Bacterial Histidine Kinases as Novel Antibacterial Drug Targets
Bem, A.E. ; Velikova, N.R. ; Pellicer, M.T. ; Baarlen, P. van; Marina, A. ; Wells, J.M. - \ 2015
Acs Chemical Biology 10 (2015)1. - ISSN 1554-8929 - p. 213 - 224.
2-component signal-transduction - structure-based discovery - staphylococcus-aureus - mycobacterium-tuberculosis - escherichia-coli - multidrug-resistance - response regulator - vancomycin resistance - antibiotic-resistance - streptococcus-mutans
Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In this review, we discuss the biological importance of TCSs and bacterial HKs for the discovery of novel antibacterials, as well as published TCS and HK inhibitors that can be used as a starting point for structure-based approaches to develop novel antibacterials.
Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,,12:i:- Strains Circulating in Europe.
Garcia, P. ; Hopkins, K.L. ; García, V. ; Beutlich, J. ; Mendoza, M.C. ; Threlfall, J. ; Mevius, D.J. ; Helmuth, R. ; Rodicio, M.R. ; Guerra, B. - \ 2014
PLoS ONE 9 (2014)2. - ISSN 1932-6203
antimicrobial resistance - multidrug-resistance - spanish clone - health-risk - determinants - serotype - region - genes
Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,,12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,,12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,,12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacE¿1-sul1 3' conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and blaTEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,,12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.
Salmonella enterica Serovar Typhimurium Virulence-Resistance Plasmids Derived from the pSLT Carrying Nonconventional Class 1 Integrons with dfrA12 Gene in Their Variable Region and sul3 in the 3' Conserved Segment
Beutlich, J. ; Rodicio, M.R. ; Mendoza, M.C. ; Garcia, P. ; Kirchner, M. ; Luzzi, I. ; Mevius, D.J. ; Threllfall, J. ; Helmuth, R. ; Guerra, B. - \ 2013
Microbial Drug Resistance-Mechanisms Epidemiology and Disease 19 (2013)6. - ISSN 1076-6294 - p. 437 - 445.
antimicrobial resistance - multidrug-resistance - drug-resistance - antibiotic-resistance - phage type - serotype - identification - determinants - sequence - strains
Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.
Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against gram-positive pathogenic bacteria.
Fiamegos, Y.C. ; Kastritis, P.L. ; Exarchou, V. ; Han, H. ; Bonvin, A.M. ; Vervoort, J.J.M. ; Lewis, K. ; Hamblin, M.R. ; Tegos, G.P. - \ 2011
PLoS ONE 6 (2011)4. - ISSN 1932-6203 - 12 p.
staphylococcus-aureus - multidrug-resistance - antibiotic-activity - chlorogenic acids - nora - transporter - derivatives - berberine - analogs - protein
Background Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings In this study we report the identification and characterization of 4',5'-O-dicaffeoylquinic acid (4',5'-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3',5'-ODCQA, 4',5'-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4',5'-ODCQA with pump inhibitory activity whereas 3',5'-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4',5'-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.
Antimicrobial resistance and virulence determinants in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins.
Beutlich, J. ; Jahn, S. ; Malorny, B. ; Hauser, E. ; Hühn, S. ; Schroeter, A. ; Rosario Rodicio, M. ; Appel, B. ; Thelfall, J. ; Mevius, D.J. ; Helmuth, R. ; Guerra, B. - \ 2011
Applied and Environmental Microbiology 77 (2011)16. - ISSN 0099-2240 - p. 5655 - 5664.
multiple-antibiotic-resistance - serovar typhimurium dt104 - multidrug-resistance - drug-resistance - gene-cluster - variant - region - sequence - salmonella-genomic-island-1 - integrons
Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla(PSE-1), floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings
Multiresistant Salmonella enterica serovar 4,,12:i:- in Europe: a new pandemic strain?
Hopkins, K.L. ; Kirchner, M. ; Guerra, B. ; Granier, A. ; Lucarelli, C. ; Porrero, M.C. ; Jakubczak, A. ; Threlfall, J. ; Mevius, D.J. - \ 2010
Eurosurveillance 15 (2010)22. - ISSN 1025-496X - p. 1 - 9.
antimicrobial resistance genes - molecular characterization - multidrug-resistance - monophasic variant - serotype typhimurium - 4,5,12-i - identification - infections - emergence - carcasses
A marked increase in the prevalence of S. enterica serovar 4,,12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,,12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,,12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,,12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.
The effect of co-administered flavonoids on the metabolism of hesperetin and the disposition of its metabolites in Caco-2 cell monolayers
Brand, W. ; Padilla, B. ; Bladeren, P.J. van; Williamson, G. ; Rietjens, I. - \ 2010
Molecular Nutrition & Food Research 54 (2010)6. - ISSN 1613-4125 - p. 851 - 860.
cancer resistance protein - dependent glucose-transporter - line caco-2 - multidrug-resistance - orange juice - (abcg2)-mediated transport - intestinal-absorption - mediated inhibition - abc transporters - biochanin-a
Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers in a two-compartment transwell system. Flavonoids may interfere with hesperetin metabolism and can also inhibit the apically located ABC transporter breast cancer resistance protein (ABCG2) which was previously shown to be responsible for the apical transport of hesperetin metabolites. Co-exposure of Caco-2 cell monolayers to hesperetin with specific flavonoids reduced the ratio of apical efflux to basolateral transport of hesperetin metabolites, and in some cases, also reduced the amount of hesperetin metabolites detected extracellularly. As intracellular accumulation of hesperetin metabolites did not account for this decrease, inhibition of metabolism of hesperetin is likely the underlying mechanism for the reduced metabolite formation and excretion. In spite of the reduction in metabolism the amount of hesperetin metabolites transported to the basolateral side significantly increased upon co-exposure with specific flavonoids and therefore co-administration of specific flavonoids could be a strategy to improve the bioavailability of hesperetin
Effects of flavonoid mixtures on the transport of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through Caco-2 monolayers: an in vitro and a kinetic modeling approach to predict the combined effects on transporter inhibition
Schutte, M.E. ; Boersma, M.G. ; Verhallen, D.A.M. ; Groten, J.P. ; Rietjens, I.M.C.M. - \ 2008
Food and Chemical Toxicology 46 (2008)2. - ISSN 0278-6915 - p. 557 - 566.
cancer resistance protein - multidrug-resistance - p-glycoprotein - mediated inhibition - (abcg2)-mediated transport - oral bioavailability - dietary flavonoids - drug permeability - efflux proteins - cell-lines
This study describes and kinetically models the effect of flavonoid mixtures on PhIP transport through Caco-2 monolayers. Previously it was shown that quercetin, luteolin, naringenin and myricetin increase the apical to basolateral PhIP transport in Caco-2 monolayers. In this study, apigenin was shown to exert a similar effect with an apparent Ki value of 10.8 ¿M. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing all five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model previously developed to describe the effect of the individual flavonoids on PhIP transport, could be extended and adequately describes the experimental values obtained for the flavonoid mixtures. We conclude that combinations of flavonoids increase the transport of PhIP and do so by interacting in an additive way with the active transport of PhIP. This flavonoid-mediated increase in PhIP transport through Caco-2 monolayers may point at a possible increased bioavailability of PhIP in the presence of flavonoid mixtures in the in vivo situation. This would imply an adverse effect of these supposed beneficial food ingredients.
Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers
Brand, W. ; Wel, P.A.I. van der; Rein, M.J. ; Barron, D. ; Williamson, G. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2008
Drug Metabolism and Disposition 36 (2008)9. - ISSN 0090-9556 - p. 1794 - 1802.
cancer resistance protein - messenger-rna expression - multidrug-resistance - p-glycoprotein - cyclosporine-a - drug transporters - abc-transporters - slc-transporters - culture model - orange juice
Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied hesperetin (10 µM) was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm2), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 µM), the apical efflux of hesperetin conjugates was 1.9-fold reduced (p 0.01), and transport to the basolateral side was 3.1-fold increased (p 0.001). These findings elucidate a novel pathway of hesperetin metabolism and transport and show that BCRP-mediated transport could be a limiting step for hesperetin bioavailability.
The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin
Roohparvar, R. ; Mehrabi, R. ; Nistelrooy, J.G.M. van; Zwiers, L.H. ; Waard, M.A. de - \ 2008
Pest Management Science 64 (2008)7. - ISSN 1526-498X - p. 685 - 693.
qo inhibitor fungicides - natural toxic compounds - mycosphaerella-graminicola - multidrug-resistance - genetic-variation - candida-albicans - abc transporters - mechanisms - respiration - pathosystem
BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola. (c) 2008 Society of Chemical Industry
Cloning and functional characterization of BcatrA, a gene encoding an ABC transporter of the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea)
Sorbo, G. Del; Ruocco, M. ; Schoonbeek, H. ; Scala, F. ; Pane, C. ; Vinale, F. ; Waard, M.A. de - \ 2008
Mycological Research 112 (2008)6. - ISSN 0953-7562 - p. 737 - 746.
binding-cassette transporter - natural toxic compounds - saccharomyces-cerevisiae - multidrug-resistance - mycosphaerella-graminicola - drosophila-melanogaster - aspergillus-nidulans - efflux pump - cutinase-a - yeast
BcatrA was cloned from the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) and sequenced. Sequence analysis revealed that BcatrA encodes a protein composed of 1562 amino acid residues displaying high similarity with various fungal ATP-binding cassette (ABC) transporters having the (NBF-TM6)2 topology. Expression of BcatrA is barely detectable during normal vegetative growth in liquid substrates. Transcript levels of BcatrA are enhanced in a dose- and time-dependent manner after treatment with cycloheximide or catechol, but not by a number of other drugs or fungicides, including fludioxonil, fenarimol, imazalil, and the plant defense compounds pisatin and resveratrol. Quantitative analysis of BcatrA during the synchronized infection of bean leaves revealed an overaccumulation of the gene transcript at 6, 12 and 24 h post-inoculation, suggesting an involvement of the gene in the first steps of pathogenesis. Functional analysis of BcatrA was performed by targeted gene replacement in a wild-type strain of the fungus, and by overexpression in a mutant of Saccharomyces cerevisiae carrying multiple non-functional multidrug-resistance genes. BcatrA replacement mutants did not show any significant increase in sensitivity to drugs, including inducers of BcatrA transcription, and displayed an unaltered virulence on several common host plants of B. cinerea. However, when expressed in the heterologous system, BcatrA reduced sensitivity to cycloheximide and catechol, thus indicating the ability of the BcatrA product to function as a multidrug transporter.
Occurance and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands
Essen-Zandbergen, A. van; Smith, H.E. ; Veldman, K.T. ; Mevius, D.J. - \ 2007
Journal of Antimicrobial Chemotherapy 59 (2007)4. - ISSN 0305-7453 - p. 746 - 750.
multidrug-resistance - determinants - bacteria
Objectives: To determine the occurrence and transmission of class 1, 2 and 3 integrons in multidrug-resistant or sulfamethoxazole-resistant Salmonella from human and animal sources and in Campylobacter spp. and Escherichia coli from broilers isolated in the Netherlands in 2004. Methods: PCR, restriction fragment length polymorphism (RFLP) and DNA sequencing were used to detect integrase genes and gene cassettes within 234 E. coli isolates, 40 Campylobacter isolates and 228 Salmonella isolates. Results: Class 1 integrons were found in 76% of the E. coli and in 43% of the Salmonella isolates. Class 2 integrons were found in 11% of the E. coli and 1% of the Salmonella isolates. No class 1 or 2 integrons were detected in the Campylobacter isolates, and no class 3 integrons were detected in any of the bacterial species examined. The 22 different integrons detected harboured 20 different gene cassettes. The cassette arrays dfrA1-aadA1 and dfrA1-sat2-aadA1 were most frequently associated with class 1 and 2 integrons, respectively. For the first time linF was found to be associated with a class 2 integron as part of the linF-sat2-aadA1 cassette. The gene cassettes found within the integrons explain only a part of the resistance profile of the isolates. Conjugation experiments demonstrated transfer of class 1 and 2 integrons. Conclusions: Our data demonstrate the importance of integrons for the occurrence and transmission of multidrug resistance. Identical predominant class 1 and 2 integrons in E. coli and Salmonella serovars indicate horizontal transfer between these species.
MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides
Roohparvar, R. ; Waard, M.A. de; Kema, G.H.J. ; Zwiers, L.H. - \ 2007
Fungal Genetics and Biology 44 (2007)5. - ISSN 1087-1845 - p. 378 - 388.
multidrug-resistance - saccharomyces-cerevisiae - abc transporters - aspergillus-nidulans - filamentous fungi - drug-resistance - gene - cercosporin - virulence - efflux
MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H+ antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.
MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis
Zwiers, L.H. ; Roohparvar, R. ; Waard, M.A. de - \ 2007
Fungal Genetics and Biology 44 (2007)9. - ISSN 1087-1845 - p. 853 - 863.
major facilitator superfamily - atp-binding cassette - multidrug-resistance - siderophore biosynthesis - filamentous fungi - toxic compounds - gene-cluster - spore wall - wheat - virulence
The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.
Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters
Roohparvar, R. ; Huser, A. ; Zwiers, L.H. ; Waard, M.A. de - \ 2007
Applied and Environmental Microbiology 73 (2007)15. - ISSN 0099-2240 - p. 5011 - 5019.
major facilitator superfamily - natural toxic compounds - multidrug-resistance - abc transporters - botrytis-cinerea - efflux pump - fungicide sensitivity - virulence - plant - reversal
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.
Flavonoid-mediated inhibition of intestinal ABC transporters may affect the oral bioavailability of drugs, food-borne toxic compounds and bioactive ingredients
Brand, W. ; Schutte, M.E. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2006
Biomedicine and Pharmacotherapy 60 (2006)9. - ISSN 0753-3322 - p. 508 - 519.
cancer resistance protein - epithelial caco-2 cells - neuronal signal-transduction - grapefruit juice components - regulating cellular-levels - organic anion transporter - p-glycoprotein activity - seville orange juice - rat small-intestine - multidrug-resistance
The transcellular transport of ingested food ingredients across the intestinal epithelial barrier is an important factor determining bioavailability upon oral intake. This transcellular transport of many chemicals, food ingredients, drugs or toxic compounds over the intestinal epithelium can be highly dependent on the activity of membrane bound ATP binding cassette (ABC) transport proteins, able to export the compounds from the intestinal cells. The present review describes the ABC transporters involved in the efflux of bioactive compounds from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, reducing bioavailability. The role of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The present paper focuses on the role of flavonoids as important modulators or substrates of intestinal ABC transport proteins. Several examples of such an effect of flavonoids are presented. It can be concluded that flavonoid-mediated inhibition of ABC transporters may affect the bioavailability of drugs, bioactive food ingredients and/or food-borne toxic compounds upon oral uptake. All together it appears that the flavonoid-mediated interactions at the level of the intestinal ABC transport proteins may be an important mechanism for unexpected food¿drug, food¿toxin or food¿food interactions. The overview also indicates that future studies should focus on i) in vivo validation of the flavonoid-mediated effects on bioavailability of drugs, toxins and beneficial bioactive food ingredients detected in in vitro models, and on ii) the role of flavonoid phase II metabolism in modulating the activity of the flavonoids to act as ABC transporter inhibitors and/or substrates.
An in vitro and in silico study on the flavonoid mediated modulation of the transport of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through Caco-2 monolayers
Schutte, M.E. ; Freidig, A.P. ; Sandt, J.J.M. van de; Alink, G.M. ; Rietjens, I.M.C.M. ; Groten, J.P. - \ 2006
Toxicology and Applied Pharmacology 217 (2006)2. - ISSN 0041-008X - p. 204 - 215.
cancer resistance protein - multidrug-resistance - p-glycoprotein - cell-lines - biochanin-a - absorption - carcinogen - expression - quercetin - efflux
The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.
Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis
Hoek, A.H.A.M. van; Scholtens-Toma, I.M.J. ; Cloekaert, A. ; Aarts, H.J.M. - \ 2005
Journal of Microbiological Methods 62 (2005)1. - ISSN 0167-7012 - p. 13 - 23.
typhimurium dt104 - dna microarrays - multidrug-resistance - mycobacterium-tuberculosis - antimicrobial resistance - nucleic-acids - identification - integrons - sequence - strains
In this study the feasibility of 50- and 60-mer oligonucleotides in microarray analysis for the detection and identification of antibiotic resistance genes in various Salmonella strains was assessed. The specificity of the designed oligonucleotides was evaluated, furthermore the optimal spotting concentration was determined. The oligonucleotide microarray was used to screen two sets of Salmonella strains for the presence of several antibiotic resistance genes. Set 1 consisted of strains with variant Salmonella Genomic Island 1 (SGI1) multidrug resistance (MDR) regions of which the antibiotic resistance profiles and genotypes were known. The second set contained strains of which initially only phenotypic data were available. The microarray results of the first set of Salmonella strains perfectly matched with the phenotypic and genotypic information. The microarray data of the second set were almost completely in concordance with the available phenotypic data. It was concluded that the microarray technique in combination with random primed genomic labeling and 50- or 60-mer oligonucleotides is a powerful tool for the detection of antibiotic resistance genes in bacteria.
Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides
Sesink, A.L.A. ; Arts, I.C.W. ; Boer, V.C.J. de; Breedveld, P. ; Schellens, J.H.M. ; Hollman, P.C.H. ; Russel, F.G.M. - \ 2005
Molecular pharmacology 67 (2005)6. - ISSN 0026-895X - p. 1999 - 2006.
lactase-phlorhizin hydrolase - multidrug-resistance - oral bioavailability - dietary quercetin - biliary-excretion - fumitremorgin-c - heart-disease - in-vitro - transporter - cells
The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug resistance-associated protein 2 (Mrp2)/Abcc2. To study the possible involvement of Mrp2, we compared intestinal uptake of quercetin-3-glucoside between control and Mrp2-deficient rats, using an in situ intestinal perfusion system. The contribution of Bcrp1 was determined using the specific inhibitor fumitremorgin C (FTC) in Mrp2-deficient rats. Furthermore, vectorial transport of quercetin was studied in Madin-Darby canine kidney (MDCK)II cells transfected with either human MRP2 or murine Bcrp1. In these MDCKII cells, we showed an efficient efflux-directed transport of quercetin by mouse Bcrp1, whereas in control and MRP2-transfected cells no vectorial transport of quercetin was observed. In Mrp2-deficient rats, intestinal uptake of quercetin from quercetin-3-glucoside, efflux of quercetin glucuronides to the gut lumen, and plasma concentration of quercetin were similar to that in control rats. When intestinal Bcrp1 was inhibited by FTC in Mrp2-deficient rats, total plasma concentrations of quercetin and its methylated metabolite isorhamnetin after 30 min of perfusion were more than twice that of controls (12.3 ± 1.5 versus 5.6 ± 1.3 µM; p <0.01), whereas uptake of free quercetin from the intestinal lumen was not affected. Instead, inhibition of Bcrp1 lowered the efflux of quercetin glucuronides into the perfusion fluid by approximately 4-fold. In conclusion, Bcrp1 limits net intestinal absorption of quercetin by pumping quercetin glucuronides back into the lumen.
Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin
Zanden, J.J. van; Mul, A. de; Wortelboer, H.M. ; Usta, M. ; Bladeren, P.J. van; Rietjens, I.M.C.M. ; Cnubben, N.H.P. - \ 2005
Biochemical Pharmacology 69 (2005)11. - ISSN 0006-2952 - p. 1657 - 1665.
organic anion transporter - multidrug-resistance - protein mrp1 - substrate-specificity - drug-resistance - p-glycoprotein - binding sites - cmoat mrp2 - glutathione - cells
In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5 ± 1.7 µM for MRP1 and 24.6 ± 1.3 µM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1 ± 0.1 µM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1 ± 1.9 and 22.2 ± 1.4 µM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 µM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6 ± 0.5 and 5.8 ± 0.5 µM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.