Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.

Le traitement des infections dues aux bactéries multirésistantes aux antibiotiques est un défi médical majeur du 21ème siècle. Ce défi a incité la recherche de cibles ayant des fonctions essentielles pour le développement de nouveaux antibiotiques. Les enzymes de la voie FASII, responsables de la synthèse des acides gras (AG), sont considérées comme essentielles et de nombreux antibiotiques, appelés anti-FASII, ont été développés pour lutter contre des pathogènes du phylum des Firmicutes. Cependant, notre laboratoire a montré que plusieurs pathogènes contournent l’inhibition des anti-FASII par l’utilisation des AGs exogènes abondants chez l’hôte (sang, organes, aliments). Ce contournement compromet l’utilisation des anti-FASII en traitement. Le statut du pathogène majeur, Staphylococcus aureus, est néanmoins resté en débat. Il synthétise un AG non disponible chez l’hôte, et donc, d’après la littérature, ne pourrait pas être compensé. La question de cette thèse est de comprendre les mécanismes utilisés par S. aureus lui permettant de contourner les anti-FASII. Deux mécanismes sont mis en évidence: I- Des mutations à haute fréquence du gène fabD surviennent et permettent à la bactérie d’utiliser des AGs exogènes. Ce type de mutation favorise la disponibilité de la protéine ACP permettant l’utilisation des AGs exogènes. II- Une stratégie sans mutation décelable survient en présence de fluides hôtes tels que le sérum. Elle comprend une première étape de "dormance" d’environ 8 à 10 heures pendant laquelle des AGs sont incorporés. Durant cette adaptation les bactéries semblent bloquées dans la division cellulaire, et subissent des changements morphologiques. Cette étape est suivie par une reprise de croissance « normale » où S. aureus utilise librement des AGs exogènes et reste insensible aux anti-FASII. Dans nos conditions, une étude microscopique « time-lapse », a permis de visualiser qu’environ 3% de la population bactérienne adaptée aux anti-FASII émerge. Nos résultats pointent vers un mécanisme d’adaptation dans lequel le sérum diminuerait le stress bactérien et augmenterait ainsi la disponibilité de l’ACP et des AGs exogènes, facilitant leur utilisation pour la synthèse des phospholipides. Les AGs exogènes peuvent donc remplacer totalement les endogènes. Ce résultat va à l’encontre de l’hypothèse couramment acceptée qu’un AG endogène de S. aureus est conservé et essentiel. Nous avons poursuivi cette étude par des analyses protéomiques et par le criblage d’une banque de 2000 mutants de S. aureus en cherchant des loci impliqués dans l’adaptation aux anti-FASII. Des fonctions de la réponse au stress, la division cellulaire et le métabolisme des lipides semblent être impliqués dans cette adaptation. Pour conclure, cette étude a permis de clarifier les étapes impliquées dans la réponse adaptative de S. aureus aux anti-FASII. Même si nos résultats prouvent que S. aureus contourne les anti-FASII, une approche combinatoire pourrait être envisagée où l’anti-FASII serait couplé avec un deuxième inhibiteur qui bloquerait sa capacité à sortir de la dormance. / Treatment of infections caused by multidrug-resistant bacteria is a major medical challenge of the 21st century, which has stimulated the search for essential bacterial functions as potential antimicrobial drug targets. The fatty acid synthesis (FASII) pathway enzymes are considered essential, and numerous antibiotics called (anti-FASII), have been developed to eliminate pathogens of the Firmicutes phylum. However, our laboratory has shown that several pathogens bypass FASII inhibitors by incorporating exogenous fatty acids (FAs), which are abundant in the host (in blood, organs and foods). FASII bypass thus compromises the use of FASII-based antibiotics. The status of the major pathogen, Staphylococcus aureus, has remained in debate. S. aureus synthesizes an FA not produced by the host and according to the literature, is required and would not be available in the host. However, work in my lab showed that indeed bypasses FASII. The goal of my research project is to understand the mechanisms used by S. aureus to bypass FASII antibiotics. Two mechanisms are highlighted: I- High frequency mutations of the fabD gene allow S. aureus to use exogenous FAs; our study indicates that higher availability of ACP in these mutants facilitates FA utilization. II- A strategy without detectable mutation occurs in the presence of host fluids such as serum. It comprises a first "dormancy" step of about 8 to 10 hours, followed by outgrowth; FAs are incorporated throughout these steps. The latency phase appears to be due to a division block, during which cells undergo morphological changes. During "normal" growth recovery, S. aureus freely uses exogenous FAs and remains insensitive to anti-FASII. Using « time-lapse » microscopic study, we showed in our test conditions that about 3% of the bacterial population adapted to FASII antibiotics. Ours results point to an adaptation mechanism in which serum decreases bacterial stress, leading to increased availability of ACP and exogenous FAs. These substrates can then be used for phospholipid synthesis. These results resolve the debate by showing that S. aureus can replace endogenous FAs with exogenous FAs when FASII is blocked. Contrary to current dogma, FASII is not essential in S. aureus. To identify the loci involved in adaptation to anti-FASII, we performed proteomic analyses and also screened a S. aureus mutant library. Functions of stress response, cell division, and lipid metabolism appear to be involved in this adaptation. To conclude, this study clarified the steps leading to S. aureus adaptation to FASII antibiotics. Although our results show that S. aureus bypasses anti-FASII, a combinatorial approach could be considered in which FASII antibiotics could be coupled to a second inhibitor that would prevent exit from the dormancy.