title: Structural and functional characterization of conjugative transposition for the design of novel antibiotic resistance transfer inhibitors
creator: Rojas Cordova, Carlos Alberto
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
description: Antibiotic resistance (ABR) is currently one of the most significant global health challenges. In addition to the rapid development of resistance against new antibiotics, the transfer of existing ABR genes between bacteria leads to the growth of difficult-to-treat multidrug-resistant opportunistic pathogens, causing millions of infections and thousands of deaths every year worldwide. Mobile genetic elements (MGEs) provide a powerful mechanism to transfer ABR genes, because they can move across bacterial cells and species and carry ABR cargos within their sequence. However, their mechanisms of transfer are incompletely understood. Therefore, I investigated the molecular mechanism of a prominent but poorly characterized MGE, the vancomycin resistance carrying conjugative transposon (CTn) Tn1549 from Enterococcus spp. This element is responsible for propagating resistance to this last-resort antibiotic in a wide range of intestinal bacteria. I focused my work on the integrase (Int) and excisionase (Xis) proteins, which are responsible for performing all DNA cleavage and joining reactions during Tn1549 transposition.    In the first part, I reconstituted the complex of Int with a four-way Holliday Junction (HJ) DNA molecule in vitro and solved its crystal structure at 3.3 Å resolution. This is the first CTn integrase structure trapped with this reaction intermediate, showing that these enzymes assemble a stable tetramer to recombine two DNA substrates, in a similar way as the site- specific tyrosine recombinases. Comparison of both enzyme families shows that a cyclic exchange of the C-terminal protein segments promotes tetramerization in all cases. I further characterized the structure of the accessory Xis protein at 1.5 Å resolution. In the second part of my work, I validated the structure by performing HJ resolution experiments in vitro. I found that Int can resolve HJ intermediates both to products and to substrates, likely due to missing regulatory factors. This reaction leads to DNA products containing up to 3 nt long unpaired regions, reflecting Int’s ability to insert its cargo DNA at diverse genomic sequences. In the third part of my work, I show that novobiocin, an aminocoumarin antibiotic, can inhibit Int tetramerization and HJ resolution in vitro, highlighting the importance of the tetrameric state for the transposition reaction.    This work sheds light on an essential step of Tn1549 transposition and its regulation. Moreover, it highlights crucial similarities with site-specific recombinases, increasing our understanding of conjugative transposition. As a proof-of-principle, inhibition of Int activity by novobiocin may open doors to develop potent CTn inhibitors as a new strategy to limit ABR spreading among bacteria.
date: 2022
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/29453/1/CarlosRojas_Thesis.pdf
identifier: DOI:10.11588/heidok.00029453
identifier: urn:nbn:de:bsz:16-heidok-294532
identifier:   Rojas Cordova, Carlos Alberto  (2022) Structural and functional characterization of conjugative transposition for the design of novel antibiotic resistance transfer inhibitors.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/29453/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng