Structure of BrlR reveals a potential pyocyanin binding site

Drug Tolerance ◽

Binding Partner ◽

Direct Binding ◽

AbstractThe transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3′,5′-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections

10.1101/2020.10.23.352104 ◽

2020 ◽

Author(s):

Isabella Santi ◽

Pablo Manfredi ◽

Enea Maffei ◽

Adrian Egli ◽

Urs Jenal

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Tolerance ◽

Antibiotic Tolerance ◽

Bacterial Survival ◽

Chronic Infections ◽

Tolerance Mechanisms ◽

Resistance Development ◽

Evolutionary Trajectories ◽

AbstractThe widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multi-drug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.

OsaR (PA0056) functions as a repressor of the gene fleQ encoding an important motility regulator in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00145-21 ◽

2021 ◽

Author(s):

Yibing Ma ◽

Yujie Liu ◽

Yutong Bi ◽

Xiao Han ◽

Yongxin Jin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Site ◽

Biofilm Formation ◽

Regulatory Mechanisms ◽

Chronic Obstructive ◽

Transcriptional Level ◽

Obstructive Pulmonary Disease

FleQ plays a crucial role in motility and biofilm formation by regulating flagellar and exopolysaccharide biosynthesis in Pseudomonas aeruginosa . It has been reported that the expression of FleQ is transcriptionally downregulated by the virulence factor regulator Vfr. Herein we demonstrated that a LysR-type transcriptional regulator, OsaR, is also capable of binding to the promoter region of fleQ and repressing its transcription. Through gel shift and DNase I footprinting assays, the OsaR binding site was identified and characterized as a dual LysR-type transcriptional regulator box (AT-N 11 -AT-N 7 -A-N 11 -T). Mutation of the A-T palindromic base pairs in fleQ promoter not only reduced the binding affinity of OsaR in vitro , but also de-repressed fleQ transcription in vivo . The OsaR binding site was found to cover the Vfr binding site; knockout of osaR or vfr separately exhibited no effect on the transcriptional level of fleQ ; however, fleQ expression was repressed by overexpression of osaR or vfr . Furthermore, simultaneously deleting both osaR and vfr resulted in an upregulation of fleQ , but it could be complemented by the expression of either of the two repressors. In summary, our work revealed that OsaR and Vfr function as two transcriptional repressors of fleQ that bind to the same region of fleQ but work separately. IMPORTANCE Pseudomonas aeruginosa is a widespread human pathogen, which accounts for serious infections in the hospital, especially for lung infection in cystic fibrosis and chronic obstructive pulmonary disease patients. P. aeruginosa infection is closely associated with its motility and biofilm formation, which are both under the regulation of the important transcription factor FleQ. However, the upstream regulatory mechanisms of fleQ have not been fully elucidated. Therefore, our research identifying a novel regulator of fleQ as well as new regulatory mechanisms controlling its expression will be significant for better understanding the intricate gene regulatory mechanisms related to P. aeruginosa virulence and infection.

Efficacy of a ciprofloxacin/amikacin combination against planktonic and biofilm cultures of susceptible and low-level resistant Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz355 ◽

2019 ◽

Vol 74 (11) ◽

pp. 3252-3259 ◽

Cited By ~ 3

Author(s):

Anaïs Soares ◽

Kévin Alexandre ◽

Fabien Lamoureux ◽

Ludovic Lemée ◽

François Caron ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Biofilm ◽

The Other ◽

Clinical Strain ◽

Bacterial Reduction ◽

Low Level ◽

Mechanical Dispersion ◽

Resistant Mutants ◽

Abstract Background Eradicating bacterial biofilm without mechanical dispersion remains a challenge. Combination therapy has been suggested as a suitable strategy to eradicate biofilm. Objectives To evaluate the efficacy of a ciprofloxacin/amikacin combination in a model of in vitro Pseudomonas aeruginosa biofilm. Methods The antibacterial activity of ciprofloxacin and amikacin (alone, in combination and successively) was evaluated by planktonic and biofilm time–kill assays against five P. aeruginosa strains: PAO1, a WT clinical strain and three clinical strains overexpressing the efflux pumps MexAB-OprM (AB), MexXY-OprM (XY) and MexCD-OprJ (CD), respectively. Amikacin MIC was 16 mg/L for XY and ciprofloxacin MIC was 0.5 mg/L for CD. The other strains were fully susceptible to ciprofloxacin and amikacin. The numbers of total and resistant cells were determined. Results In planktonic cultures, regrowth of high-level resistant mutants was observed when CD was exposed to ciprofloxacin alone and XY to amikacin alone. Eradication was obtained with ciprofloxacin or amikacin in the other strains, or with the combination in XY and CD strains. In biofilm, bactericidal reduction after 8 h followed by a mean 4 log10 cfu/mL plateau in all strains and for all regimens was noticed. No regrowth of resistant mutants was observed whatever the antibiotic regimen. The bacterial reduction obtained with a second antibiotic used simultaneously or consecutively was not significant. Conclusions The ciprofloxacin/amikacin combination prevented the emergence of resistant mutants in low-level resistant strains in planktonic cultures. Biofilm persister cells were not eradicated, either with monotherapy or with the combination.

Tolerance of Pseudomonas aeruginosa in in-vitro biofilms to high-level peracetic acid disinfection

Journal of Hospital Infection ◽

10.1016/j.jhin.2017.06.024 ◽

2017 ◽

Vol 97 (2) ◽

pp. 162-168 ◽

Cited By ~ 23

Author(s):

A.B. Akinbobola ◽

L. Sherry ◽

W.G. Mckay ◽

G. Ramage ◽

C. Williams

Keyword(s):

Pseudomonas Aeruginosa ◽

Peracetic Acid ◽

NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase

Microbiology ◽

10.1099/mic.0.076042-0 ◽

2014 ◽

Vol 160 (4) ◽

pp. 789-794 ◽

Cited By ~ 11

Author(s):

Amin Omairi-Nasser ◽

Carla V. Galmozzi ◽

Amel Latifi ◽

M. Isabel Muro-Pastor ◽

Ghada Ajlani

Keyword(s):

Binding Site ◽

Translation Initiation ◽

Growth Conditions ◽

Gene Encoding

In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform.

Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00585-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 11

Author(s):

Paulo Juarez ◽

Katy Jeannot ◽

Patrick Plésiat ◽

Catherine Llanes

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Redox Homeostasis ◽

Significant Loss ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Content Type ◽

Constitutive Overexpression

ABSTRACT The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa206 ◽

2020 ◽

Vol 75 (9) ◽

pp. 2508-2515 ◽

Cited By ~ 1

Author(s):

María A Gomis-Font ◽

Gabriel Cabot ◽

Irina Sánchez-Diener ◽

Pablo A Fraile-Ribot ◽

Carlos Juan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Infection Model ◽

Mechanisms Of Resistance ◽

Resistance Development ◽

C Elegans ◽

Imipenem Resistance ◽

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

14-3-3 proteins are required for the inhibition of Ras by exoenzyme S

Biochemical Journal ◽

10.1042/bj3490697 ◽

2000 ◽

Vol 349 (3) ◽

pp. 697-701 ◽

Cited By ~ 25

Author(s):

Maria Lena HENRIKSSON ◽

Ulrika TROLLÉR ◽

Bengt HALLBERG

Keyword(s):

Signal Transduction ◽

Pseudomonas Aeruginosa ◽

Binding Site ◽

Phage Display Library ◽

Checkpoint Control ◽

Exoenzyme S ◽

Interaction Site ◽

Killing Activity

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where ‘Xaa’ represents ‘any residue’, and ‘pSer’ is ‘phosphoserine’) . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibα and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

The Journal of Cell Biology ◽

10.1083/jcb.201412127 ◽

2015 ◽

Vol 210 (5) ◽

pp. 753-769 ◽

Cited By ~ 49

Author(s):

Nan Hyung Hong ◽

Aidong Qi ◽

Alissa M. Weaver

Keyword(s):

Membrane Trafficking ◽

Membrane Curvature ◽

Actin Dynamics ◽

Complex Activity ◽

Actin Networks ◽

Binding Partner ◽

Late Endosomes ◽

Actin Turnover ◽

Direct Binding

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

fleQ, the Gene Encoding the Major Flagellar Regulator of Pseudomonas aeruginosa, Is σ70 Dependent and Is Downregulated by Vfr, a Homolog of Escherichia coli Cyclic AMP Receptor Protein

Journal of Bacteriology ◽

10.1128/jb.184.19.5240-5250.2002 ◽

2002 ◽

Vol 184 (19) ◽

pp. 5240-5250 ◽

Cited By ~ 74

Author(s):

Nandini Dasgupta ◽

Evan P. Ferrell ◽

Kristen J. Kanack ◽

Susan E. H. West ◽

Reuben Ramphal

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Binding Site ◽

Caulobacter Crescentus ◽

Bacterial Species ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Receptor Protein ◽

Regulatory Cascade

ABSTRACT The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a σ70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5′-AATTGACTAATCGTTCACATTTG-3′. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative −10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli σ70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this −10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the −10 region to initiate transcription.