We developed a new approach to facilitate identification of natural products with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. We have now developed a complimentary approach that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. Ths approach, which we call rapid inhibition profiling (R.I.P.), entails the rapid, inducible depletionof a target protein, followed by cytological profiling. Our preliminary data demonstrate that depletion of a drug target by R.I.P. produces cytological effects identical to those produced by the corresponding drug. Furthermore, depletion of essential proteins that are not targeted by current antibacterial drugs produces novel cytological profiles that can be subsequently used to identify molecules that inhibit these new targets. We here propose to more fully develop the R.I.P. technology by employing it on a genome wide basis in E. coli and B. subtilis. This will create a comprehensive reference set of profiles associated with the inhibition of essential cellular pathways that are not the targets of current antibacterial drugs. The genome-wide R.I.P. analysis will also provide insight into the function of conserved genes and our preliminary data suggests that it will provide insight into proteins that coordinate two or more biosynthetic pathways, providing interesting starting points for future basic research. We will then use this more complete reference data set with BCP to screen a unique and diverse collection of natural product extracts to identify those that inhibit these new drug targets and kill multidrug resistant bacteria. Together with our collaborators at Fundaci?n Medina, we will then purify and characterize our highest priority lead molecules (those that kill multidrug resistant bacteria) wit a goal of advancing them into toxicity trials.

Public Health Relevance

We have developed an approach for rapidly identifying natural products with antibacterial activities in crude natural product extracts and for rapidly discriminating between different mechanisms of action, detecting detect nuisance compounds and multiple activities in partially purified or crude natural product extracts. We have now developed a complimentary approach (R.I.P.) that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. We here propose to fully develop the R.I.P. technology and use it screen for antibacterial molecules and identify those that inhibit new drug targets, allowing the more rapid discovery of antibiotics to combat drug resistant bacterial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI113295-01
Application #
8767927
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Xu, Zuoyu
Project Start
2014-05-12
Project End
2019-04-30
Budget Start
2014-05-12
Budget End
2015-04-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Kumaraswamy, Monika; Do, Carter; Sakoulas, George et al. (2018) Listeria monocytogenes endocarditis: case report, review of the literature, and laboratory evaluation of potential novel antibiotic synergies. Int J Antimicrob Agents 51:468-478
Peters, Christine E; Lamsa, Anne; Liu, Roland B et al. (2018) Rapid Inhibition Profiling Identifies a Keystone Target in the Nucleotide Biosynthesis Pathway. ACS Chem Biol :
Lopez-Garrido, Javier; Ojkic, Nikola; Khanna, Kanika et al. (2018) Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology. Cell 172:758-770.e14
Hall Snyder, Ashley D; Werth, Brian J; Nonejuie, Poochit et al. (2016) Fosfomycin Enhances the Activity of Daptomycin against Vancomycin-Resistant Enterococci in an In Vitro Pharmacokinetic-Pharmacodynamic Model. Antimicrob Agents Chemother 60:5716-23
Lamsa, Anne; Lopez-Garrido, Javier; Quach, Diana et al. (2016) Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials. ACS Chem Biol 11:2222-31
Nonejuie, Poochit; Trial, Rachelle M; Newton, Gerald L et al. (2016) Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis. J Antibiot (Tokyo) 69:353-61
Quach, D T; Sakoulas, G; Nizet, V et al. (2016) Bacterial Cytological Profiling (BCP) as a Rapid and Accurate Antimicrobial Susceptibility Testing Method for Staphylococcus aureus. EBioMedicine 4:95-103
Ojkic, Nikola; López-Garrido, Javier; Pogliano, Kit et al. (2016) Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation. Elife 5:
Kumaraswamy, Monika; Lin, Leo; Olson, Joshua et al. (2016) Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia. J Antimicrob Chemother 71:1264-9
Hindler, Janet A; Wong-Beringer, Annie; Charlton, Carmen L et al. (2015) In vitro activity of daptomycin in combination with ?-lactams, gentamicin, rifampin, and tigecycline against daptomycin-nonsusceptible enterococci. Antimicrob Agents Chemother 59:4279-88

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