The ability of bacteria to successfully adapt to changing environments allows for increased resistance to antibiotics, an increasing problem in drug development. Efflux via the cellular membrane is one of the major and most significant mechanisms bacteria employ for defense against antimicrobials. The outer membrane protein, TolC, is a nonspecific channel with broad substrate specificity and is a fundamental constituent of a number of multidrug resistance (MDR) efflux systems. TolC, which interacts with membrane bound antiporters to export various substrates, including antimicrobials, has been a focus for antibiotic development. The capacity to expel a wide range of compounds such as toxins, bile, dyes and detergents, further establishes the significance of these systems in aiding bacterial survival. Continued investigation and discovery of compounds secreted via TolC is required to fully understand the role of efflux systems. In addition to exporting compounds out of cells, TolC has been researched in connection with a number of metabolic processes including virulence, motility and quorum sensing. This work contributes to knowledge of interactions between these pathways by analysing phenotypic changes and alterations in gene expression in the tolC- mutant within Vibrio furnissii. Vibrio species are strongly associated with a number of reported phenotypic changes induced by mutations in tolC, particularly virulence and quorum sensing; the emerging pathogen V. furnissii is therefore a model organism for this study. Notably, this bacterium has become significant within the bio-fuel industry due to reports that it is able to synthesise large quantities of hydrocarbons. The potential for bacteria to produce hydrocarbons and fatty acids suitable for use in bio-fuels has considerable industrial applications. This work establishes the quantities of hydrocarbons and lipids present in V. furnissii and examines the role of TolC in the secretion of these compounds. Resistance nodulation division efflux pumps are a class of MDR systems that have been identified as targets for antimicrobial drug development. Understanding the consequence of disrupting the integral outer membrane component TolC, is therefore of significant interest. This work examines the connection between pathways disrupted within tolC- mutants, showing that quorum sensing gene regulation is altered within the mutant and subsequently, expression of the virulence factor AphB is increased. Reduced motility is also observed in the tolC- mutant, a phenotype that appears to result from disruption of the membrane and mis-assembled flagella. Investigation into cellular stress, recently reported to occur within tolC- mutants, was performed by comparing the capability of wild type and mutant cells to metabolise selenite, and cope with the subsequent increase in superoxides generated. Results show that tolC- mutants employ mechanisms which cope with the inevitable intracellular build up of substrates, particularly those that may be toxic to the cell.

L’objet de ces recherches était de trouver de nouveaux EPIs du système d’efflux AcrAB-TolC dans les groupes de dérivés d’amine de la 5-arylidenehydantoine et de la 5-arylideneimidazolone. Dans la première étape de recherche, 32 nouveaux dérivés ont été obtenus après modification de la structure lead P2.Un screening théorique du risque toxique et la prédiction des propriétés médicamenteuses des composés ont été réalisés en utilisant le programme OSIRIS qui calcule différentes propriétés médicamenteuses pertinentes basées sur la structure planaire de la molécule.Dans l’étape suivante de la recherche, l’activité des composés a été évaluée par des études microbiologiques sur souches d’Enterobacter aerogenes avec différentes expressions de la pompe d’efflux AcrAB. La première étude effectuée était un test de sensibilité pour déterminer les CMI des composés afin de trouver une concentration à laquelle les utiliser avec des antibiotiques sans avoir l’influence de leur activité intrinsèque. Puis l’effet des composés sur la sensibilité des antibiotiques tels que l’acide nalidixique, le chloramphénicol, la doxycycline et l’érythromycine a été étudié. Après, le type de coopération avec les antibiotiques a été déterminé par la construction d’isobologrammes et le calcul de l’index de FIC. Les dernières études microbiologiques réalisées sont des tests d’efflux en temps réel qui utilisent un colorant fluorescent 1,2’-dinaphthylamine et ont permis de suivre directement le fonctionnement de la pompe. L’étude des relations structure-activité souligne le rôle essentiel de la nature amphiphile des EPIs dérivés de groupes arylidene de l’hydantoine et de l’imidazolone. / The purpose of this research was to find new EPIs of the AcrAB-TolC efflux system in groups of amine derivatives of 5-arylidenehydantoin and 5-arylideneimidazolone. In the first stage of the research 32 new derivatives of 5-arylidenehydantoin were obtained as a result of modifications of the lead structure P2. Theoretical screening of the toxicity risk as well as the prediction of drug-like properties of hydantoins/imidazolones synthesised were performed by using the OSIRIS program which calculates various drug-relevant properties based on a planar structure of the molecule.In the next stage of the research the activity of compounds was evaluated in microbiological studies. Strains of Enterobacter aerogenes with different expressions of the AcrAB efflux pump were used. The first study carried out was a susceptibility test determining the MICs of compounds in order to find a concentration that could be used in combination with antibiotics without the influence of an intrinsic antibacterial activity of the compounds. Then the effect of the compounds on bacterial susceptibility to antibiotics such as nalidixic acid, chloramphenicol, doxycycline and erythromycin was examined. After, the type of cooperation with antibiotics was determined based on isobolograms and the FIC index calculated. The last of microbiological studies was the real-time efflux (RTE) assay which used the fluorescent dye 1,2’-dinaphthylamine and allowed the functioning of the pump to be monitored directly. The structure-activity relationship (SAR) analysis emphasized the essential role of the amphiphilic nature of the EPIs from the group of arylidene derivatives of hydantoin and imidazolone.

abstract: Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2018

Master of Science / Department of Plant Pathology / Revathi Govind / Clostridium difficile, a gram-positive, anaerobic bacterium, is a major cause of antibiotic-related diarrhea and pseudomembraneous colitis. In the last decades, C. difficile has emerged as a major threat because of its tendency to cause frequent and severe disease. Because of the severity of the infection and its high rate of recurrence, there is a significant financial burden on healthcare systems. Antibiotic treatments are a primary risk factor for the development of C. difficile infection because they disrupt the normal gut flora in the host, enabling the antibiotic resistant bacterium to colonize the colon. Most of the resistance mechanisms in C. difficile reported to date can be classified as either antibiotic-degrading enzymes or modification of target sites. Another mechanism that can contribute to antibiotic resistance in C. difficile is the extrusion of antimicrobial compounds by efflux pumps. The goal of this project was to provide initial insights into the roles and mechanisms of a putative efflux pump complex. To do this, a number of experiments were designed to provide information about the structures, localization, and functions of this protein complex. It was determined that acidic pH conditions and a small number of antimicrobials, including inorganic compounds, organic compounds, fungicides, and antibiotics, inhibit growth of a C. difficile mutant lacking this pump system. Interestingly, higher NaCl in the medium and alkaline pH seem to promote the growth of a C. difficile mutant lacking this pump or, surprisingly, only inhibit growth of the wild type strain. The experiments performed in this project suggest that this efflux pump might have an essential role in C. difficile physiology, possibly by serving as an efflux pump for toxic metabolites.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:02Z No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:47Z (GMT) No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Made available in DSpace on 2018-04-16T14:40:47Z (GMT). No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) Previous issue date: 2018-03-06 / Klebsiella pneumoniae is one of the main bacterial agents that may cause infections related to health care assistance. K. pneumoniae frequently carries the resistance gene K. pneumoniae carbapenemase (KPC). Currently, tigecycline can be considered one of the last therapeutic options for KPC, but reports of tigecycline-resistant KPC isolates are on the rise, being indicated as most common mechanism the increased AcrAB-TolC efflux pump system expression. However, molecular tigecycline resistance mechanisms associated to AcrAB-TolC still remains obscure. Thus, the main goal of this study was to verify if tigecycline resistance can be related to the presence of mutations in the regulatory genes of AcrAB-TolC, AcrR and RamR. Therefore, 32 K. pneumoniae isolates were used, identification and antibiogram performed using Vitek 2 systems. The minimum inhibitory concentrations were confirmed using E-test. Primers were designed in order to verify mutations within AcrR and RamR genes. PCR analysis showed that the mutations found within these genes were transversions (94% for AcrR and 90% for RamR) and transitions (6% for AcrR and 10% for RamR). Nevertheless among the mutations, no distinction between tigecycline susceptible and resistant isolates was found. Some of the transversions caused change in the amino acid encoding 6 in AcrR and 15 in RamR. Presence of these types of mutations evaluation can be seen as the first bacterial resistance study step, as it may be caused by oxidative damage for bacterial DNA, frequently caused by antibiotic selective pressure. Tigecycline resistance found in this study`s clinical isolates may be associated to alterations in another genes that can trigger mechanisms associated to this antibiotic. / Klebsiella pneumoniae consiste em um dos principais agentes bacterianos causadores de infec????es relacionadas ?? assist??ncia ?? sa??de (IRAS). K. pneumoniae carrega frequentemente o gene de resist??ncia K. pneumoniae carbapenemase (KPC). Atualmente, a tigeciclina pode ser considerada uma das ??ltimas op????es terap??uticas para KPC, mas os relatos de isolados de KPC resistentes a tigecilina est??o em ascens??o, sendo a hiperexpress??o da bomba de efluxo AcrAB-TolC indicado como mecanismo mais comum. No entanto, os mecanismos moleculares de resist??ncia ?? tigeciclina associada ao AcrAB-TolC permanecem obscuros. Desta forma o objetivo deste trabalho foi verificar se a resist??ncia a tigeciclina pode estar relacionada ?? presen??a de muta????es nos genes ArcR e RamR, reguladores de AcrAB-TolC. Para tanto, 32 isolados de K. pneumoniae foram utilizados, sendo a identifica????o e o antibiograma feitos utilizando o sistema Vitek 2. A confirma????o das concentra????es inibit??rias m??nimas (CIMs) foram realizadas por E-test. Iniciadores foram desenhados para verifica????o de muta????es nos genes (AcrR e RamR). As an??lises por PCR mostraram que as muta????es encontradas nos genes AcrR e RamR foram substitui????es por transvers??o (94% e 90% para AcrR e RamR respectivamente) e transi????o (6% e 10% para AcrR e RamR respectivamente), por??m n??o foi identificada distin????o da presen??a de muta????es entre isolados sens??veis e resistentes a tigeciclina. Algumas tranvers??es ocasionaram mudan??a na codifica????o do amino??cido, sendo 6 em AcrR e 15 em RamR. A avalia????o da presen??a desses tipos de muta????es consiste em um primeiro passo para o estudo da resist??ncia bacteriana, j?? que pode ser causada por dano oxidativo ao DNA bacteriano, frequentemente ocasionado por press??o seletiva dos antibi??ticos. A resist??ncia a tigeciclina encontrada nos isolados cl??nicos do presente estudo, provavelmente pode estar associada a altera????es em outros genes desencadeadores de mecanismos de resist??ncia a tigeciclina.

abstract: Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression. Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression. To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A. / Dissertation/Thesis / Masters Thesis Biology 2016

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.