CrpP Is Not a Fluoroquinolone-Inactivating Enzyme.
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ABSTRACT:
SUBMITTER: Zubyk HL
PROVIDER: S-EPMC8373257 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP, for ciprofloxacin resistance protein, plasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.
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Project description:The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Biologics used to modulate hC5a are predominantly monoclonal antibodies. Here we present data to support an alternative therapeutic approach based on the specific inactivation of hC5a by ScpA in studies using recombinant hC5a (rhC5a). Initial characterization of ScpA confirmed activity in human serum and against rhC5a desArg (rhC5adR), the predominant hC5a form in blood. A new FRET based enzyme assay showed that ScpA cleaved rhC5a at near physiological concentrations (K m 185 nM). Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) studies established a high affinity ScpA-rhC5a interaction (K D 34 nM, KDITC 30.8 nM). SPR analyses also showed that substrate binding is dominated (88% of ΔG°bind) by interactions with the bulky N-ter cleavage product (PN, 'core' residues 1-67) with interactions involving the C-ter R74 contributing most of the remaining ΔG°bind. Furthermore, reduced binding affinity following mutation of a subset of positively charged Arginine residues of PN and in the presence of higher salt concentrations, highlighted the importance of electrostatic interactions. These data provide the first in-depth study of the ScpA-C5a interaction and indicate that ScpA's ability to efficiently cleave physiological concentrations of C5a is driven by electrostatic interactions between an exosite on the enzyme and the 'core' of C5a. The results and methods described herein will facilitate engineering of ScpA to enhance its potential as a therapeutic for excessive immune response to infectious disease.
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Project description:Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2022-12-05 | GSE206610 | GEO
Project description:The quinolone resistance crpP genes can mediate decreased susceptibility to quinolones. However, diversification and prevalence of crpP genes and crpP-carrying integrative and conjugative elements (ICEs) still need to be elucidated. In this study, genome sequencing was conducted for 200 Chinese Pseudomonas aeruginosa isolates, 16 of which were fully sequenced. All the 37 available CrpP variants were collected for phylogenetic analysis, 10 CrpP enzymes were chosen to conduct cloning and antimicrobial susceptibility test, and 22 crpP-carrying Tn6786-related ICEs were selected for detail genetic dissection analysis. Then, typing/nomenclature schemes for crpP variants and crpP-carrying ICEs were established for the first time. The 10 representative CrpP enzymes were confirmed to mediate decreased susceptibility to one to three quinolones. Tn6786-related ICEs displayed high-level diversification in both nucleotide sequences and modular structures. Mainly, massive gene acquisition/loss occurred across the whole genomes of Tn6786-related ICEs. 53.5% (107/200) of the tested clinical P. aeruginosa isolates from China carried crpP genes, which were exclusively located within chromosome-borne Tn6786-related ICEs. The crpP-carrying ICEs were at active stages of evolution and had the high potential to be an important vector for the dissemination of resistance genes besides crpP. The present study furthered the understanding of the bioinformatics and epidemiology of crpP genes and crpP-carrying ICEs.
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Project description:Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatus SMF13 by ammonium sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 degrees C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation.
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Project description:The ciprofloxacin-modifying crpP gene was recently identified in a plasmid isolated from a Pseudomonas aeruginosa clinical isolate. Homologues of this gene were also identified in Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. We set out to explore the mobile elements involved in the acquisition and spread of this gene in publicly available and complete genomes of Pseudomonas spp. All Pseudomonas complete genomes were downloaded from NCBI's Refseq library and were inspected for the presence of the crpP gene. The mobile elements carrying this gene were further characterized. The crpP gene was identified only in P. aeruginosa, in more than half of the complete chromosomes (61.9%, n = 133/215) belonging to 52 sequence types, of which the high-risk clone ST111 was the most frequent. We identified 136 crpP-harboring integrative and conjugative elements (ICEs), with 93.4% belonging to the mating-pair formation G (MPFG) family. The ICEs were integrated at the end of a tRNALys gene and were all flanked by highly conserved 45-bp direct repeats. The crpP-carrying ICEs contain 26 core genes (2.2% of all 1193 genes found in all the ICEs together), which are present in 99% or more of the crpP-harboring ICEs. The most frequently encoded traits on these ICEs include replication, transcription, intracellular trafficking and cell motility. Our work suggests that ICEs are the main vectors promoting the dissemination of the ciprofloxacin-modifying crpP gene in P. aeruginosa.
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