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Thiopeptides are a family of antimicrobial peptides that are characterized for having sulfur-containing heterocycles, and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The antimicrobial peptide micrococcin is a thiopeptide that is synthesized by the ribosome and undergoes several post-translational modifications (PTMs). Micrococcin is formed from a precursor peptide, TclE. TclE comprises an N-terminal leader (35-AA) that is crucial for recognition of the PTM machinery, alongside a C-terminal core sequence (14-AA) that undergoes multiple PTMs to acquire its antimicrobial activity. In the first series of modifications, the scaffold protein TclI binds the leader of TclE and presents the core of TclE to the modifying enzymes TclJ and TclN, facilitating the conversion of 6 cysteine residues into thiazoles. The work of this dissertation focuses on understanding the key interactions between the TclIJN protein complex and the precursor peptide TclE. By carrying out mutagenesis analysis on the leader peptide, I determined a minimal region of TclE that is required for thiazole installation. By doing bioinformatic analysis and copurification experiments, I determined that the TclI scaffold protein binds to the enzymes TclJ and TclN one at a time in dynamic equilibrium. I also further characterized the region of TclI that is important for coordinating these interactions and determined key residues that play a role for binding to its enzymatic partners. During my PhD, I had the opportunity to work on a few side projects that came up as I was working on plasmid construction for the Tcl project and working as a teaching assistant for the Microbial Genetics class (MMBIO360). During plasmid construction for protein expression of Tcl proteins, we recognized that there was room for improvement on transcriptional terminators, especially for the widely used T7 RNA polymerase. We engineered a set T7 terminators that are shorter and more efficient compared to previously reported T7 terminators, both in vivo and in vitro. As a teaching assistant for the MMBIO360 class, I had the opportunity to coordinate the work of undergraduate students in research-driven projects. We used the genetically tractable organism Agrobacterium fabrum to investigate flagellar motility. We carried out a near-saturating screen that led to the finding of four previously undescribed genes that are essential for motility in this organism. Another side project that also emerged from this class is investigating the genetics of streptomycin resistance in A. fabrum. Once paper from each of these three side projects are reprinted in Chapters 3, 4 and 5, respectively.

Thiopeptides are posttranslationally-processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogenous ring, thioazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, produced by Streptomyces laurentii ATCC 31255, is one of the more structurally complex thiopeptides, containing a second macrocycle bearing a quinaldic acid. Thiostrepton A and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens, in addition to their antimalarial and anticancer properties. The ribosomal origins for thiopeptides have been established, however, few details are known concerning the posttranslational modification steps. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton A posttranslational machinery. The information gathered from current studies can also be used to refine thiostrepton’s structure-activity relationship, providing insight into the key features of its scaffold that impart specificity toward each biological target. A fosmid-dependent biosynthetic engineering platform for thiostrepton A was developed and a series of thiostrepton analogs were successfully produced adapting this method. The seventh residue of thiostrepton A is predicted to be critical for the metabolite’s antibacterial activity. Our results were consistent this hypothesis and demonstrated that substitution of Thr7 in the thiostrepton A precursor peptide disrupts both biological activity and successful biosynthesis of the analogs. The thiostrepton biosynthetic machinery’s tolerances toward structural variation at the second and fourth positions of the TsrA core peptide were probed by the saturation mutagenesis of Ala2 and Ala4, respectively. Eight thiostrepton Ala2 variants were isolated with two analogs truncated at the N-terminus by one amino acid, bearing a shortened quinaldic acid-containing macrocycle. Our results suggested that the identity of the core peptide second residue influences the biosynthesis of a thiostrepton analog, however, not essential for the antibacterial and proteasome inhibitory activities of the full-length variants. Additionally, the quinaldic acid loop size affects thiostrepton’s antibacterial potency, but is not critical for the proteasome inhibitory activity. Sixteen thiostrepton analogs were isolated from Ala4 mutagenesis studies. We demonstrated that the identity of the amino acid residue at the fourth position in the thiostrepton scaffold is not critical to inhibit either the ribosome or the proteasome in vitro.

Face à l’apparition alarmante et continue de résistance massive des bactéries à l’arsenal actuel d’antibiotiques, la recherche de nouveaux agents antibactériens est actuellement un enjeu sociétal de tout premier ordre. Bien que connus depuis plus de 50 ans, les thiopeptides antibiotiques suscitent actuellement un très fort regain d’intérêt de la communauté scientifique internationale en raison de leurs propriétés antibactériennes remarquables, notamment contre les staphylococcus areus résistants à la méthiciline et les enterococci résistants à la vancomycine, impliquées dans de nombreuses infections qui engagent la vie des patients, et qui s’exercent de façon très intéressante selon deux modes d’action d’inhibition de la synthèse protéique originaux et encore inexploités en thérapie antibiotique humaine. Le travail développé s’inscrit dans ce programme international de valorisation pharmacologique qui repose pour une grande part sur le développement d’approches synthétiques rapides et modulables. Le projet a été centré en particulier sur la conception et la mise en oeuvre de nouveaux plans de synthèse des unités centrales di- ou trithiazolylpyridines, appelées coeurs hétérocycliques, communs à de nombreux thiopeptides de la série d et qui représentent les principaux défis synthétiques. Dans le cadre d’un programme de recherche du laboratoire ciblé sur l’étude de nouvelles méthodes de fonctionnalisation directe d’aromatiques et d’hétérocyclique, un premier travail méthodologique d’étude de la fonctionnalisation directe dans deux séries structurellement représentatives, thiazole-4-carboxylate et 2-cétothiazole, a été réalisé. Ainsi, une nouvelle méthodologie originale d’hétéroarylation directe pallado-catalysée régioselective du thiazole-4-carboxylate de tert-butyle avec une large gamme d’halogéno(hétéro)aromatiques a été développée avec succès. Une seconde méthodologie originale d’hétéroarylation directe de 4-bromo-2-cétothiazoles selon une séquence réactionnelle de boroylation pallado-catalysée suivie d’un couplage de Suzuki-Miyaura (BSC) a également été développée. Un second travail a porté sur l’exploitation des deux méthodologies de fonctionnalisation directe en série thiazolique développées et associées à la méthodologie de construction thiazolique de Hantzsch pour proposer et mettre en oeuvre un nouveau plan de synthèse expéditif et général d’accès aux coeurs hétérocycliques communs à une grande majorité des thiopeptides de la série d basé sur la fonctionnalisation séquencée d’un précurseur pyridinique aisément accessible. En particulier, la stratégie envisagée a permis, à partir des esters 5-bromopicolinates, la préparation tout d’abord d’un analogue thiazolique du coeur hétérocyclique des sulfomycines puis celle des coeurs hétérocycliques des micrococcines et des amithiamicynes. / Facing the alarmist and constant emergence of novel resistances of bacteria to the current arsenal of antibiotics, the research of novel antibacterial agents is of main importance in our modern society. Though being well-known since 50 years, the thiopeptide family display a high renewed of interest due to their high antibacterial properties against the Gram-positive bacteria, including the multi-drugs resistant staphylococcus areus strains (MRSA) and vancomycinresistant enterococci (VRE), through interestingly two original modes of action of the inhibition of the protein synthesis, yet unexploited by human therapeutics. The present work takes part of the current active international research program of pharmalogical valorisation of thiopeptides as novel human antibacterial therapeutics and one of the major points is the development of innovative and versatile synthetic approaches. Toward this direction, the project is mainly based upon the design of original synthetic strategy to the most synthetically challenging di- or trithiazolypyridines heterocyle cores of thiopeptide of serie d. In the course of a current research program on the development of metallo-catalyzed direct C-H(X) functionnalization in heteroaromatics series, the first part of this work had been focused on the setting of two innovative direct C-H(X) functionnalization methodologies in two structurally representative scaffolds, thiazole-4-carboxylate and 2- ketothiazoles. Thus, the direct regioselective C-H heteroarylation of thiazole-4-carboxylate as well as the direct C-Br heteroarylation of 4-bromo-2-ketothiazole through a boroylation Suzuki coupling sequence (BSC) have been examined. The last part of the work has been focused on the use of both novel direct C-H(Br) functionalization methodologies in thiazole series combined with the Hantzsch thiazole synthesis to propose a novel general and versatile synthetic approach toward the common heterocyclic cores of several thiopeptides of serie d through a pyridine modulation strategy from a ready available pyridine precursor. In particular, the novel developed strategy has been sucessfully applied to the preparation of the sulfomycinamate thio-analog as well as the heterocyclic cores of micrococcins and amithiamycins.