitro to define their interaction profile. Sixty five percent (n=34) of SA/PA pairs coexist. Using univariate and multivariate analysis, we confirm that SA patients have a clinical condition less severe than others, and PA induce a poor outcome independently of the presence of SA. FEV1 is lower in patients infected by competition strain pairs than in those infected by coexisting strain pairs compared to SA mono-infection. Coexistence between SA and PA may be an important step in the natural history of lung bacterial colonization within CF patients.

scholarly journals Analysis of the gene coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46,XY disorders of sex development

2014 ◽  
Vol 170 (5) ◽  
pp. 759-767 ◽  
Author(s):  
Sally Tantawy ◽  
Inas Mazen ◽  
Hala Soliman ◽  
Ghada Anwar ◽  
Abeer Atef ◽  
...  
Keyword(s):  
Adrenal Insufficiency ◽  
Direct Sequencing ◽  
Disorders Of Sex Development ◽  
Missense Mutations ◽  
Coding Region ◽  
Steroidogenic Factor 1 ◽  
Sex Development ◽  
Gene Coding ◽  
Future Fertility

ObjectiveSteroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamic–pituitary–gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD.DesignClinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum.MethodsMolecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected.ResultsThree novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Müllerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism.ConclusionWe identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5–15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

scholarly journals Case report of a successful pregnancy in a cystic fibrosis patient with the c.1521_1523delCTT/c.3718-2477C>t genotypes

2020 ◽  
Vol 23 (2) ◽  
pp. 103-106
Author(s):  
VL Spasova ◽  
LI Koleva ◽  
DI Toncheva ◽  
VI Karamisheva
Keyword(s):  
Cystic Fibrosis ◽  
Case Report ◽  
Bacterial Colonization ◽  
Genetic Disorder ◽  
Vital Signs ◽  
Forced Expiratory Volume ◽  
Rare Mutation ◽  
Normal Limits ◽  
The Right ◽  
Common Genetic Disorder

Abstract The aim of this case report was to show the consequences of pregnancy in a cystic fibrosis (CF) patient with a rare mutation. We present a case of a patient with CF, pregnant for the second time, who gave birth to a healthy child. Her mutation status revealed the presence of relatively rare mutation c.3718-2477C>T that is associated with a milder phenotype of the disease. During pregnancy, her vital signs were within normal limits. She had no exacerbations after the third gestational month. Cystic fibrosis is the most common genetic disorder among Caucasians. Over the last few decades, the survival rate and the lifespan of patients with CF have increased progressively. This is why more affected women are choosing to become pregnant. Predictive factors for the pregnancy outcome are basal pulmonary function [measured by forced expiratory volume/1 second (FEV1)], nutritional status [measured by body mass index (BMI)], diabetes and bacterial colonization. The report of our case emphasizes the need for establishing the exact mutations in CF patients who plan to become pregnant in order to predict the possible outcomes of this specific period of life. Moreover, genetic counseling is strongly recommended for the right understanding of the pregnancy risks in such cases.

A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations

2020 ◽  
Vol 56 (5) ◽  
pp. 2000205 ◽  
Author(s):  
Huibi Cao ◽  
Hong Ouyang ◽  
Onofrio Laselva ◽  
Claire Bartlett ◽  
Zhichang Peter Zhou ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Adenoviral Vector ◽  
Genetic Disorder ◽  
Ussing Chamber ◽  
Reproductive Organs ◽  
Gene Replacement ◽  
Class I ◽  
Multiple Organs ◽  
Healthy Control

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose–response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

scholarly journals Specialized Pro-Resolving Lipid Mediators in Cystic Fibrosis

2018 ◽  
Vol 19 (10) ◽  
pp. 2865 ◽  
Author(s):  
Réginald Philippe ◽  
Valerie Urbach
Keyword(s):  
Cystic Fibrosis ◽  
Structural Damage ◽  
Bacterial Colonization ◽  
Airway Disease ◽  
Lipid Mediators ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Persistent Inflammation ◽  
Gene Coding ◽  
Cystic Fibrosis Transmembrane

In cystic fibrosis (CF), impaired airway surface hydration (ASL) and mucociliary clearance that promote chronic bacterial colonization, persistent inflammation, and progressive structural damage to the airway wall architecture are typically explained by ion transport abnormalities related to the mutation of the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. However, the progressive and unrelenting inflammation of the CF airway begins early in life, becomes persistent, and is excessive relative to the bacterial burden. Intrinsic abnormalities of the inflammatory response in cystic fibrosis have been suggested but the mechanisms involved remain poorly understood. This review aims to give an overview of the recent advances in the understanding of the defective resolution of inflammation in CF including the abnormal production of specialized pro-resolving lipid mediators (lipoxin and resolvin) and their impact on the pathogenesis of the CF airway disease.

Prc protease promotes mucoidy in mucA mutants of Pseudomonas aeruginosa

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2251-2261 ◽  
Author(s):  
S. A. Reiling ◽  
J. A. Jansen ◽  
B. J. Henley ◽  
S. Singh ◽  
C. Chattin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Cluster ◽  
Sigma Factor ◽  
Respiratory Infections ◽  
Negative Regulator ◽  
Coding Region ◽  
Extreme Stress ◽  
Alginate Production ◽  
Stress Response System ◽  
Mutant Forms

Mucoid strains of Pseudomonas aeruginosa that overproduce the exopolysaccharide alginate are a frequent cause of chronic respiratory infections in cystic fibrosis (CF) patients. The overproduction of alginate by these strains is often caused by mutations within mucA of the algU mucABCD gene cluster. This gene cluster encodes an extreme stress response system composed of the ECF alternative sigma factor AlgU, the anti-sigma factor MucA located in the inner membrane and the negative regulator MucB located in the periplasm. Most of the mutations in mucA found in mucoid strains cause a truncation of the C-terminal, periplasmic domain of MucA. The most significant effect of these mutations appears to be to reduce the levels of MucA. PA3257 (prc) was identified as a regulator of alginate production in P. aeruginosa through the isolation and study of mutations that partially suppressed the mucoid phenotype of a mucA22 strain. The suppressor of mucoidy (som) mutants isolated produced very little alginate when grown on LB medium, but were still mucoid when grown on Pseudomonas isolation agar. These som mutations and another previously isolated suppressor mutation were complemented by cosmids or plasmids carrying PA3257. PA3257 is predicted to encode a periplasmic protease similar to Prc or Tsp of Escherichia coli. Sequencing of prc from three strains with som suppressor mutations confirmed that each had a mutation within the prc coding region. The authors propose that Prc acts to degrade mutant forms of MucA. Additional evidence in support of this hypothesis is: (1) transcription from the AlgU-regulated algD reporter was reduced in som mutants; (2) inactivation of prc affected alginate production in mucoid strains with other mucA mutations found in CF isolates; (3) inactivation or overexpression of prc did not affect alginate production in strains with wild-type MucA.

scholarly journals Blocking the alternative sigma factor RpoN reduces virulence ofPseudomonas aeruginosaisolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

2018 ◽  
Author(s):  
MG Lloyd ◽  
JL Vossler ◽  
CT Nomura ◽  
JF Moffat
Keyword(s):  
Cystic Fibrosis ◽  
Sigma Factor ◽  
Antimicrobial Agents ◽  
Laboratory Strain ◽  
Infection Model ◽  
Alternative Sigma Factor ◽  
Beta Lactam ◽  
Cis Acting

AbstractMultidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria.Pseudomonas aeruginosais an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections.P. aeruginosais inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment.P. aeruginosahas virulence factors that are regulated by sigma factors in response to the tissue microenvironment. The alternative sigma factor, RpoN (σ54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a “molecular roadblock”, binding RpoN consensus promoters at the −24 site and blocking transcription. RpoN* reduces virulence ofP. aeruginosalaboratory strains bothin vitroandin vivo,but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically variedP. aeruginosastrains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in aP. aeruginosa – C. elegansinfection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strainP. aeruginosaPA19660Xen5. Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

scholarly journals The Pseudomonas aeruginosa Flagellar Cap Protein, FliD, Is Responsible for Mucin Adhesion

1998 ◽  
Vol 66 (3) ◽  
pp. 1000-1007 ◽  
Author(s):  
Shiwani K. Arora ◽  
Bruce W. Ritchings ◽  
Ernesto C. Almira ◽  
Stephen Lory ◽  
Reuben Ramphal
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Promoter Region ◽  
Transcriptional Regulator ◽  
Adhesion Assay ◽  
Multicopy Plasmid ◽  
Binding Sequence ◽  
Adhesion Process

ABSTRACT Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosaPAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of thefliD gene was independent of the flagellar sigma factor ς28. Consistent with this finding, no ς28binding sequence could be identified in the fliD promoter region. The results of the β-galactosidase assays suggest that thefliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor ς54 (RpoN).

scholarly journals Mixed Communities of Mucoid and NonmucoidPseudomonas aeruginosaExhibit Enhanced Resistance to Host Antimicrobials

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Sankalp Malhotra ◽  
Dominique H. Limoli ◽  
Anthony E. English ◽  
Matthew R. Parsek ◽  
Daniel J. Wozniak
Keyword(s):  
Cystic Fibrosis ◽  
Extracellular Dna ◽  
Cationic Antimicrobial Peptide ◽  
Alginate Production ◽  
Enhanced Resistance ◽  
Extracellular Release ◽  
Mucoid Conversion ◽  
Mixed Populations

ABSTRACTPseudomonas aeruginosacauses chronic pulmonary infections in patients with cystic fibrosis (CF).P. aeruginosamucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype bothin vitroandin vivo. Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H2O2). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H2O2stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent onlys, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions ofP. aeruginosamucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung.IMPORTANCEP. aeruginosamucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities ofP. aeruginosademonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversifiedP. aeruginosacommunitiesin vivo, as these variants can differentially shield one another from components of the host response.

scholarly journals RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

2013 ◽  
Vol 305 (1) ◽  
pp. C111-C120 ◽  
Author(s):  
Laura Smith ◽  
Paul Litman ◽  
Ekta Kohli ◽  
Joseph Amick ◽  
Richard C. Page ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Genetic Disorder ◽  
Surface Expression ◽  
Epithelial Cell Line ◽  
Common Mutation ◽  
Airway Epithelial ◽  
Filamin A ◽  
Cystic Fibrosis Transmembrane

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+ exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization.

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