Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

AbstractThe opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

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Neutron crystallography reveals novel mechanisms used by Pseudomonas aeruginosa for host-cell binding

10.1101/2021.09.24.461693 ◽

2021 ◽

Author(s):

Lukas Gajdos ◽

Matthew P Blakeley ◽

Michael Haertlein ◽

V Trevor Forsyth ◽

Juliette M Devos ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Calcium Ions ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Host Cells ◽

Carbohydrate Binding ◽

Hydrogen Atoms ◽

High Affinity ◽

Structural Details ◽

Neutron Crystallography

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

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Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains

Biosensors ◽

10.3390/bios11040124 ◽

2021 ◽

Vol 11 (4) ◽

pp. 124

Author(s):

Liliam K. Harada ◽

Waldemar Bonventi Júnior ◽

Erica C. Silva ◽

Thais J. Oliveira ◽

Fernanda C. Moreli ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infections ◽

Research Effort ◽

Acquired Resistance ◽

Integrated Approach ◽

Healthcare Services ◽

Host Cells ◽

Color Changes ◽

Metabolic Versatility ◽

The Matrix

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet’s biosphere, killing between 4–50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13–15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

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Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters

Molecules ◽

10.3390/molecules23123073 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3073 ◽

Cited By ~ 3

Author(s):

Lucie Dupin ◽

Mathieu Noël ◽

Silvère Bonnet ◽

Albert Meyer ◽

Thomas Géhin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Biofilm Formation ◽

Selective Advantage ◽

Host Cells ◽

Host Defenses ◽

Negative Bacterium ◽

High Affinity ◽

Structure Relationship ◽

Relationship Of

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

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PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

Infection and Immunity ◽

10.1128/iai.01388-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1638-1647 ◽

Cited By ~ 28

Author(s):

Ziyu Sun ◽

Jing Shi ◽

Chang Liu ◽

Yongxin Jin ◽

Kewei Li ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Bacterial Colonization ◽

Mrna Level ◽

Sos Response ◽

Opportunistic Pathogen ◽

Host Cells ◽

Chronic Infections ◽

Mobility Shift ◽

Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

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Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients

Microbiology ◽

10.1099/mic.0.26822-0 ◽

2004 ◽

Vol 150 (5) ◽

pp. 1315-1326 ◽

Cited By ~ 101

Author(s):

Julianne V. Kus ◽

Elizabeth Tullis ◽

Dennis G. Cvitkovitch ◽

Lori L. Burrows

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Host Cells ◽

Phylogenetic Groups ◽

Allele Distribution ◽

Specific Pcr ◽

Type Iv ◽

Accessory Genes ◽

Group I

Type IV pili (TFP) are important colonization factors of the opportunistic pathogen Pseudomonas aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic fibrosis (CF) isolates of P. aeruginosa. Based on the results, a new system of nomenclature is proposed, in which P. aeruginosa TFP are divided into five distinct phylogenetic groups. Each pilin allele is stringently associated with characteristic, distinct accessory genes that allow the identification of the allele by specific PCR. The invariant association of the pilin and accessory genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin genes from CF compared with non-CF human sources (P<0·0001), suggesting this particular pilin type, which can be post-translationally modified by glycosylation via the action of TfpO (PilO), may confer a colonization or persistence advantage in the CF host. This allele was also predominant in paediatric CF isolates (29 of 43; 67·4 %), showing that this bias is apparent early in colonization. Group I pilins were also the most common type found in environmental isolates tested. To the authors' knowledge, this is the first example of a P. aeruginosa virulence factor allele that is strongly associated with CF isolates.

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An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

eLife ◽

10.7554/elife.30205 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 28

Author(s):

Jeanyoung Jo ◽

Krista L Cortez ◽

William Cole Cornell ◽

Alexa Price-Whelan ◽

Lars EP Dietrich

Keyword(s):

Pseudomonas Aeruginosa ◽

Cytochrome C ◽

Opportunistic Pathogen ◽

Bacterial Pathogenesis ◽

Cytochrome Oxidase Subunit ◽

Biofilm Growth ◽

Unique Role ◽

High Affinity ◽

Biofilm Model ◽

Biofilm Development

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.

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Syndecan 1 Shedding Contributes to Pseudomonas aeruginosa Sepsis

Infection and Immunity ◽

10.1128/iai.73.12.7914-7921.2005 ◽

2005 ◽

Vol 73 (12) ◽

pp. 7914-7921 ◽

Cited By ~ 45

Author(s):

Allan Haynes ◽

Frank Ruda ◽

Jeffrey Oliver ◽

Abdul N. Hamood ◽

John A. Griswold ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Thermal Injury ◽

Opportunistic Pathogen ◽

Multiple Organ ◽

Innate Immune ◽

Host Cells ◽

Ectodomain Shedding ◽

Systemic Spread ◽

Immunocompromised State ◽

First Time

ABSTRACT The innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many of these factors, and the opportunistic pathogen Pseudomonas aeruginosa takes advantage of this condition, making it one of the leading causes of morbidity and mortality in the setting of thermal injury. P. aeruginosa is extremely efficient at colonizing burn wounds, spreading systemically, and causing sepsis, which often results in a systemic inflammatory response, multiple-organ failure, and death. The pathogenicity of P. aeruginosa is due to the arsenal of virulence factors produced by the pathogen and the immunocompromised state of the host. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity. Here we show for the first time that thermal injury results in shedding of syndecan 1 from host tissue. Our data show that syndecan 1 null mice are significantly less susceptible to P. aeruginosa infection than their wild-type counterparts, as demonstrated by (i) significantly lower mortality; (ii) absence of systemic spread of P. aeruginosa; and (iii) significant reductions in some proinflammatory cytokines. These results suggest that shed syndecan 1 plays an important role in the pathogenesis of P. aeruginosa infection of thermal injury and that syndecan 1-neutralizing agents may be effective supplements to current P. aeruginosa treatments.

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Global Analysis of the Zinc Homeostasis Network in Pseudomonas aeruginosa and Its Gene Expression Dynamics

Frontiers in Microbiology ◽

10.3389/fmicb.2021.739988 ◽

2021 ◽

Vol 12 ◽

Author(s):

Verena Ducret ◽

Melina Abdou ◽

Catarina Goncalves Milho ◽

Sara Leoni ◽

Oriane Martin--Pelaud ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infections ◽

Global Analysis ◽

Opportunistic Pathogen ◽

Zinc Homeostasis ◽

Protein Levels ◽

Its Gene ◽

Zn Homeostasis ◽

Gene Expression Dynamics ◽

Nanostring Technology

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

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Energy-Optimized Pharmacophore Coupled Virtual Screening in the Discovery of Quorum Sensing Inhibitors of LasR Protein of Pseudomonas aeruginosa

10.20944/preprints201910.0074.v1 ◽

2019 ◽

Author(s):

Zulkar Nain ◽

Sifat Bin Sayed ◽

Mohammad Minnatul Karim ◽

Md. Ariful Islam ◽

Utpal Kumar Adhikari

Keyword(s):

Pseudomonas Aeruginosa ◽

Virtual Screening ◽

Quorum Sensing ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Vital Role ◽

Energy Calculation ◽

Quorum Sensing Inhibitors ◽

Binding Energy Calculation ◽

Standard Precision

Pseudomonas aeruginosa is an emerging opportunistic pathogen responsible for cystic fibrosis and nosocomial infections. In addition, empirical treatments are become inefficient due to their multiple-antibiotic resistance and extensive colonizing ability. Quorum sensing (QS) plays a vital role in the regulation of virulence factors in P. aeruginosa. Attenuation of virulence by QS inhibition could be an alternative and effective approach to control infections. Therefore, we sought to discover new QS inhibitors (QSIs) against LasR receptor in P. aeruginosa using chemoinformatics. Initially, a structure-based high-throughput virtual screening was performed using the LasR active site that identified 61404 relevant molecules. E-pharmacophore (ADAHH) screening of these molecules rendered 72 QSI candidates. In standard-precision docking, only 7 compounds were found as potential QSIs due to their higher binding affinity to LasR receptor (-7.53 to -10.32 kcal/mol compared to -7.43 kcal/mol of native ligands). The ADMET properties of these compounds were suitable to be QSIs. Later, extra-precision docking and binding energy calculation suggested ZINC19765885 and ZINC72387263 as the most promising QSIs. The dynamic simulation of the docked complexes showed good binding stability and molecular interactions. The current study suggested that these two compounds could be used in P. aeruginosa QS inhibition to combat bacterial infections.

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A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.075291-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1488-1500 ◽

Cited By ~ 12

Author(s):

Ronan R. McCarthy ◽

Marlies J. Mooij ◽

F. Jerry Reen ◽

Olivier Lesouhaitier ◽

Fergal O’Gara

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptional Repression ◽

Transcriptional Regulators ◽

Opportunistic Pathogen ◽

Host Cells ◽

Cell Contact ◽

Virulence Determinants ◽

Surface Attachment ◽

Global Regulators ◽

Virulence Regulator

LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogen Pseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role in P. aeruginosa pathogenicity within the Caenorhabditis elegans acute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associated cupA and alg genes. This highlights the importance of BvlR as a new virulence regulator in P. aeruginosa with a central role in modulating key events in the pathogen–host interactome.

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