scholarly journals Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity

2016 ◽  
Vol 21 (6) ◽  
pp. 590-595 ◽  
Author(s):  
Ana Filipa Pinto ◽  
Mahsa Ebrahimi ◽  
Michael Saleeb ◽  
Åke Forsberg ◽  
Mikael Elofsson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Starting Point ◽  
Rho Family Gtpases ◽  
Cytoskeletal Dynamics ◽  
Important Object ◽  
Adp Ribosyltransferase ◽  
Further Development

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1, N6-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β–stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development.

scholarly journals Bacteria-Host Crosstalk: Sensing of the Quorum in the Context of Pseudomonas aeruginosa Infections

2018 ◽  
Vol 11 (3) ◽  
pp. 263-279 ◽  
Author(s):  
Maria V. Turkina ◽  
Elena Vikström
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Current Knowledge ◽  
Single Species ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Future Perspective ◽  
Mammalian Host ◽  
Acylhomoserine Lactone ◽  
Cytoskeletal Dynamics ◽  
Biofilm Development

Cell-to-cell signaling via small molecules is an essential process to coordinate behavior in single species within a community, and also across kingdoms. In this review, we discuss the quorum sensing (QS) systems used by the opportunistic pathogen Pseudomonas aeruginosa to sense bacterial population density and fitness, and regulate virulence, biofilm development, metabolite acquisition, and mammalian host defense. We also focus on the role of N-acylhomoserine lactone-dependent QS signaling in the modulation of innate immune responses connected together via calcium signaling, homeostasis, mitochondrial and cytoskeletal dynamics, and governing transcriptional and proteomic responses of host cells. A future perspective emphasizes the need for multidisciplinary efforts to bring current knowledge of QS into a more detailed understanding of the communication between bacteria and host, as well as into strategies to prevent and treat P. aeruginosa infections and reduce the rate of antibiotic resistance.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
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.

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

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1315-1326 ◽  
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.

scholarly journals Syndecan 1 Shedding Contributes to Pseudomonas aeruginosa Sepsis

2005 ◽  
Vol 73 (12) ◽  
pp. 7914-7921 ◽  
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.

scholarly journals A complex of LaoA and LaoB acts as a Tat-dependent dehydrogenase for long-chain alcohols in Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Gianna Panasia ◽  
Steffen L. Drees ◽  
Susanne Fetzner ◽  
Bodo Philipp
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alcohol Dehydrogenase ◽  
Distribution Systems ◽  
Alcohol Oxidation ◽  
Opportunistic Pathogen ◽  
Chain Alcohol ◽  
Starting Point ◽  
Long Chain Alcohol ◽  
Long Chain

The opportunistic pathogen Pseudomonas aeruginosa can utilize unusual carbon sources like sodium dodecyl sulfate (SDS) and alkanes. Whereas the initiating enzymatic steps of the corresponding degradation pathways have been characterized in detail, the oxidation of the emerging long-chain alcohols found little attention. Recently, the genes for the Lao ( l ong-chain a lcohol/ a ldehyde o xidation) system were discovered to be involved in the oxidation of long-chain alcohols derived from SDS and alkane degradation. In the Lao-system, LaoA is predicted to contain the catalytic site, however, according to genetic studies, efficient long-chain alcohol oxidation additionally required the Tat-dependent protein LaoB. In the present study, the Lao-system was further characterized. In vivo analysis revealed that the Lao-system complements the substrate spectrum of the well-described Exa-system, which is required for growth with ethanol and other short-chain alcohols. Mutational analysis revealed that the Tat-site of LaoB was required for long-chain alcohol oxidation activity strongly suggesting a periplasmic localization of the process. Purified LaoA was only fully active when co-purified with LaoB. Interestingly, in vitro activity of the purified LaoAB-complex also depended on the presence of the Tat-site. The co-purified LaoAB-complex contained a flavin co-factor and preferentially oxidized a range of saturated, unbranched primary alcohols. Furthermore, the LaoAB-complex could reduce cytochrome c 550 type redox carriers like ExaB, a subunit of the Exa alcohol dehydrogenase system. LaoAB-complex activity was stimulated by rhamnolipids in vitro . In summary, LaoAB constitutes an unprecedented protein complex with specific properties apparently required for oxidizing long-chain alcohols. IMPORTANCE Pseudomonas aeruginosa is a major threat to public health. Its ability to thrive in clinical settings, water distribution systems or even jet fuel tanks is linked to detoxification and degradation of diverse hydrophobic substrates that are metabolized via alcohol intermediates. Our study illustrates a novel flavoprotein long-chain alcohol dehydrogenase consisting of a facultative two-subunit complex, which is unique among related enzymes while the homologs of the corresponding genes are found in numerous bacterial genomes. Understanding the involved catalytic and compartmentalization processes is of great interest for biotechnological and hygiene research as it may be a potential starting point for rationally designing novel antibacterial substances with high specificity against this opportunistic pathogen.

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

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1488-1500 ◽  
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.

scholarly journals Exoenzyme Y Contributes to End-Organ Dysfunction Caused by Pseudomonas aeruginosa Pneumonia in Critically Ill Patients: An Exploratory Study

Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 369
Author(s):  
Brant M. Wagener ◽  
Naseem Anjum ◽  
Sarah C. Christiaans ◽  
Morgan E. Banks ◽  
Jordan C. Parker ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Organ Dysfunction ◽  
Virulence Factors ◽  
Opportunistic Pathogen ◽  
Lavage Fluid ◽  
Host Cells ◽  
Clinical Isolates ◽  
Icu Patients ◽  
Edema Factor ◽  
Host Infection

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY+/ExoU−) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY−/ExoU+). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

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

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.

scholarly journals Molecular Cloning, Expression and FPLC Purification of Lectin A from Pseudomonas aeruginosa

2014 ◽  
Vol 2 (4) ◽  
pp. 529-536
Author(s):  
Peyman Ghoraishizadeh ◽  
Shraddha Raikar ◽  
Mahsa Takhtechian
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Molecular Cloning ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Related Protein ◽  
Burn Victims ◽  
Gene Coding ◽  
Lectin Protein

Pseudomonas aeruginosa (PA) as an opportunistic pathogen infects the pulmonary tract, bladder, cystic fibrosis patients and burn victims. PA infections treatment is challenging because of its ability to rapidly develop resistance to multiple classes of antibiotics. Lectin is protein that isexpressed in cell of PA and cause of infection by attaching to the host cells. Lectin A gene coding lectin protein so we cloned and expressedthis gene then purified of related protein, that can be used in preparation of vaccine to treat PA infections.DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11559 Int J Appl Sci Biotechnol, Vol. 2(4): 529-536 

scholarly journals A simple mung bean infection model for studying the virulence of Pseudomonas aeruginosa

2017 ◽  
Author(s):  
Sneha Garge ◽  
Sheyda Azimi ◽  
Stephen P. Diggle
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
Mung Bean ◽  
Opportunistic Pathogen ◽  
Mortality Rates ◽  
Infection Model ◽  
Wild Type ◽  
Antimicrobial Drugs ◽  
Bean Model

AbstractHere we highlight the development of a simple and high throughput mung bean model to study virulence in the opportunistic pathogen Pseudomonas aeruginosa. The model is easy to setup and infection and virulence can be monitored for up to 10 days. In a first test of the model, we found that mung bean seedlings infected with PAO1 showed poor development of roots and high mortality rates compared to un-infected controls. We also found that a quorum sensing (QS) mutant was significantly less virulent when compared with the PAO1 wild type. Our work introduces a new tool for studying virulence in P. aeruginosa, that will allow for high throughput virulence studies of mutants, and for testing the in vivo efficacy of new therapies at a time when new antimicrobial drugs are desperately needed.

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