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Discovery and characterization of the antimetabolite action of thioacetamide-linked 1,2,3-triazoles as disruptors of cysteine biosynthesis in Gram-negative bacteria

ABSTRACT: Increasing rates of drug-resistant Gram-negative (GN) infections combined with a lack of new GN-effective antibiotic classes is driving the need for the discovery of new agents. As bacterial metabolism represents an underutilized mechanism targeted in current antimicrobial therapies, we sought to identify novel antimetabolites and explore the specific impacts of these inhibitors on key pathways in bacterial metabolism. This study describes the successful application of this approach to discover N-(phenyl) thioacetamide-linked 1,2,3-triazoles (TAT) that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis revealed a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-L-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics.

ORGANISM(S): Escherichia coli

PROVIDER: GSE142164 | GEO | 2020/01/01

REPOSITORIES: GEO

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Project description:This study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress. Conclusions Overall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival. two connected loops, both containing samples derived on a single day (sample 1-6, sample 7-13)

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Discovery and characterization of the antimetabolite action of thioacetamide-linked 1,2,3-triazoles as disruptors of cysteine biosynthesis in Gram-negative bacteria

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