<i>Pseudomonas aeruginosa</i> is a notorious nosocomial opportunist. Planktonic forms of this pathogen have been traditionally studied for its pathogenicity. Such studies have shown that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are able to negatively modulate pathogenicity. However, more recent findings suggest a biofilm basis of infection. In this study, monospecies and binary biofilms of <i>Pseudomonas aeruginosa</i> ATCC 15692 (PAOl) and <i>Escherichia coli</i> ATCC 10000 were investigated for their pathogenic potential using resistance and virulence as key pathogenic determinants, in the presence of sub-MICs of selected antibiotics (Ampicillin, Nalidixic acid and Streptomycin). MICs of biofilms were observed to be at least 7-fold greater than those of the corresponding planktonic form of the same species (as judged from results obtained from MIC experiments). SDS-PAGE and 2D-PAGE analysis indicate alteration of outer membrane proteins (OMPs) within the envelope of the pathogen in sub-MIC antibiotic treated samples. The observed rearrangement of lipopolysaccharide (LPS; as observed in LPS gel experiments) may also contribute to the pathogens increased tolerance to antibiotics within the biofilm state. While LPS changes may possibly help the biofilm bacteria escape host immune system <i>in vivo</i>, more direct evidence of increases in virulence of the pathogen comes from investigation of its secreted proteases and cytotoxins (leucocidin). Virulence-specific azocasein and micro-culture tetrazolium (MTT) assays against both monospecies and binary biofilms of <i>Pseudomonas aeruginosa</i> indicate significant increases in virulence potential of proteases and cytotoxins, respectively. These results were further substantiated in phase contrast microscopy images showing advanced stages of oncosis in tissue cultured mouse spleen myeloma (Sp2) cells treated with leucocidin isolated from <i>Ps. aeruginosa</i> treated with sub-MIC of ampicillin (8 pg mL'1). The results reported in this thesis provide evidence of observed increases in virulence and pathogenicity in biofilm cells of <i>Pseudomonas aeruginosa</i> in the presence of sub-MICs of selected antibiotics, <i>in vitro</i>. Although these findings are those of <i>in vitro</i> experiments, they may have significant implications regarding the usage and therapeutic control of antibiotics in clinical situations.

No description available.

Microorganisms are becoming increasingly resistant to current antimicrobial agents and therefore new strategies and agents are being developed to combat infection. One strategy is to target and block the first step of microbial infection (adhesion of the microbe to the host tissue) by using molecules that mimic (or antibodies against) the microbial adhesin or its complementary host cell receptor. Plants have also been shown to provide natural sources of antimicrobial substances as well as inhibit microbial adhesion. One major problem in adhesion-inhibition studies has been the accurate quantification of adhesion. Most investigators have relied on manual counting while a few have used automated methods using image analysis software. The first aim of this study was therefore to compare the effectiveness of several current software packages, to develop the most accurate method of quantification and to use this method to test potential inhibitors of microbial adhesion. The organisms under investigation in this project were Candida albicans and Helicobacter pylori, both of which are becoming resistant to available antibiotic treatments. A new and accurate method of quantification was developed for assessing microbial adhesion using 'Metamorph' image analysis software. Aided by this system, several domain antibodies, carbohydrates and plant extracts were found to be successful inhibitors of C. albicans and H.pylori adhesion in vitro and therefore have the potential to form the basis of new and alternative therapies to treat infection caused by these microorganisms. Additionally, because it is not fully known why H. pylori preferentially colonises specific topographical regions of the human stomach, the second aim was to compare its adhesion to different topographical regions of the stomach, in an attempt to explain this phenomenon. No difference was found between the receptors present in the antrum or fundus of inflamed human stomachs. However, further investigations involving both inflamed and non-inflamed stomachs are warranted.

Please note: Abstract contains images which cannot be displayed. The total synthesis of lactonamycin (1), a natural product isolated from a soil sample, by Matsomoto and co-workers1 is currently under investigation. Lactonamycin (1) possesses antimicrobal activity against Gram-Positive bacteria including MRSA and VRE as well as appreciable levels of cytotoxicity against tumor cell lines. A highly oxygenated fused perhydrofuran-furanone structural motif and a naphtha[e]isoindole ring system make up the hexacylcic core of lactonamycin (1). Adding to the structural complexity, a 2,3,6-trideoxy sugar unit is connected to the core structure via a highly hindered tertiary glycosidic linkage. Various strategies for glycosidation, using the known ABCD alcohol (±)-2 as a model for the tertiary alcohol of lactonamycin (1) are discussed. Of these include, the use of classical glycosidation approaches and more novel strategies that employ open chain glycosyl donors. In particular, the synthesis of glycoside 4 and subsequent investigations towards target compound 5 are described.

The peptidyl transferase centre (PTC) of 23S ribosomal RNA is the target for a number of antibiotics which inhibit protein synthesis. The precise mode of binding of these antibiotics is largely unknown and hence is an active area of research in structural biology. The NMR solution structures of three PT antibiotics, bamicetin, sparsomycin and anisomycin have been successfully characterised using a range of two-dimensional NMR techniques and restrained molecular dynamics. The NMR structures of the these antibiotics provided valuable first hand insight into their conformations, since no X-ray crystal structures of the antibiotics in their free states have been determined so far. Bamicetin adopts a folded conformation possibly held by intramolecular hydrogen bonds and similar to the published NMR structure of amicetin. These antibiotics generate spontaneous single nucleotide mutants upon prolonged exposure and bamicetin and sparsomycin are universal PT inhibitors, interacting with all three evolutionary domains of 23S rRNAs. The amicetin antibiotic produces a spontaneous single mutation U2457C in the Halobacterium halobium (H.hal) 23S rRNA and the binding site is predicted to be very close to this nucleotide. The similarity in chemical structure with amicetin, suggests bamicetin to target the same binding site on the 23S rRNA. Both bamicetin and sparsomycin show exchange retarded amide proton resonances in the NMR spectrum, akin to other amicetin family antibiotics, indicating the retarded exchange to be a characteristic feature in the native solution state. The Bacillus subtilis (B.subtilis) 70S ribosomes have strong affinity for bamicetin and so a highly conserved 27mer RNA motif containing the possible binding site was selected for NMR structure determination and bamicetin binding studies. The greater number of imino proton resonances observed together with the high quality of the determined structure of the motif proved that B.subtilis rRNA is more stable than E.coli and H.hal rRNAs. The B.subtilis 27mer rRNA-bamicetin interaction studies revealed a fast exchange, weak binding system and careful analysis of line width and chemical shifts indicated changes at the local conformation of the RNA after binding. To probe the cross-hypersensitivity phenomenon, a 25mer RNA corresponding to the thiostrepton-resistant mutant (G1159) residing in the domain II of H.hal 23S rRNA was chosen for NMR structure determination and amicetin binding. Discrete chemical shift changes and NOESY experiments using ultrahigh field 1GHz NMR revealed weak interactions. The structures of the antibiotics and analysis of their dynamics as well as interactions with the RNA motifs of different organisms have yielded important information in understanding their binding and inhibitory activities at the atomic level. The results can be used for generating new or hybrid antibiotics to tackle the escalating problem of antibiotic resistance.

The discovery of antibiotics in the early 20th century revolutionised medicine, but quickly bacteria began to demonstrate resistance to these agents. Antibiotic resistance is still on the increase, and soon, if this trend continues, bacterial infections may not be treatable with antibiotics. In an effort to prevent this occurring, the search for novel antibiotics is under way and new antibiotic targets are being considered. One target that has not been studied in depth is the partitioning systems of bacterial plasmids. Disruption of plasmid partition would prevent effective plasmid inheritance, which, in the case of resistance plasmids, would render the host cell antibiotic sensitive. The aim of this study was to determine whether plasmid partitioning is a viable target for a new antibiotic. A series of plasmids that contained genetically altered partitioning systems was used, which provided a range of plasmid stabilities. The plasmids all conferred antibiotic resistance on the host cell, allowing the effect of plasmid instability on a population grown in the presence of antibiotics to be determined. Several different methods of cell culture were used. Simple batch culture experiments allowed the observation that few plasmid-free cells were produced from cells containing plasmids that had a functional system. In contrast, plasmids containing a faulty system were found to be rapidly lost from the host cells. Steady-state chemostat culture was used to provide a simple model of a clinical infection. The formation of equilibria between plasmid-free and plasmid-bearing cells was observed, and the cultures contained a large proportion of plasmid-free cells when the experiments involved unstable plasmids. The slow growth rate of cultures in the chemostat was seen to dramatically affect the inheritance of plasmids relying on random distribution. Finally, cultures were subjected to washout in order to determine their maximum specific growth rate (µmax). While the results from these experiments are not entirely conclusive, there is a strong indication that the growth rate of cultures containing unstable plasmids grown in the presence of antibiotics is reduced.

The emergence of multidrug resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii has been recognized worldwide and some clinical isolates of these bacteria are now resistant to most of the antibiotics currently available. Unfortunately, there are at present not enough new antimicrobial drugs being produced by the pharmaceutical industry to keep pace with the continuing development of antibiotic resistance. Colistin is an old antibiotic and in the form of colistin methanesulphonate sodium (CMS) has re-emerged as a major treatment for burn patients colonised with Acinetobacter baumannii. However, this antibiotic can lead to profound toxicity and for this reason the blood levels of CMS should be carefully monitored. Unfortunately the methods available for measuring it in serum are not robust and are not able to differentiate between the two forms of colistin i.e. colistin sulphate (CLS) and CMS. In this study the chemical (Thin Layer Chromatography and High Performance Liquid Chromatography) and microbiological methods for CLS and CMS analysis were investigated to develop a methodology for reproducible quantification of CLS and CMS in water or serum. Since CMS in aqueous solution has the potential to hydrolyze to produce a complex mixture of colistin sulphomethylated derivatives as well as colistin base, the optimized chemical and microbiological methods were used to determine the degradation of CLS or CMS in aqueous solution and serum. The bacteriostatic and bactericidal activity of CLS and CMS were probed by calculations of minimum inhibitory concentrations and time survivor studies. Depending on their concentrations, both antibiotics were found to exhibit bacteriostatic and bactericidal properties against a range of Gram-negative bacteria. Membrane damage caused by both forms of colistin was investigated using Acinetobacter lwoffii R46383 anddetennining intracellular potassium leakage and 260nm absorbing materials leakage. CLS was shown to cause substantial membrane damage, indicated by rapid, gross potassium leakage, while the effect of CMS on the membrane appeared to be more subtle, with cells exhibiting a more concentration dependent loss of potassium. Adsorption isotherms ofCLS gave results that were indicative of high affmity isotherm (H- shape), while adsorption isotherms of CMS were indicative of co-operative sorption (S- shape). Investigations into CLS and CMS cytotoxicity were performed using normal rat kidney (NRK-52E) cell line; the investigations revealed that neither forms of colistin has a major adverse effect on the rat renal cells even at concentrations higher than the therapeutic doses.

Many eukaryotic organisms produce membrane interactive, a-helical antimicrobial peptides (a-AMPs) and a database of such peptides, together with selected physiochemical parameters was established. This database was divided into four groups according to the a-AMPs target organism(s) (active against Gram-positive bacteria {G+}; active against Gram-negative bacteria {G-}; active against Grampositive and Gram-negative bacteria {G+, G-}; or active against Gram-positive bacteria, Gram-negative bacteria and fungi {G+, G-, F}). Analysis of the database showed that there was no statistically significant correlation between specificity and p1 (range 4.2 to 12.7) or net charge (range -5 to +16). The peptides exhibited variable hydrophobicity, < H > (range -0.8 to +0.7) whilst amphiphilicity (measured by the hydrophobic moment, < jAH >) ranged from 0.2 to 1.1. A statistically significant negative correlation between < pH > and < H > was noted for each group of a-AMPs and this may relate to the amphiphilic balance required for antimicrobial activity, a-AMPs showed some differences in amino acid composition compared to the McCaldon and Argos dataset of unrelated oligopeptides, suggesting functional relevance of some amino acid residues. The groups {G+, G-} and {G+, G-, F}, for example are characterised by being rich in weakly hydrophobic or hydrophilic amino acids. Minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) can provide a measure of AMP potency. A statistical analysis of MIC's for peptides from {G+, G-} group, showed no significant differences in the potency of these peptides when directed against either Gram-positive or Gram-negative bacteria. In contrast, a statistical analysis of MIC's for peptides from the {G+, 0-, F} group showed that peptides from this group were effective at lower concentrations against bacterial targets as compared to fungal targets. Increases in hydrophobic arc size were generally accompanied by increases in peptide antimicrobial potency and in addition, a negative correlation between MIC and net charge was observed. Regression analyses indicated that an appropriate amphiphilicity/hydrophobicity balance was required for the antimicrobial action of a-AMPs and this may indicate a general structure/function relationship underlying both the efficacy and specificity of these peptides. Oblique orientated a-helices are highly specialised protein structural elements that penetrate membranes at a shallow angle and are used to promote membrane destabilisation by a number of protein classes. Here, the use of extended < pH > methodology showed that over 50% of the a-AMPs are candidate oblique ahelices providing some insight into possible modes of action. Peptides VP I and BYDV-MP were identified here as candidate AMPs based on amino acid composition and the potential to form oblique orientation. The biological activity of VPI and BYDV-MP was confirmed in vivo when an MLC of 3 mM was demonstrated on both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Monolayer studies using lipid extract from these target organisms and parallel studies using mimetic monolayers confirmed a high level of peptide membrane interaction. The single lipid monolayer results suggested that VPI and BYDV-MP have a lower affinity for zwitterionic lipid (DMPE surface pressure increase of 4 mNm') but a high affinity for anionic lipid (DMPS surface pressure increase of 7 to 9 mN m') and may have a requirement for this specific lipid or anionic lipids in general to achieve higher levels of membrane penetration. To test the ability of VP! to penetrate membranes protonated and deuterated homologues were analysed by neutron diffraction, in the presence of POPC: POPS (10:1 molar ratio). The data analysed from these studies showed the protonated homologue to penetrate the membrane core but the deuterated homologue showed no significant levels of membrane interaction. Monolayer studies confirmed that the protonated homologue interacted strongly with anionic and zwitterionic membranes (surface pressure increase 4 mNm'), however, the deuterated homologue did not have the ability to interact with POPC:POPS monolayers. FTIR conformational analysis showed the protonated homologue to adopt high levels of a-helical stmcture (65 %) and in contrast the deuterated homologue exhibited low levels of a-helical structure (C 20 %). These results support the original predictions and also appear to show that deuteration has directly or indirectly, affected the ability of the VP! peptide to interact with membranes, possibly by inhibiting a-helix formation by the peptide or decreasing structural stability.

Τα ριβοσώματα, οι μακρομοριακές μεταφραστικές μηχανές που είναι υπεύθυνες για την πρωτεϊνική σύνθεση, αποτελούν έναν από τους κυριότερους κυτταρικούς στόχους των αντιβιοτικών, που χορηγούνται για αντιμικροβιακή θεραπεία. Μελέτες για περισσότερο από 40 χρόνια δείχνουν ότι το κατάλληλο ιοντικό περιβάλλον (μονοσθενή, δισθενή κατιόντα και πολυαμίνες) είναι απαραίτητο για τη σωστή ριβοσωματική λειτουργία, ενώ παράλληλα επηρεάζει τις αλληλεπιδράσεις του με διάφορους προσδέτες. Παρόλα αυτά η μοριακή βάση της επίδρασης του ιοντικού περιβάλλοντος στο μηχανισμό δράσης των αντιβιοτικών δεν έχει ενδελεχώς μελετηθεί. Στόχος της παρούσας διατριβής είναι η διερεύνηση του μηχανισμού δράσης αντιβιοτικών που αναστέλλουν την πρωτεϊνική σύνθεση σε συνθήκες που προσομοιάζουν με τις φυσιολογικές του κυττάρου και η μελέτη της επίδρασης του ιοντικού περιβάλλοντος στη δράση αυτών. Τα αντιβιοτικά που μελετήθηκαν ήταν: α) η βλαστισιδίνη, ως κλασικός αναστολέας της πεπτιδυλοτρανσφεράσης (ΡΤάσης), β) το μακρολίδιο τυλοσίνη που αναστέλλει την ΡΤάση, αλλά παράλληλα προσδένεται στην αρχή του τούνελ εξόδου και παρεμποδίζει την πολυπεπτιδική αλυσίδα να εξέλθει από το ριβόσωμα, και γ) τα μακρολίδια ερυθρομυκίνη (πρώτης γενεάς), αζιθρομυκίνη (δεύτερης γενεάς) και τελιθρομυκίνη (μακρολίδιο τρίτης γενεάς ή κετολίδιο), η δράση των οποίων έγκειται στην παρεμπόδιση του τούνελ εξόδου. Εξίσου σημαντική φαίνεται να είναι η επίδραση των μακρολιδίων στη συγκρότηση της 50S ριβοσωματικής υπομονάδας. Ο μηχανισμός δράσης των αντιβιοτικών και η επίδραση του ιοντικού περιβάλλοντος στη δράση τους έγινε αρχικά με κινητικές μελέτες. Το πειραματικό σύστημα που χρησιμοποιήθηκε ήταν η αντίδραση πουρομυκίνης, η οποία μας έδωσε τη δυνατότητα τιτλοδότησης των ενεργών ριβοσωμάτων. Βάσει αυτού μελετήθηκαν τα αντιβιοτικά βλαστισιδίνη και τυλοσίνη που αναστέλλουν άμεσα την ΡΤάση, ενώ για τη μελέτη των υπολοίπων μακρολιδίων διεξήχθησαν πειράματα συναγωνιστικής αναστολής. Ως γνωστό, τα μακρολίδια ερυθρομυκίνη, αζιθρομυκίνη και τελιθρομυκίνη μοιράζονται κοινές θέσεις πρόσδεσης στο ριβόσωμα με την τυλοσίνη. Έτσι, για την εύρεση των σταθερών πρόσδεσης αυτών στο ριβόσωμα έγινε συναγωνισμός με τυλοσίνη. Τα πειράματα συναγωνισμού πραγματοποιήθηκαν, επωάζοντας το ριβόσωμα με μείγμα μακρολιδίου και τυλοσίνης, και τιτλοδοτώντας την απομένουσα δραστικότητα του ριβοσώματος με την αντίδραση πουρομυκίνης απομακρύνοντας την περίσσεια αντιβιοτικών. Σε παράλληλα πειράματα, το ριβόσωμα προεπωάστηκε αρχικά με το μακρολίδιο και στη συνέχεια προστέθηκε τυλοσίνη, η οποία ανιχνεύει το εναπομείναν ριβοσωματικό σύμπλοκο. Επειδή η σταθερά συγγένειας στη δεύτερη περίπτωση βρέθηκε μικρότερη (ισχυρότερη πρόσδεση αντιβιοτικού) συμπεράναμε, ότι ο μηχανισμός πρόσδεσης του αντιβιοτικού είναι βραδύς και ακολουθεί δυο στάδια. Βασιζόμενοι στις τιμές των σταθερών συγγένειας σε πειράματα αναγέννησης του ριβοσωματικού συμπλόκου από την απενεργοποιημένη μορφή του, προσδιορίστηκαν ξεχωριστά όλες οι κινητικές παράμετροι που χαρακτηρίζουν την πρόσδεση του αντιβιοτικού στο ριβόσωμα. Συγκρίναμε τις παραμέτρους αυτές και γενικότερα την ισχύ πρόσδεσης των αντιβιοτικών στο ριβόσωμα σε πέντε ιοντικές συνθήκες: (α) 4,5 mM Mg2+, 150 mM NH4+, (β) 4,5 mM Mg2+, 150 mM NH4+, 100 μΜ σπερμίνη, (γ) 4,5 mM Mg2+, 150 mM NH4+, 50 μΜ σπερμίνη και 2 mM σπερμιδίνη, (δ) 4,5 mM Mg2+, 150 mM NH4+ ριβοσωματικό σύμπλοκο φωτοσημασμένο με 100 μΜ ΑΒΑ-σπερμίνη, και (ε) 10 mM Mg2+, 100 mM NH4+. Τα αποτελέσματα έδειξαν ότι οι πολυαμίνες βελτιώνουν την πρόσδεση της βλαστισιδίνης, αλλά μειώνουν την πρόσδεση των μακρολιδίων. Η επίδραση των ιόντων Mg2+ προσομοιάζει εκείνης των πολυαμινών, αλλά είναι λιγότερο αποτελεσματική, αφού 100 μΜ σπερμίνης επιφέρουν μεγαλύτερη αναστολή πρόσδεσης, από ότι 10 mM Mg2+. Για να ερμηνευτεί σε μοριακό επίπεδο η επίδραση της σπερμίνης στη συγγένεια των αντιβιοτικών έναντι του ριβοσώματος, οι θέσεις πρόσδεσης των πολυαμινών στο ριβόσωμα προσδιορίστηκαν με φωτοσήμανση συγγένειας, χρησιμοποιώντας ως μοριακό ανιχνευτή ένα φωτοδραστικό ανάλογο της σπερμίνης, την ΑΒΑ-σπερμίνη. Οι θέσεις αυτές αποκάλυψαν ότι οι πολυαμίνες προσδένονται σε γειτονικές θέσεις προς τα αντιβιοτικά, επηρεάζοντας την τοπική διαμόρφωση και το φορτίο. Επιβεβαίωση του μηχανισμού δράσης και της επίδρασης των πολυαμινών έγινε με ανάλυση αποτυπώματος. Σύμφωνα με την τεχνική αυτή, τα μακρολίδια προσδενόμενα στο ριβόσωμα προστατεύουν ορισμένα νουκλεοτίδια από την επίδραση χημικών τροποποιητών. Τα αποτελέσματα έδειξαν ότι τα αντιβιοτικά διέρχονται μια ενδιάμεση κατάσταση πρόσδεσης στο ριβόσωμα δεσμευόμενα αρχικά στην είσοδο του τούνελ εξόδου και στη συνέχεια εισχωρώντας βαθύτερα σε αυτό. Η φύση της ενδιάμεσης κατάστασης εξαρτάται από τα ιδιαίτερα χαρακτηριστικά του κάθε μακρολιδίου. Η επίδραση των πολυαμινών στο μηχανισμό πρόσδεσης ελέγχθηκε επαναλαμβάνοντας τα πειράματα χημικής προστασίας παρουσία αυτών. Τα αποτελέσματα έδειξαν ότι η μείωση της πρόσδεσης των μακρολιδίων στο ριβόσωμα επιτελείται κυρίως μέσω της επίδρασης των πολυαμινών στη δέσμευση του υδρόφοβου λακτονικού δακτυλίου. Τα ιδιαίτερα χαρακτηριστικά του κάθε αντιβιοτικού επηρεάζουν ποικιλοτρόπως το μέγεθος της επίδρασης αυτής. Στο τελευταίο κομμάτι της διατριβής μελετήθηκε η ισχύς των μακρολιδίων, υπολογίζοντας την αναστολή που προκαλούν στο συζευγμένο σύστημα μεταγραφής-μετάφρασης του γονιδίου της GFP πρωτεΐνης, και τα αποτελέσματα επιβεβαίωσαν τα κινητικά δεδομένα πρόσδεσης των μακρολιδίων στο ριβόσωμα-στόχο. Σε υψηλή συγκέντρωση ιόντων Mg2+ η τυλοσίνη έχει μεγαλύτερη ισχύ, ενώ σε χαμηλή συγκέντρωση ιόντων απουσία ή παρουσία πολυαμινών η αζιθρομυκίνη. Η τελιθρομυκίνη παρουσίασε τη χαμηλότερη ισχύ πρόσδεσης στο ριβόσωμα και αναστολής της πρωτεϊνικής σύνθεσης. Επιπρόσθετα, ελέγχθηκε πιθανή επίδραση των μακρολιδίων στην πρόσδεση των υποστρωμάτων (tRNAs) στην Α-, Ρ- και Ε- θέση του ριβοσώματος, στη μετατόπιση αυτών από την Α- στην Ρ- θέση και στη μεταφραστική πιστότητα του ριβοσώματος. Βρήκαμε ότι τα μακρολίδια δεν μπορούν να επηρεάσουν αυτά τα στάδια της ριβοσωματικής λειτουργίας. / Ribosomes, the macromolecular translating machines responsible for protein biosynthesis, are the most common targets for many antibacterial agents. Experiments for more than 40 years have demonstrated that a distinct ionic environment (monovalent, divalent cations and polyamines) is essential for ribosomal functions and their interactions with the ligands. Nevertheless, the molecular basis of the ionic environment’s influence on antibiotic mechanism of action has never been precisely elucidated. The aim of this thesis was first to investigate the mechanism of action of several antibiotics –inhibitors of protein synthesis, under ionic conditions close to the cell environment and second, to clarify the role of the ionic environment on their mechanism of action. The antibiotics studied were: a) blasticidin-S, a classic inhibitor of peptidyl tranferase (PTase) activity, b) tylosin which inhibits PTase, but in parallel binds at the entrance of exit tunnel and blocks the passage of the nascent polypeptide chain, and c) erythromycin (a first generation macrolide), azithromycin (a second generation macrolide), and telithromycin (a third generation macrolide, ketolide), that blocks the exit tunnel. The mechanism of action of antibiotics and the influence of ionic environment on antibiotic potency was studied primarily with kinetic methods. The experimental procedure was based on the puromycin reaction, performed under conditions allowing the estimation of the catalytic rate constant. Using this experimental approach we studied the mechanism of action of blasticidin and tylosin which directly inhibit PTase. For studying the other macrolides, experiments employing competitive kinetics were performed. Erythromycin, azithromycin and telithromycin share common binding sites on ribosomes with tylosin. Thus, to estimate the kinetic constants of their interactions with ribosomes, competitive kinetic experiments were carried out in the presence of tylosin. Namely, a posttranslocation ribosomal complex formed from Escherichia coli 70S ribosomes bearing tRNAPhe at the E-site and AcPhe-tRNA at the P-site (complex-C) was incubated with a mixture of each macrolide and tylosin for the desired time intervals. The rest of ribosomal activity was titrated by the puromycin reaction. In parallel experiments, complex-C was pre-incubated with each one of the macrolides and then reacted with tylosin. The rest of complex-C activity was again titrated with the puromycin reaction. Since the affinity constant obtained by the second series of experiments was less than that obtained by the first series of experiments, we concluded that the mechanism of action of antibiotics follows a slow onset inhibition process, which includes two steps. Based on secondary plots and on kinetic plots derived from regeneration of complex-C, we measured the kinetic parameters participating in the kinetic model. Thus, the potency of each antibiotic was determined under five different ionic conditions: (a) 4,5 mM Mg2+, 150 mM NH4+, (b) 4,5 mM Mg2+, 150 mM NH4+, 100 μΜ spermine, (c) 4,5 mM Mg2+, 150 mM NH4+, 50 μΜ spermine and 2 mM spermidine, (d) 4,5 mM Mg2+, 150 mM NH4+, and ribosomal complex photolabelled with 100μΜ ΑΒΑ-spermine, and (e) 10 mM Mg2+, 100 mM NH4+. Processing of the data led us to the conclusion that polyamines and Mg2+ ions increase the potency of blasticidin, but decrease the potency of macrolides. To explain the diverse action of polyamines and of the ionic environment in general on antibiotic potency, the binding sites of spermine in ribosomes were localized by photoaffinity labeling, using a photoactive analogue of spermine, ABA-spermine. These experiments revealed that polyamines bind at the vicinity of antibiotics, influencing the ionic charge and the local conformation of rRNA. Confirmation of the macrolide mechanism of action and verification of the influence of polyamines on their potency was achieved by footprinting analysis. According to this technique, macrolides bind to ribosomes and protect specific nucleotides from modification by chemical reagents like DMS, CMCT and kethoxal. The results demonstrated that the antibiotics (I) form an encounter complex with complex-C (CI), in which the antibiotics occupy the entrance of the exit tunnel. This intermediate complex is then isomerized slowly to a tighter complex (C*I) with which antibiotics move deeply in the exit tunnel. The exact interactions stabilizing the intermediate complex depend on the characteristic groups of each macrolide. The influence of polyamines was checked by repeating the experiment in the presence of polyamines. The results showed that polyamines reduce the macrolide binding to ribosomes, by affecting mainly the interactions of the hydrophobic lactone ring with the ribosome. The special characteristic groups of each macrolide affect the polyamine action. The potency of macrolides action was also estimated using a coupled transcription-translation system for GFP expression. The results obtained were consistent with those produced by kinetic analysis. In addition, we check for possible macrolide effects on tRNA binding at the A-, P- and E- sites of the ribosome, on translocation, and on translational fidelity. No strong effects were identified excluding the macrolide from these ribosomal functions.

Les peptides antimicrobiens (PAMs) représentent des composants importants de l`immunité innée. Ils sont présents dans la plupart des organismes multicellulaires et constituent la première ligne de défense contre les infections. Ils possèdent un large éventail d`activités, une non-toxicité contre les cellules de l`hôte et des effets synergiques avec les antibiotiques conventionnels. Par conséquent, ils peuvent être d`excellents candidats dans le développement de nouveaux antibiotiques pour lutter contre la résistance de microorganismes. Concernant les PAMs dérivés de la chromogranine A (CgA), la cateslytine (Ctl) présente des activités antimicrobiennes directes et des propriétés immunomodulatrices. Dans ma thèse, j`ai cherché à caractériser l`épipeptide D-Ctl, où tous les résidus en conformation-L ont été remplacés par des résidus en conformation-D. Tout d`abord, la stabilité dans les surnageants bactériens et des dosages de l`activité antimicrobienne ont été réalisés, ainsi que l`analyse de viabilité des cellules et des dosages des cytokines libérées par les cellules immunitaires. L`efficacité de D-Ctl a été comparée à celle de L-Ctl contre des souches bactériennes, puis les CMIs ont été déterminées et comparées dans le cas de combinaisons avec des antibiotiques conventionnels, afin de montrer un effet synergique et/ou additif. De plus, D-Ctl ne déclenche pas de résistance chez E. coli. Des tests de cytotoxicité ont été effectués sur plusieurs types de lignées cellulaires et de PBMCs. Les effets inflammatoires aussi ont été testés. Ensuite, le modèle bactérien E. coli MDR a été utilisé pour des analyses physico-chimiques, telles que la microscopie à épifluorescence, la spectroscopie ATR-FTIR et la microscopie à force atomique. Enfin, le brevet D-Ctl a été déposé en 2016 sous le numéro EP 16306539.4 « Nouveau peptide de cateslytine en conformation D ». En conclusion, D-Ctl est capable de tuer rapidement un large spectre de micro-organismes, et il pourrait potentialiser l`effet antimicrobien de plusieurs antibiotiques. / Antimicrobial peptides (AMPs) represent important components of innate immunity. They are present in most multicellular organisms and constitute the first line of defense against infections. They exhibit a large spectrum of activities, a non-toxicity against host cells and synergistic effects with conventional antibiotics. Therefore, they can be as excellent candidates in the development of new antibiotics to fight pathogens resistance. Concerning to AMPs derived from chromogranin A (CgA), Cateslytin (Ctl) represents a new antibiotic, which displays direct antimicrobial activities and immunomodulatory properties. In my thesis, I aimed to characterize the epipeptide D-Ctl, where all (L-conformation) residues were replaced by (D-conformation) residues. Firstly, antimicrobial assays were performed, cells viability, immune assays, and the stability in bacterial supernatant was tested. The efficiency of D-Ctl was compared with L-Ctl against bacterial strains, then MICs were determined and compared with combinations in presence of classical antibiotics in order to show synergistic or/and additive effect. Moreover, D-Ctl does not trigger resistance in E. coli. Also, cytotoxicity assays were performed on several types of cell line and PBMCs. Inflammatory effects were tested too. Then, bacterial model E. coli MDR was used for physicochemical analysis such as epifluorescence microscopy, ATR-FTIR spectroscopy and atomic force microscopy. Finally, D-Ctl patent has been deposited in 2016 under the number EP 16306539.4 “New D-configured cateslytin peptide”. To conclude: D-Ctl is able to rapidly kill a broad spectrum of microorganisms, and it could potentiate the antimicrobial effect of several antibiotics.