scholarly journals Tetraphenylporphyrin Enters the Ring: First Example of a Complex Between Highly Bulky Porphyrins and a Protein

Author(s):  
Yuma Shisaka ◽  
Erika Sakakibara ◽  
Kazuto Suzuki ◽  
Joshua Kyle Stanfield ◽  
Hiroki Onoda ◽  
...  

Tetraphenylporphyrin (TPP) is a synthetic porphyrin whose properties can be readily modified, endowing it with significant benefits over naturally occurring porphyrins. Yet, their insolubility in water and/or steric bulk have rendered them incompatible with biological systems. Herein, we report the first example of a native biomolecule capturing TPP as well as its derivatives. The haemoprotein HasA, secreted by certain pathogens to scavenge haem from their hosts, can capture various metal- and meso-substituted TPPs. The rapid crystallisation of TPP derivatives captured by HasA revealed the binding mode of TPP at excellent resolutions. A single-site mutation (L85A) of HasA enlarged the binding pocket, allowing the incorporation of a bulkier derivative of TPP. HasA binding TPP derivatives was also demonstrated to inhibit proliferation of the opportunistic pathogen Pseudomonas aeruginosa. This study not only represents a simple method for the complexation of TPP derivatives with a native protein, but also opens the door for the future use of TPP derivatives as biological tools.

2007 ◽  
Vol 189 (19) ◽  
pp. 6870-6881 ◽  
Author(s):  
Gregory T. Robertson ◽  
Timothy B. Doyle ◽  
Qun Du ◽  
Leonard Duncan ◽  
Khisimuzi E. Mdluli ◽  
...  

ABSTRACT Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.


Author(s):  
F. Helfrich ◽  
Axel J. Scheidig

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.


2017 ◽  
Vol 2 (3) ◽  
pp. 150-163
Author(s):  
Ekajayanti Kining ◽  
Syamsul Falah ◽  
Novik Nurhidayat

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.


2020 ◽  
Vol 16 (6) ◽  
pp. 761-773
Author(s):  
Huda K. Mahmoud ◽  
Hanadi A. Katouah ◽  
Marwa F. Harras ◽  
Thoraya A. Farghaly

Background: One of the most successful reagents used in the synthesis of the reactive enaminone is DMF-DMA, but it is very expensive with harmful effects on the human health and reacts with special compounds to generate the enaminone such as active methylene centers. Aim: In this article, we synthesized a new ketenaminal by simple method with inexpensive reagents (through desulfurization in diphenylether). Methods: Thus, a novel reactive ketenaminal (enaminone) was synthesized from the desulfurization of 2-((2-(4-chlorophenyl)-2-oxoethyl)thio)-5,7-bis(4-methoxyphenyl)pyrido[2,3-d]pyrimidin- 4(3H)-one with diphenylether. The starting keteneaminal was coupled with diazotized anilines via the known coupling conditions to give a new series of 2-(4-chlorophenyl)-1-(2-(arylhydrazono)-2- oxoethyl)-5,7-bis(4-methoxy-phenyl)pyrido[2,3-d]pyrimidin-4(1H)-ones. Results: The structures of the new compounds were elucidated based on their IR, 1H-NMR, 13CNMR, and Mass spectra. Moreover, the potency of these compounds as antimicrobial agents has been evaluated. The results showed that some of the products have high activity nearly equal to that of the used standard antibiotic. Additionally, the docking study was done to get the binding mode of the synthesized compounds with the binding site of the DHFR enzyme. The results of molecular docking of the synthesized arylhydrazono compounds are able to fit in DHFR binding site with binding energies ranging from -4.989 to -8.178 Kcal/mol. Conclusion: Our goal was achieved in this context by the synthesis of new ketenaminal from inexpensive reagents, which was utilized in the preparation of bioactive arylhydrazone derivatives.


2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Wei Wang ◽  
Xiaoya Wang

AbstractBackgroundPseudomonas aeruginosa is an opportunistic pathogen which is associated with nosocomial infections and causes various diseases including urinary tract infection, pneumonia, soft-tissue infection and sepsis. The emergence of P. aeruginosa-acquired metallo-β-lactamase (MBL) is most worrisome and poses a serious threat during treatment and infection control. The objective of this study was to identify antibiotic susceptibility, phenotypic detection of MBL production and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples.MethodsA total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from two hospitals in China between September 2017 and March 2019 were included in this study. Phenotypic detection of MBL was performed by the combined detection method using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) discs. MBL-encoding genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaSIM, blaNDM-1 and blaGIM were detected by polymerase chain reaction (PCR).ResultsOf the 329 P. aeruginosa, majority of the isolates were resistant to imipenem (77.5%) followed by meropenem (64.7%). Of the 270 P. aeruginosa isolates tested, 149 (55.2%) isolates were found to be positive for MBL detection. Of the different samples, 57.8% (n = 26) of P. aeruginosa isolated from blood were found to be positive for MBL production. Of the various MBL genes, blaIMP-1 (28.2%) was the most predominant gene detected followed by blaVIM-2 (18.8%), blaVIM-1 (16.1%), blaNDM-1 (9.4%), blaIMP-2 (6.7%), blaSIM (6.0%), blaSPM-1 (4.0%) and blaGIM (1.3%) genes.ConclusionsThe high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of blaIMP-1 and blaVIM-2 set the alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.


2008 ◽  
Vol 190 (8) ◽  
pp. 2790-2803 ◽  
Author(s):  
Matthew A. Oberhardt ◽  
Jacek Puchałka ◽  
Kimberly E. Fryer ◽  
Vítor A. P. Martins dos Santos ◽  
Jason A. Papin

ABSTRACT Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jennifer M. Peña ◽  
Samantha M. Prezioso ◽  
Kirsty A. McFarland ◽  
Tracy K. Kambara ◽  
Kathryn M. Ramsey ◽  
...  

AbstractIn Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen.


Author(s):  
Chiara Luise ◽  
Dina Robaa ◽  
Wolfgang Sippl

AbstractSome of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.


2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.


Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2612-2619 ◽  
Author(s):  
Lisa K. Nelson ◽  
Genevieve H. D'Amours ◽  
Kimberley M. Sproule-Willoughby ◽  
Douglas W. Morck ◽  
Howard Ceri

Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.


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