Targeted Chemoenzymatic Synthesis of Sugar Nucleotide Probes Reveal an Inhibitor of the GDP-D-Mannose Dehydrogenase from Pseudomonas Aeruginosa

Sufferers of the autosomal recessive genetic disorder cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime one bacterial strain in particular, Pseudomonas aeruginosa, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a phenomenon known as mucoid conversion, resulting in copious secretion of alginate, a carbohydrate exopolysaccharide. Strategies that can stop the production of alginate in mucoid Pseudomonas aeruginosa infections are therefore of paramount importance. To aid in this we developed a series of sugar nucleotide chemical tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD). This enzyme catalyses the irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy we accessed a series of modified sugar nucleotides, identifying a C6-amide derivative of the native substrate as a micromolar inhibitor of GMD. This discovery will provide a framework for wider inhibition strategies against GMD to be developed.

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Targeted Chemoenzymatic Synthesis of Sugar Nucleotide Probes Reveal an Inhibitor of the GDP-D-Mannose Dehydrogenase from Pseudomonas Aeruginosa

10.26434/chemrxiv.12369758 ◽

2020 ◽

Author(s):

Laura Beswick ◽

Eleni Dimitriou ◽

Sanaz Ahmadipour ◽

Ayesha Zafar ◽

Martin Rejzek ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Autosomal Recessive ◽

Genetic Disorder ◽

Loss Of Function ◽

Bacterial Strains ◽

Sugar Nucleotide ◽

Guanosine Diphosphate ◽

Mannuronic Acid ◽

Mucoid Conversion ◽

Lung Infections

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The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

Journal of Bacteriology ◽

10.1128/jb.01490-08 ◽

2009 ◽

Vol 191 (7) ◽

pp. 2285-2295 ◽

Cited By ~ 53

Author(s):

F. Heath Damron ◽

Dongru Qiu ◽

Hongwei D. Yu

Keyword(s):

Pseudomonas Aeruginosa ◽

Response Regulator ◽

Negative Regulator ◽

Loss Of Function ◽

Cystic Fibrosis Mutation ◽

Strain Pao1 ◽

Alginate Production ◽

Mucoid Conversion ◽

Σ Factor ◽

Lung Infections

ABSTRACT Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative σ factor RpoN (σ54) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

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Potentiation of Aminoglycoside Lethality by C4-Dicarboxylates Requires RpoN in Antibiotic-Tolerant Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01313-19 ◽

2019 ◽

Vol 63 (10) ◽

Author(s):

Clayton W. Hall ◽

Eszter Farkas ◽

Li Zhang ◽

Thien-Fah Mah

Keyword(s):

Pseudomonas Aeruginosa ◽

Carbon Sources ◽

Aminoglycoside Antibiotic ◽

Loss Of Function ◽

Mixed Genotype ◽

Content Type ◽

Promising Strategy ◽

Transport Chain ◽

Suitable Treatment ◽

Lung Infections

ABSTRACT Antibiotic tolerance contributes to the inability of standard antimicrobial therapies to clear the chronic Pseudomonas aeruginosa lung infections that often afflict patients with cystic fibrosis (CF). Metabolic potentiation of bactericidal antibiotics with carbon sources has emerged as a promising strategy to resensitize tolerant bacteria to antibiotic killing. Fumarate (FUM), a C4-dicarboxylate, has been recently shown to resensitize tolerant P. aeruginosa to killing by tobramycin (TOB), an aminoglycoside antibiotic, when used in combination (TOB+FUM). Fumarate and other C4-dicarboxylates are taken up intracellularly by transporters regulated by the alternative sigma factor RpoN. Once in the cell, FUM is metabolized, leading to enhanced electron transport chain activity, regeneration of the proton motive force, and increased TOB uptake. In this work, we demonstrate that a ΔrpoN mutant displays impaired FUM uptake and, consequently, nonsusceptibility to TOB+FUM treatment. RpoN was also found to be essential for susceptibility to other aminoglycoside and C4-dicarboxylate combinations. Importantly, RpoN loss-of-function mutations have been documented to evolve in the CF lung, and these loss-of-function alleles can also result in TOB+FUM nonsusceptibility. In a mixed-genotype population of wild-type and ΔrpoN cells, TOB+FUM specifically killed cells with RpoN function and spared the cells that lacked RpoN function. Unlike C4-dicarboylates, both d-glucose and l-arginine were able to potentiate TOB killing of ΔrpoN stationary-phase cells. Our findings raise the question of whether TOB+FUM will be a suitable treatment option in the future for CF patients infected with P. aeruginosa isolates that lack RpoN function.

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Frequency of quorum sensing mutations in Pseudomonas aeruginosa strains isolated from different environments

10.1101/2021.02.22.432365 ◽

2021 ◽

Author(s):

Kathleen O’Connor ◽

Conan Y. Zhao ◽

Stephen P. Diggle

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Opportunistic Pathogen ◽

Human Infection ◽

Multiple Infection ◽

Loss Of Function ◽

Mutational Signatures ◽

Amino Acid Length ◽

Lung Infections

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. lasR QS mutations have been shown to frequently arise in cystic fibrosis (CF) lung infections, however, there has been far less emphasis on determining whether QS system mutations arise across other environments. To test this, we utilized 852 publicly available sequenced P. aeruginosa genomes from the Pseudomonas International Consortium Database (IPCD) to study P. aeruginosa QS mutational signatures. We found that across all isolates, LasR is the most variable protein sequence compared to other QS proteins. In order to study isolates by source, we focused on a subset of 654 isolates collected from CF, wounds, and non-infection environmental isolates, where we could clearly identify their source. Using this sub-set analysis, we found that LasR mutations are not specific to CF lungs, but are common across all environments. We then used amino acid length as a proxy for observing loss of function in LasR proteins among the strains. We found that truncated LasR proteins are more abundant in P. aeruginosa strains isolated from human infection than the environment. Overall, our findings suggest that the evolution of lasR QS mutations in P. aeruginosa are common and not limited to infection environments.ImportancePseudomonas aeruginosa is an opportunistic pathogen which is often isolated from infection and environmental sources. P. aeruginosa uses quorum sensing (QS) to establish and adapt to infection environments. QS in P. aeruginosa is controlled by a complex hierarchical gene network in which the transcriptional regulator LasR has traditionally been thought to play a major controlling role. Despite this, lasR mutants are frequently isolated from chronic infection sites including the cystic fibrosis lung. Using an online P. aeruginosa strain database, we determined the frequency of mutation in key QS genes in multiple infection and non-infection environments and found that mutations and truncations in the lasR gene is more common than in other QS genes. Further, we found that lasR mutants are common in both infection and environmental strains. These findings further our understanding of QS in P. aeruginosa and have implications for the development of future therapies designed to inhibit QS.

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Instantaneous Within-Patient Diversity of Pseudomonas aeruginosa Quorum-Sensing Populations from Cystic Fibrosis Lung Infections

Infection and Immunity ◽

10.1128/iai.00755-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5631-5639 ◽

Cited By ~ 84

Author(s):

Cara N. Wilder ◽

Gopal Allada ◽

Martin Schuster

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Loss Of Function ◽

Selective Forces ◽

Lung Infections

ABSTRACT In the opportunistic pathogen Pseudomonas aeruginosa, acyl-homoserine lactone (acyl-HSL) quorum sensing (QS) regulates biofilm formation and expression of many extracellular virulence factors. Curiously, QS-deficient variants, often carrying mutations in the central QS regulator LasR, are frequently isolated from infections, particularly from cystic fibrosis (CF) lung infections. Very little is known about the proportion and diversity of these QS variants in individual infections. Such information is desirable to better understand the selective forces that drive the evolution of QS phenotypes, including social cheating and innate (nonsocial) benefits. To obtain insight into the instantaneous within-patient diversity of QS, we assayed a panel of 135 concurrent P. aeruginosa isolates from eight different adult CF patients (9 to 20 isolates per patient) for various QS-controlled phenotypes. Most patients contained complex mixtures of QS-proficient and -deficient isolates. Among all patients, deficiency in individual phenotypes ranged from 0 to about 90%. Acyl-HSL, sequencing, and complementation analyses of variants with global loss-of-function phenotypes revealed dependency upon the central QS circuitry genes lasR, lasI, and rhlI. Deficient and proficient isolates were clonally related, implying evolution from a common ancestor in vivo. Our results show that the diversity of QS types is high within and among patients, suggesting diverse selection pressures in the CF lung. A single selective mechanism, be it of a social or nonsocial nature, is unlikely to account for such heterogeneity. The observed diversity also shows that conclusions about the properties of P. aeruginosa QS populations in individual CF infections cannot be drawn from the characterization of one or a few selected isolates.

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Quorum Sensing as Antivirulence Target in Cystic Fibrosis Pathogens

International Journal of Molecular Sciences ◽

10.3390/ijms20081838 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1838 ◽

Cited By ~ 19

Author(s):

Scoffone ◽

Trespidi ◽

Chiarelli ◽

Barbieri ◽

Buroni

Keyword(s):

Cystic Fibrosis ◽

Quorum Sensing ◽

Burkholderia Cepacia ◽

Autosomal Recessive ◽

Genetic Disorder ◽

Burkholderia Cepacia Complex ◽

Mucus Layer ◽

Emerging Pathogens ◽

Alternative Pathways ◽

Lung Infections

Cystic fibrosis (CF) is an autosomal recessive genetic disorder which leads to the secretion of a viscous mucus layer on the respiratory epithelium that facilitates colonization by various bacterial pathogens. The problem of drug resistance has been reported for all the species able to colonize the lung of CF patients, so alternative treatments are urgently needed. In this context, a valid approach is to investigate new natural and synthetic molecules for their ability to counteract alternative pathways, such as virulence regulating quorum sensing (QS). In this review we describe the pathogens most commonly associated with CF lung infections: Staphylococcus aureus, Pseudomonas aeruginosa, species of the Burkholderia cepacia complex and the emerging pathogens Stenotrophomonas maltophilia, Haemophilus influenzae and non-tuberculous Mycobacteria. For each bacterium, the QS system(s) and the molecules targeting the different components of this pathway are described. The amount of investigations published in the last five years clearly indicate the interest and the expectations on antivirulence therapy as an alternative to classical antibiotics.

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Evolution and adaptation of Pseudomonas aeruginosa in the paranasal sinuses of people with cystic fibrosis

10.1101/2020.10.29.359844 ◽

2020 ◽

Author(s):

Catherine R. Armbruster ◽

Christopher W. Marshall ◽

Jeffrey A. Melvin ◽

Anna C. Zemke ◽

Arkadiy I. Garber ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Disease ◽

Respiratory Infections ◽

Genetic Disorder ◽

List Type ◽

Host Restriction ◽

Single Clone ◽

Lung Infections ◽

End Stage

AbstractPeople with the genetic disorder cystic fibrosis (CF) harbor lifelong respiratory infections, with morbidity and mortality frequently linked to chronic lung infections dominated by the opportunistically pathogenic bacterium Pseudomonas aeruginosa. During chronic CF lung infections, a single clone of P. aeruginosa can persist for decades and dominate end-stage CF lung disease due to its propensity to adaptively evolve to the respiratory environment, a process termed “pathoadaptation”. Chronic rhinosinusitis (CRS), chronic inflammation and infection of the sinonasal space, is highly prevalent in CF and the sinuses may serve as the first site in the respiratory tract to become colonized by bacteria that then proceed to seed lung infections. We identified three evolutionary genetic routes by which P. aeruginosa evolves in the sinuses of people with CF, including through the evolution of mutator lineages and proliferative insertion sequences and culminating in early genomic signatures of host-restriction. Our findings raise the question of whether a significant portion of the pathoadaptive phenotypes previously thought to have evolved in response to selective pressures in the CF lungs may have first arisen in the sinuses and underscore the link between sinonasal and lung disease in CF.Graphical abstract and highlightsPseudomonas aeruginosa undergoes adaptive evolution in the sinuses of people with CFOver time, pathoadapted strains display early signatures of genome degradation consistent with recent host restrictionMutations previously thought to occur in CF lungs may have first evolved in sinuses

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Kindler's Syndrome with Recurrent Neutropenia: Report of Two Cases from Saudi Arabia

Journal of Pediatric Genetics ◽

10.1055/s-0040-1721077 ◽

2020 ◽

Author(s):

Yousef Binamer ◽

Muzamil A. Chisti

Keyword(s):

Autosomal Recessive ◽

Common Mechanism ◽

Sequencing Analysis ◽

Loss Of Function ◽

Skin Atrophy ◽

Whole Exome ◽

Infancy And Childhood ◽

Genetics And Genomics ◽

New Feature ◽

Generation Sequencing

AbstractKindler syndrome (KS) is a rare photosensitivity disorder with autosomal recessive mode of inheritance. It is characterized by acral blistering in infancy and childhood, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility. Besides these major features, many minor presentations have also been reported in the literature. We are reporting two cases with atypical features of the syndrome and a new feature of recurrent neutropenia. Whole exome sequencing analysis was done using next-generation sequencing which detected a homozygous loss-of-function (LOF) variant of FERMT1 in both patients. The variant is classified as a pathogenic variant as per the American College of Medical Genetics and Genomics guidelines. Homozygous LOF variants of FERMT1 are a common mechanism of KS and as such confirm the diagnosis of KS in our patients even though the presentation was atypical.

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