Chemical genomics informs antibiotic and essential gene function in Acinetobacter baumannii
Jennifer Suzanne Tran, Ryan David Ward, Rubén Iruegas-López, Ingo Ebersberger, Jason Matthew Peters
Abstract
The Gram-negative pathogen, Acinetobacter baumannii, poses a serious threat to human health due to its role in nosocomial infections that are resistant to treatment with current antibiotics. Despite this, our understanding of fundamental A. baumannii biology remains limited, as many essential genes have not been experimentally characterized. These essential genes are critical for bacterial survival and, thus, represent promising targets for drug discovery.
Introduction
Systems biology provides a robust framework for deciphering the complex networks that govern cellular functions. This approach is especially pertinent in the study of infectious diseases and antibiotic resistance as it offers sophisticated tools to analyze the multifaceted interactions between pathogens and their environments, including their responses to therapeutic interventions. The Gram-negative, hospital-acquired pathogen Acinetobacter baumannii is categorized as an ‘urgent threat’ due to certain clinical strains having developed resistance to all known therapeutics and its ability to persist on surfaces that are typically adverse to cellular life, such as stainless steel [1,2]. Despite the critical dangers posed by A. baumannii, our understanding of how the fundamental elements of its biology interact with antibiotics or other inhibitors remains limited.
Materials and method
Bacterial strains and growth
All strains, plasmids, and oligos used in this study are listed in S1–S3 Tables. Strains were grown in EZ Rich Defined Media (Teknova) supplemented 40 mM sodium succinate as the carbon source (AbRDM) or AbRDM + 1.5% agar at 37°C unless otherwise noted. Selective antibiotics were used when necessary: for E. coli, 100 µg/mL ampicillin (amp) or carbenicillin (carb), 15 µg/mL gentamicin (gent), or 30 µg/mL kanamycin (kan); and for A. baumannii, 10 µg/mL polymyxin B (pmb), 150 µg/mL gentamicin (gent), 60 µg/mL kanamycin (kan). Diaminopimelic acid (DAP) was added at 300 µM to support growth of E. coli dap- donor strains. IPTG (isopropyl b-D-1-thiogalactopyranoside) (0 to 1 mM) was added where indicated in the figures or figure legends. Strains were preserved in 15% glycerol at -80°C.
Results
The vast majority of A. baumannii essential genes show significant chemical-gene interactions
To identify phenotypes for essential gene knockdown strains in the context of chemical treatment (i.e., chemical-gene interactions) in A. baumannii, we conducted a large-scale chemical genomics screen using our established inducible CRISPRi library in A. baumannii strain ATCC19606 (19606 throughout) [20]. This library consists of pooled CRISPRi strains with guides targeting 406 putatively essential genes derived from Tn-seq data in other strains [3,4], along with 1000 non-targeting control sgRNAs. For each targeted gene, we cloned four sgRNA spacers that exactly match the target sequence (perfect-match) and ten mismatch spacers with single-base variations from the target sequence that allow for titration of knockdown [22].
Discussion
Essential cellular processes are the current and likely future targets of mono- and poly-therapies against antibiotic resistant pathogens. Despite the biological and clinical importance of essential genes, few studies have comprehensively characterized their functions, especially in Gram-negative pathogens. This work advances our understanding of gene and antibiotic function in A. baumannii through large-scale, chemical-genomic profiling of an essential gene knockdown library. The breadth and diversity of our chemical perturbations provided functional insights that could not be obtained with a smaller scope.
Acknowledgments
We thank the UW-Madison Biotech Center for their assistance with sequencing. Thanks to Stephen Trent and Brent Simpson for their gift of the LOS- strain, and special thanks to Rachel Salemi and Alex Goetsch for their assistance and training with microscopy. We thank Warren Rose and Tim Bugni for helpful comments.
Citation: Tran JS, Ward RD, Iruegas-López R, Ebersberger I, Peters JM (2025) Chemical genomics informs antibiotic and essential gene function in Acinetobacter baumannii . PLoS Genet 21(3): e1011642. https://doi.org/10.1371/journal.pgen.1011642
Editor: Jan-Willem Veening,, University of Lausanne: Universite de Lausanne, SWITZERLAND
Received: December 31, 2024; Accepted: March 3, 2025; Published: March 28, 2025
Copyright: © 2025 Tran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets and computer code produced in this study are available in the following databases: • Raw sequencing data: NCBI BioProject (PRJNA1190454) • Scripts: Github (https://github.com/jasonpeterslab)
Funding: o This work (JMP lab) is supported by the National Institutes of Health (https://www.nih.gov/grants-funding) under award numbers K22AI137122 and R35GM150487. JST is supported by the Biotechnology Training Program (NIH 5T32GM135066; https://btp.wisc.edu/), SciMED Graduate Research Scholars (https://scimedgrs.wisc.edu/), and a National Science Foundation GRFP (https://www.nsfgrfp.org/). RDW is supported by the Predoctoral Training Program in Genetics (NIH 5T32GM007133) and SciMED Graduate Research Scholars. The work in IE lab was supported by a grant by the German Research Foundation (DFG; https://www.dfg.de/) in the scope of the Research Group FOR2251 “Adaptation and persistence of A. baumannii.” Grant ID EB-947 285-2/2 to IE. IE was further supported by the Research Funding Program Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE; https://wissenschaft.hessen.de/forschen/landesprogramm-loewe) of the State of Hessen, Research Center for Translational Biodiversity Genomics (TBG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JMP has filed for patents related to Mobile-CRISPRi technology and bacterial promoters.
