Abstract
To streamline the elucidation of antibacterial compounds’ mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus. C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.
| Original language | English (US) |
|---|---|
| Article number | e01231-18 |
| Journal | Antimicrobial agents and chemotherapy |
| Volume | 62 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2018 |
Bibliographical note
Funding Information:This work was supported by grants from Cystic Fibrosis Canada and Research Manitoba to S.T.C., the Italian Cystic Fibrosis Foundation (grant FFC#19/2015 to G.R., adopted by Gruppo di Sostegno FFC di Como Dongo, Delegazione FFC di Olbia Tempio, Delegazione FFC di Reggio Calabria), the Cystic Fibrosis Foundation to GR (grant RICCAR17G0), and by a BlueSky research grant of the University of Pavia to S.B. A.M.H. was supported by grants from the University of Manitoba (UMGF), the Canadian Institutes of Health Research (CIHR), and Cystic Fibrosis Canada.
Funding Information:
This work was supported by grants from Cystic Fibrosis Canada and Research Manitoba to S.T.C., the Italian Cystic Fibrosis Foundation (grant FFC#19/2015 to G.R., adopted by Gruppo di Sostegno FFC di Como Dongo, Delegazione FFC di Olbia Tempio, Delegazione FFC di Reggio Calabria), the Cystic Fibrosis Foundation to GR (grant RICCAR17G0), and by a BlueSky research grant of the University of Pavia to S.B. A.M.H. was supported by grants from the University of Manitoba (UMGF), the Canadian Institutes of Health Research (CIHR), and Cystic Fibrosis Canada. We are grateful to Pavel Drevinek (University Hospital Motol, Prague, Czech Republic), Renato Fani (University of Florence, Italy), and Eshwar Mahenthiralingam (University of Cardiff, UK) for providing some of the Burkholderia clinical isolates used in this study, to Eric Brown (McMaster University) for providing ASKA clones, to Luis Galietta (TIGEM, Pozzuoli, Italy) for providing the epithelial 16HBE cells, to George Zhanel (University of Manitoba) for providing ESBL-producing E. coli and K. pneumoniae strains, to Marta Mollerach (Universi-dad de Buenos Aires) for providing S. aureus CF 225, and to Heather Adam (Diagnostic Services Manitoba) for providing isolates of M. abscessus. We also thank André Dufresne for technical support, Génome Québec for sequencing services, and Ayush Kumar, Sean McKenna, and Mazdak Khajehpour for critically reading the manuscript. S.T.C. and S.B. conceived the idea, designed the research, and edited the final version of the paper. A.M.H. designed and performed the fitness assays and microscopy experiments and wrote the paper. V.C.S. performed cloning, protein expression, and purification and enzymatic assays. V.M., O.R., and N.M. synthesized C109. A.S.G. performed preliminary experiments and edited the final version of the manuscript. H.T. and S.B. performed MIC experiments and checkerboard assays. M.S.S. performed the C. elegans assays and edited the final version of the manuscript. A.K.C.B. contributed microscopy support. M.D. and X.L. were involved in processing and analyzing the data. A.A. performed cytotoxicity experiments on human cells. M.B. performed electron microscopy experiments. L.R.C. performed enzymatic assays and analyzed data. G.R., S.B., and S.T.C supervised the work and contributed financially.
Publisher Copyright:
© 2018 Hogan et al.
Keywords
- Burkholderia
- Drug targets
- Essential genes
- Fluorescent image analysis
- FtsZ
- Mechanisms of action
- Tn-seq
