Isolation of GST isoenzymes from Phaseolus vulgaris L. and characterization of detoxifying mechanism under biotic and abiotic stress

Three Glutathione transferase (GSTs) isoenzymes have been isolated from P. vulgaris leaves after in vivo treatment with 1/250 fluazifop-p-butyl herbicide. The inducible GST isoenzymes were identified and separated by affinity chromatography. They found to belong to phi and tau classes. Moreover, the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs, termed PvGSTU2-2, PvGSTF1-1 and PvGSTU3-3) were found to catalyze a broad range of reactions and exhibit quite varied substrate specificity. Another GST isoenzyme was isolated from P. vulgaris (PvGST, termed PvGSTU3-3), after its induction with biotic stress treatment (Uromyces appendiculatus infection). PvGSTU3-3 shares high homology the tau class plant and catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance are the high antioxidant catalytic function and hydroperoxidase, thioltransferase, and dehydroascorbate reductase action of Pv-GSTU3-3. Transgenic tobacco plants over-expressing PvGSTU2-2 isoenzyme have been developed via Agrobacteriun tumefaciens in order to study their in planta potential to confer biotic and abiotic resistance as a means of plant breeding. Our results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris. Moreover, highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in P. vulgaris

Plant Glutathione Transferases: Structure, Antioxidant Catalytic Function and in planta Protective Role in Biotic and Abiotic Stress

Plant cytosolic glutathione transferases (GSTs) belong to an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stress. GSTs catalyze the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), leading to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroperoxides, biosynthesis of secondary metabolites, and exhibit thioltransferase and dehydroascorbate reductase activity. The applications of ‘omics’ technologies have allowed the classification of GSTs and the study of their evolution and sequence diversity, while enzymology has provided powerful insights into their catalytic role. This review focuses on plant GSTs, and attempts to give an overview of the new insights into their catalytic function and biological role in biotic and abiotic stress tolerance mechanisms in plants

Epidemiological study and investigation of Amp-C β-lactamases and carbapenemases in enterobacteriaceae from the greek region

The present study has been focused on two main objectives on the one hand the detection andcharacterization of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae, along withan evaluation of the effectiveness of various inhibitors in their detection and an assessment ofa modified CLSI-ESBL test and on the other hand the investigation of novel carbapenemasegenes which have recently been introduced to the Greek region.A total of 105 Enterobacteriaceae isolates (K.pneumoniae, E.coli, K.oxytoca, P.mirabilis)collected over a 9 year period from 2004 to 2012, exhibiting reduced susceptibility orresistance either to cefoxitin (FOX) and/or cefotetan (CTT) along with reduced susceptibility or resistance to at least one extended spectrum cephalosporin were further investigated for thepresence of plasmid AmpC β-lactamases. Plasmid-mediated AmpCs, ESBLs and the VIMtypecarbapenemases were sought by PCR amplification and subjected to direct sequencing.Susceptibility testing was performed with disk diffusion and ertapenem MICs were evaluatedwith the Etest method. Clonal relationship was investigated by either enterobacterial repetitiveintergenic consensus PCR (ERIC-PCR) or repetitive sequence based PCR (REP-PCR). FourAmpC inhibitors, cloxacillin (CLOX), phenyl-boronic acid (PBA), 3-acetyl-phenyl-boronic acid(APA) and 3-amino-phenyl-boronic (APBA) acid were evaluated, the latter in two finalconcentrations (300 and 600 μg/disk) with FOX, CTT, ceftazidime (CAZ) and cefotaxime (CTX)as substrates. Finally a modified CLSI ESBL test with the use of the aforementioned inhibitors andCTX and CAZ was evaluated.Altogether 59 isolates harbored plasmid-mediated AmpCs, with 35 also co-producing ESBLs.The MOX-2 β-lactamase was identified with the highest frequency followed by the CMY-2enzyme. CMY-4, CMY-13 and CMY-31 were also detected along with three novel to theregion enzymes, DHA-1 and two new AmpCs a CMY-4-like and a MIR-2-like. All P.mirabilisbelonged to one clonal type, K.oxytoca isolates presented two distinct clonal types whileK.pneumoniae and E.coli isolates presented 8 and 11 clonal types respectively.FOX resistance was found to be a discriminating parameter performing better than CTT as aninitial screening tool. In regards to the confirmatory phenotypic AmpC disk assay, FOX incombination with APBA produced the best results, successfully identifying 57/59 isolates. Theproposed modified CLSI ESBL test provided a significantly better overall performance whenperformed with CLOX (sensitivity 91,4% and specificity 75%), PBA (100%, 75%) and APBA(97,1%, 79,1%) in comparison to the standard CLSI ESBL test (85,7%, 58,3%). APA as an inhibitor failed to enhance the modified CLSI ESBL test exhibiting a suboptimal sensitivity of54,3%. Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistantEnterobacteriaceae isolates, which gave positive the modified Hodge test (ΜΗΤ) and werephenotypically suspected of metallo-β-lactamase production (MBL), were recovered frompatients hospitalized in Ioannina University Hospital. Molecular testing verified the presencein 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15,blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was locatedon an IncFII type plasmid, of c. 95kb, flanked upstream by a non-truncated ISAba125 elementand downstream by the bleMBL gene. Carbapenem resistance was transferable at a very low rateof ~ 1,3x10-7. Genotyping clustered all K. pneumoniae isolates into a single clonal type withone subtype and MLST assigned them to sequence type ST11. Two outbreaks were noted, thefirst between November-December 2011 involving four patients and the second initiated inMay 2012 and ongoing, involving the remaining patients. All but two cases were characterizedas hospital-acquired. No links to immigration or travel history to endemic areas wereestablished.From December 2011-March 2012, 13 ertapenem-resistant K. pneumoniae isolates, with apositive MHT, and negative phenotypic screening tests for MBL and KPC production, wererecovered from nine patients. All isolates harboured the blaOXA-48 gene along with blaCTX-M-15and blaOXA-1 genes. The blaOXA-48 gene was located on a self-transferable IncL/M-type plasmidof c. 62 kb, which harboured no other resistance genes. IS1999 was located upstream ofblaOXA-48 gene. Genetic disruptions of the OmpK35 and OmpK36 genes associated withpermeability defects were not detected. The isolates belonged to a unique PFGE clone andMLST assigned them to sequence type ST11. All cases were characterized as hospitalacquiredand none of them was linked to immigration or had a history of travel in endemicareas.Over a period of approximately 2,5 years initiated in 2010, three K.pneumoniae isolates were retrieved form urine samples of a single female patient, of which the first two with an intervalof 41 days, exhibiting resistance to cephamycins and extended spectrum cephalosporins, withpositive screening tests for AmpC β-lactamase production and a negative modified CLSIESBLtest. The initial two isolates exhibited carbapenem resistance, gave a positive ΜΗΤ andnegative screening tests for class A and B carbapenemase production.All isolates harbored the blaOXA-1 and blaDHA-1 genes and additionally the first two carried theblaOXA-162 gene. IS1999 was located upstream of blaOXA-162 gene. Genetic disruptions of theOmpK35 and OmpK36 genes associated with ertapenem resistance were not detected. Plasmidprofiling indicated the presence of three plasmids of ~140kb, ~62kb and 2kb in the first twowhile the latter lacked 62kb plasmid. Conjugation experiments failed to transfer β-lactamresistance. All K.pneumoniae belonged to a unique PFGE clone and MLST assigned them tosequence type ST11.Η παρούσα διατριβή περιστρέφεται γύρω από δύο κεντρικούς άξονες, αφενός την καταγραφή και διερεύνηση πλασμιδιακών AmpC β-λακταμασών σε στελέχη εντεροβακτηριακών, τη συγκριτική εκτίμηση της αποτελεσματικότητας διαφόρων αναστολέων στην ανίχνευση τους,την αξιολόγηση ενός τροποποιημένου CLSI ESBL τεστ και αφετέρου την καταγραφή καιδιερεύνηση νεωτέρων καρβαπενεμασών που έχουν ανιχνευθεί πρόσφατα στην Ελληνική επικράτεια. Για την μελέτη των πλασμιδιακών AmpC β-λακταμασών έγινε έλεγχος 105 Εντεροβακτηριακών στελεχών (K.pneumoniae, E.coli, K.oxytoca, P.mirabilis) με μειωμένη ευαισθησία ή αντοχή στην κεφοξιτίνη (FOX) ή/και στην κεφοτετάνη (CTT) σε συνδιασμό με αντοχή ή μειωμένη ευαισθησία σε μία τουλάχιστον εκτεταμένου φάσματος κεφαλοσπορίνη. Οι πλασμιδιακές AmpC, ESBL και VIM καρβαπενεμάσες ανιχνεύθηκαν με PCR και προσδιορίστηκαν με νουκλεοτιδική αλληλούχιση. Πραγματοποιήθηκαν τεστ ευαισθησίας με την μέθοδο διάχυσης δισκίων, προσδιορισμός MIC ερταπενέμης με Etest και διερεύνηση κλωνικότητας με ERIC-PCR και REP-PCR. Έγινε συγκριτική εκτίμηση τεσσάρων αναστολέων, της κλοξασιλλίνης (CLOX), του φαινύλ-βορονικού οξέος (PBA), του 3-ακετύλ-φαινύλ βορονικού οξέος (APA) και του 3-άμινο-φαινύλ βορονικού οξέως (APBA) το τελευταίο σε δύο συγκεντρώσεις με υποστρώματα την FOX, CTT, κεφταζιδίμη (CAZ) καικεφοταξίμη (CTX). Τέλος εκτιμήθηκε ένα τροποποιημένο CLSI ESBL τεστ με την χρήση των προηγούμενων αναστολέων.Συνολικά βρέθηκαν 59 στελέχη με πλασμιδιακές AmpC β-λακταμάσες, εκ των οποίων 35 έφεραν και ESBL. Η πλειονότητα των στελεχών έφερε την MOX-2 β-λακταμάση και ακολούθως την CMY-2. Σε μικρότερη συχνότητα βρέθηκαν η CMY-4, CMY-13 και CMY-31 καθώς και τρία πρωτοεμφανιζόμενα στην Ελλάδα ένζυμα η DHA-1 και δύο νεώτερες β-λακταμάσες μια CMY-4-like και μία MIR-2-like. Τα στελέχη P.mirabilis ανήκαν σε ένα κλώνο, οι K.oxytoca εμφάνιζαν δύο κλώνους με ένα διακριτό υπότυπο, ενώ οι E.coli καιK.pneumoniae διαχωρίστηκαν σε συνολικά 11 και 8 κλώνους αντίστοιχα.Η FOX υπερτερούσε σε σχέση με την CTT ως αρχικό screening. Όσον αφορά την επιβεβαιωτική φαινοτυπική μέθοδο η FOX σε συνδιασμό με το APBA δίνει τα καλύτερα αποτελέσματα αναγνωρίζοντας επιτυχώς 57/59 στελέχη. Το τροποποιημένο CLSI ESBL τεστ υπερείχε σημαντικά σε ευαισθησία και ειδικότητα με την χρήση CLOX (91,4%, 75%), PBA(100%, 75%) ή APBA (97,1%, 79,1%) σε σχέση με το κλασσικό CLSI ESBL τεστ (85,7%,58,3%). Συνολικά καλύτερα αποτελέσματα επιτεύχθηκαν με την χρήση του PBA. Το APAαπέτυχε να λειτουργήσει ικανοποιητικά εμφανίζοντας χαμηλή ευαισθησία (54,3%).Στην περίπτωση των NDM καρβαπενεμασών συνολικά έγινε έλεγχος 132 Εντεροβακτηριακών στελεχών με αντοχή στις καρβαπενέμες που είχαν συλλεχθεί από τον Ιανουάριο 2010 έως τον Ιούνιο 2013, έδιναν θετικό το τροποποιημένο Hodge τεστ (MHT) και τις φαινοτυπικές δοκιμασίες για παραγωγή μέταλλο-β-λακταμάσης (MBL). Συνολικά 78 στελέχη K.pneumoniae, που συγκεντρώθηκαν από 71 ασθενείς έφεραν το blaNDM-1 γονίδιο. Η πλειονότητα των στελεχών ήταν επίσης θετικά και για τα blaOXA-1, blaCTX-M-15 και blaTEM-1 γονίδια.. Η πλασμιδιακή ανάλυση έδειξε ότι το blaNDM-1 γονίδιο φέρεται επί ενός πλασμιδίου μεγέθους ~95kbp και ανήκει στο IncFII γκρουπ ασυμβατότητας. Ανάλυση του γενετικού περιβάλλοντος του blaNDM-1 γονιδίου έδειξε ότι του γονιδίου προηγείτο ένα μη διακεκομμένοISAba125 στοιχείο ενώ μετά το γονίδιο ακολουθεί το bleMBL γονίδιο. Η ERIC-PCR και ηPFGE ανέδειξαν ότι όλα τα στελέχη ανήκαν σε ένα διακριτό κλώνο Α, με μια υποομάδα Α1 και η MLST αντιστοίχησε τα στελέχη στο τύπο ST11. Μεταφορά της αντοχής στις καρβαπενεμάσες επιτεύχθηκε με πάρα πολύ χαμηλή συχνότητα ~ 1,3x10-7 διασυζευτικά στελέχη ανά κύτταρο δότη. Παρατηρήθηκαν δύο επιδημίες, η πρώτη από Νοέμβριο-Δεκέμβριο 2011, με 4 εμπλεκόμενους ασθενείς και η δεύτερη με έναρξη το Μάιο 2012 και συνεχιζόμενη, με τα υπόλοιπα περιστατικά. Για δύο ασθενείς θεμελιώθηκε η αυτόχθονος πρόσληψη των στελεχών από την κοινότητα. Δεν βρέθηκαν συνδετικά στοιχεία που να βεβαιώνουν έκθεση των περιστατικών σε ενδημικές περιοχές.Από τον Δεκέμβριο 2011 έως το Μάρτιο 2012 13 στελέχη K.pneumoniae με αντοχή στην ερταπενέμη, με θετικό MHT αλλά αρνητικές φαινοτυπικές δοκιμασίες για παραγωγή MBL καιKPC τύπου καρβαπενεμασών, απομονώθηκαν από 9 ασθενείς.Όλα τα στελέχη έφεραν το blaOXA-48 γονίδιο καθώς και το blaOXA-1 και blaCTX-M-15. Το blaOXA48 γονίδιο εντοπίστηκε επί συζευκτικού πλασμιδίου τάξης IncL/M μεγέθους ~62kb. Η ανάλυση του γενετικού περιβάλλοντος πιστοποίησε την παρουσία του IS1999 πριν το γονίδιο,σχηματίζοντας το μεταθετό στοιχείο Tn1999. Η ανάλυση αλληλουχιών των γονιδίων των μεμβρανικών πρωτεϊνών ompK35 και ompK36 δεν ανέδειξε μεταλλάξεις σχετιζόμενες με προβλήματα διαβατότητας. Η PFGE ομαδοποίησε τα στελέχη σε ένα διακριτό κλώνο, με δύο υπότυπους (Ια και Ιb) και η MLST αντιστοίχησε τα στελέχη στο ST11. Σε χρονικό διάστημα περίπου ίσο με 2,5 χρόνια και έναρξη το 2010 από τρία δείγματα ούρων ασθενούς, τα δύο πρώτα εκ των οποίων προσκομίσθηκαν με διαφορά 41 ημερών ενώ το τελευταίο μετά από 2,5 χρόνια, απομονώθηκαν στελέχη Κ.pneumoniae με αντοχή στις κεφαμυκίνες και στις εκτεταμένου φάσματος κεφαλοσπορίνες, θετικά φαινοτυπικά τεστ για παραγωγή AmpC β-λακταμάσης και αρνητικό τροποιημένο CLSI-ESBL τεστ. Τα δύο αρχικά στελέχη παρουσίαζαν επίσης αντοχή στις καρβαπενέμες, θετικό ΜΗΤ και αρνητικά φαινοτυπικά τεστ για παραγωγή καρβαπενεμάσης τάξης Α ή Β. Η μοριακή διερεύνηση αν έδειξε την παρουσία του blaOXA-162 γονιδίου καθώς και του blaOXA-1 και του blaDHA-1 στα δύο αρχικά στελέχη ενώ το τελευταίο ήταν αρνητικό για το blaOXA-162. Το IS1999 προηγείτο τουblaOXA-162. Δεν βρέθηκαν μεταλλάξεις σχετιζόμενες με προβλήματα διαβατότητας. Η πλασμιδιακή ανάλυση έδειξε την ύπαρξη δύο μεγάλων πλασμιδίων ~140kb και ~62kb καθώς και ενός μικρότερου μεγέθους 2kb στα δύο πρώτα στελέχη, ενώ στο τελευταίο απουσίαζε το 62kb πλασμίδιο. Τα πειράματα σύζευξης δεν οδήγησαν στην δημιουργία διασυζευκτών. ΗPFGE ομαδοποίησε τα στελέχη σε ένα διακριτό κλώνο και η MLST τα αντιστοίχησε στοST11

Terazosin treatment suppresses basic fibroblast growth factor expression in the rat ventral prostate

Purpose: Alpha1-adrenergic receptor antagonists may not act solely on smooth muscle contractility. We evaluated the in vivo effect of the alpha1 blocker, terazosin, on the expression of basic fibroblast growth factor (bFGF) in the rat ventral prostate. Methods: Wistar rats were treated with terazosin (1.2 mg/kg body weight, po, every second day) for 120 days. The expression of bFGF was assessed immuno-histochemically in tissue sections and by Western blotting in whole tissue preparations. Results: Terazosin treatment did not affect prostate weight or histomorphology. In the control group, epithelial and stromal cells demonstrated positive staining for the anti-bFGF antibody. In contrast, the same staining in terazosin-treated specimens was either absent or extremely weak. An analogous difference was observed among the corresponding immunoblots. Conclusions: These findings implicate the reduction of bFGF expression by terazosin as a potential additional molecular mechanism of its action that may include alterations in peptide growth factor mediated prostate homeostasis

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. “Plake Megalosperma Prespon” is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3–3 and Pvgstu2–2 genes. The overexpression of Pvgstu3–3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3–3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco

Overexpression of A Biotic Stress-Inducible <i>Pvgstu</i> Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. “Plake Megalosperma Prespon” is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3–3 and Pvgstu2–2 genes. The overexpression of Pvgstu3–3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3–3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco

Plant glutathione transferase-mediated stress tolerance : functions and biotechnological applications

Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural interest, because of their ability to confer tolerance against biotic and abiotic stresses and to detoxify herbicides. Here we review the biotechnological applications of GSTs towards developing plants that are resistant to biotic and abiotic stresses. We integrate recent discoveries, highlight, and critically discuss the underlying biochemical and molecular pathways involved. We elaborate that the functions of GSTs in abiotic and biotic stress adaptation are potentially a result of both catalytic and non-catalytic functions. These include conjugation of reactive electrophile species with glutathione and the modulation of cellular redox status, biosynthesis, binding, and transport of secondary metabolites and hormones. Their major universal functions under stress underline the potential in developing climate-resilient cultivars through a combination of molecular and conventional breeding programs. We propose that future GST engineering efforts through rational and combinatorial approaches, would lead to the design of improved isoenzymes with purpose-designed catalytic activities and novel functional properties. Concurrent GST–GSH metabolic engineering can incrementally increase the effectiveness of GST biotechnological deployment

In Vivo Acquisition of a Plasmid-Mediated blaKPC-2 Gene among Clonal Isolates of Serratia marcescens▿

Three patients admitted to a Greek hospital were infected with Serratia marcescens isolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniae carbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescens isolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescens that acquired a plasmid-mediated blaKPC-2 gene during the hospitalization