Successful Host Adaptation of IncK2 Plasmids
Rozwandowicz, Marta ; Brouwer, Michael S.M. ; Mughini-Gras, Lapo ; Wagenaar, Jaap A. ; Gonzalez-Zorn, Bruno ; Mevius, Dik J. ; Hordijk, Joost - \ 2019
Frontiers in Microbiology 10 (2019). - ISSN 1664-302X
chicken - conjugation - IncK2 - plasmid - sigma-32
The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ–32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates.
Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut
Fischer, Egil A.J. ; Dierikx, Cindy M. ; Essen-Zandbergen, Alieda van; Mevius, Dik ; Stegeman, Arjan ; Velkers, Francisca C. ; Klinkenberg, Don - \ 2019
Applied and Environmental Microbiology 85 (2019)17. - ISSN 0099-2240
antibiotic resistance - Bayesian model - challenge - conjugation - experiment - poultry
Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.
Effect of Glucuronidation on the Potential of Kaempferol to Inhibit Serine/Threonine Protein Kinases
Beekmann, Karsten ; Haan, Laura H.J. De; Actis-Goretta, Lucas ; Bladeren, Peter J. Van; Rietjens, Ivonne M.C.M. - \ 2016
Journal of Agricultural and Food Chemistry 64 (2016)6. - ISSN 0021-8561 - p. 1256 - 1263.
conjugation - flavonoid - glucuronidation - serine/threonine protein kinases - substrate microarray
To study the effect of metabolic conjugation of flavonoids on the potential to inhibit protein kinase activity, the inhibitory effects of the dietary flavonol kaempferol and its major plasma conjugate kaempferol-3-O-glucuronide on protein kinases were studied. To this end, the inhibition of the phosphorylation activity of recombinant protein kinase A (PKA) and of cell lysate from the hepatocellular carcinoma cell line HepG2 on 141 putative serine/threonine phosphorylation sites derived from human proteins was assessed. Glucuronidation reduced the inhibitory potency of kaempferol on the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively, but did not appear to affect the target selectivity for kinases present in the lysate. The data demonstrate that, upon glucuronidation, kaempferol retains part of its intrinsic kinase inhibition potential, which implies that K3G does not necessarily need to be deconjugated to the aglycone for a potential inhibitory effect on protein kinases.
Molecular determinants of xenobiotic metabolism: QM/MM simulation of the conversion of 1-chloro-2,4-dinitrobenzene catalyzed by M1-1 glutathione S-transferase.
Bowman, A.L. ; Ridder, L. ; Rietjens, I.M.C.M. ; Vervoort, J.J.M. ; Mulholland, A.J. - \ 2007
Biochemistry 46 (2007)21. - ISSN 0006-2960 - p. 6353 - 6363.
nucleophilic aromatic-substitution - potential free-energy - active-site - mu class - dynamics calculations - enzymatic-reactions - reaction-mechanisms - isoenzyme 3-3 - conjugation - evolution
Modeling methods allow the identification and analysis of determinants of reactivity and specificity in enzymes. The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) is widely used as a standard activity assay for glutathione S-transferases (GSTs). It is important to understand the causes of differences between catalytic GST isoenzymes and the effects of mutations and genetic polymorphisms. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations have been performed here to investigate the addition of the glutathione anion to CDNB in the wild-type M1-1 GST isoenzyme from rat and in three single point mutant (Tyr6Phe, Tyr115Phe, and Met108Ala) M1-1 GST enzymes. We have developed a specifically parameterized QM/MM method (AM1-SRP/CHARMM22) to model this reaction by fitting to experimental heats of formation and ionization potentials. Free energy profiles were obtained from molecular dynamics simulations of the reaction using umbrella sampling and weighted histogram analysis techniques. The reaction in solution has also been simulated and is compared to the enzymatic reaction. The free energies are in excellent agreement with experimental results. Overall the results of the present study show that QM/MM reaction pathway analysis provides detailed insight into the chemistry of GST and can be used to obtain mechanistic insight into the effects of specific mutations on this catalytic process.
Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with ß-casein
Boeriu, C.G. ; Oudgenoeg, G. ; Spekking, W.T.J. ; Berendsen, L.B.J.M. ; Vancon, L. ; Boumans, H. ; Gruppen, H. ; Berkel, W.J.H. van; Laane, N.C.M. ; Voragen, A.G.J. - \ 2004
Journal of Agricultural and Food Chemistry 52 (2004)21. - ISSN 0021-8561 - p. 6633 - 6639.
ferulic acid - fungal laccase - proteins - polysaccharides - conjugation - pentosans - bran
Heterologous conjugates of wheat arabinoxylan and beta-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of beta-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the beta-casein and the feruloylated arabinoxylan was reached at a protein-to-arabinoxylan ratio of 10:1, in combination with a molar ratio hydrogen peroxide to substrate of 2:1 and a molar protein-to-enzyme ratio between 10(2) and 10(4). The protein-arabinoxylan adducts were separated from the arabinoxylan homopolymers by size exclusion and anion exchange chromatography. The molar ratio protein:arabinoxylan in the purified conjugates varied between 0.1 and 5.6. This is the first report on the large-scale enzymatic preparation of heterologous protein-arabinoxylan conjugates.
Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin
Zanden, J.J. van; Hamman, O. Ben; Iersel, M.L. van; Boeren, J.A. ; Cnubben, N.H.P. ; Bello, M. Lo; Vervoort, J.J.M. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2003
Chemico-Biological Interactions 145 (2003)2. - ISSN 0009-2797 - p. 139 - 148.
site-directed mutagenesis - human placenta - quinone methide - ethacrynic-acid - active-site - pi - identification - consequences - inactivation - conjugation
In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon I h incubation with 100 muM quercetin or 2 h incubation with 25 muM quercetin, whereas 1 and 10 muM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LGMS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LGMS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.