Integrated genomics and chemical biology herald an era of sophisticated antibacterial discovery, from defining essential genes to target elucidation

Abstract

The golden age of antibiotic discovery in the 1940s-1960s saw the development and deployment of many different classes of antibiotics, revolutionizing the field of medicine. Since that time, our ability to discover antibiotics of novel structural classes or mechanisms has not kept pace with the ever-growing threat of antibiotic resistance. Recently, advances at the intersection of genomics and chemical biology have enabled efforts to better define the vulnerabilities of essential gene targets, to develop sophisticated whole-cell chemical screening methods that reveal target biology early, and to elucidate small molecule targets and modes of action more effectively. These new technologies have the potential to expand the chemical diversity of antibiotic candidates, as well as the breadth of targets. We illustrate how the latest tools of genomics and chemical biology are being integrated to better understand pathogen vulnerabilities and antibiotic mechanisms in order to inform a new era of antibiotic discovery.

Keywords: antibiotic discovery; gene essentiality; high-throughput screen; mode of action; target.

Figure 1:. Methods for gene essentiality determination.

(A) Transposon strategy: In Transposon Site Hybridization (TraSH) and transposon insertion sequencing (TnSeq) assays, transposon mutant libraries are subjected to growth selection in certain in vitro or in vivo conditions. Genomic DNA is extracted from the surviving population and undergoes library construction, which typically comprises of genome fragmentation or digestion, followed by adaptor ligation such that productive PCR of the transposon junctions with transposon/adaptor primer pairs only occurs in DNA fragments containing a transposon insertion. TraSH then involves in vitro transcription and microarray hybridization of amplified products while TnSeq utilizes next generation sequencing to enumerate amplicons. (B) CRISPRi strategy: In CRISPRi assays, mutant libraries are first engineered to express dCas9 and a comprehensive panel of sequence-specific small-guide RNAs (sgRNAs), and then subjected to growth selection. Plasmids are then recovered from surviving mutants, the sgRNAs are PCR amplified, and amplicons are enumerated by next generation sequencing. In all methods, essential genes are defined as those in which a disruption in expression, either from a transposon or from dCas9, leads to lack of viability and thus drop out in signal output, whether it is fluorescence on a microarray or sequencing reads.

Figure 2.. Merging whole cell screening with pathway or target-based discovery.

(A) Cell-based, pathway-specific screen and counter screening approaches chemically screen a wildtype (WT) strain of interest for hits that inhibit bacterial growth. Hits identified in the primary screen are then counter screened against a strain deficient in a pathway of interest (e.g., WTA biosynthesis, the Rod system, or LPS biogenesis). Hits that permit growth following counter screening are followed up as pathway specific inhibitors. (B) In target-based, whole cell screening, a hypomorphic strain where an essential gene target is depleted is constructed and both the hypomorph and WT strains are chemically screened for hits that preferentially inhibit the growth of the hypomorph over the WT strain. Identified hits are candidates as gene or pathway specific inhibitors. (C) PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets (PROSPECT) expands on the target-based, whole cell screening depicted in (B) with the screening of many hypomorphs in multiplex. A pool of engineered hypomorphs each carrying a unique genetic barcode is subjected to chemical screening and the census of each strain in the pool following exposure to small molecule treatment is measured by isolating DNA from all bacteria in each well, amplifying the bar code of each strain and counting the representation within the pool using NGS. Hypomorphs that are underrepresented in each pool are more sensitive to small molecule treatment relative to other members of the pool, suggesting that the hit may be targeting either the corresponding depleted gene product or an interacting pathway.

Figure 3:. Target identification and/or MOA prediction methods for a novel inhibitor.

(ABX = novel inhibitor; X = cognate target) (A) Genetic methods include isolation and whole genome sequencing (WGS) of resistant clones, activity profiles against gene dosage libraries with over- or under-expression of specific genes of interest, and transcriptomics-based generation of gene expression signature profiles with a panel of antibiotics, which includes antibiotics with known targets for reference-based analysis and novel small molecules, to aid MOA prediction. (B) Proteomic methods include affinity-based pull down of a target by tagged, immobilized small molecule followed by quantitative mass spectrometry and thermal proteome profiling of stabilization of a putative target across a temperature gradient by the small molecule detected by quantitative mass spectrometry. (C) Metabolomics-based detection of substrate accumulation and product depletion after treatment can point to the putative target or target pathway. D) Confocal microscopy-based analysis of morphology points to the broader, macromolecular process impacted by the small molecule and is also used for a profiling strategy for MOA prediction.