EvaluatingaroAgene essentiality and EPSP synthase vulnerability inMycobacterium smegmatisunder different nutritional conditions

AbstractThe epidemiological importance of bacteria from the genusMycobacteriumis indisputable and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important metabolites in bacteria, represents a target for inhibitors ofMycobacterium tuberculosisgrowth. ThearoA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene knockout, gene knockdown and kinetic assays to evaluatearoAgene essentiality and the vulnerability of its protein product, EPSPS synthase fromMycobacterium smegmatis(MsEPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality ofMsEPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions ofaroAgene, together with kinetic assays using WT and mutant recombinant proteins, we show thataroAgene essentiality depends onMsEPSPS activity. To evaluateMsEPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces foraroAgene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating thatMsEPSPS vulnerability depends on the environment conditions.ImportanceWe evaluated both gene essentiality and target vulnerability of the enzyme that catalyzes the sixth step of the shikimate pathway, thearoA-encoded 5-enolpyruvylshikimate-3-phosphate synthase fromMycobacterium smegmatis(MsEPSPS). Combining gene knockout experiments and kinetic assays, we established a causal link betweenaroAgene essentiality and the biological function of EPSPS protein, which we advocate is an indispensable step for target validation. Moreover, we characterizedMsEPSPS vulnerability under different nutritional conditions and found it is a vulnerable target only whenM. smegmatisis grown under poor nutritional conditions without supplementation with aromatic amino acids. Based on our findings, we suggest that gene essentiality information should be obtained from gene knockout experiments and not knockdown approaches, as even low levels of a protein after gene silencing can lead to a different growth phenotype when compared to that under its complete absence, as was the case witharoAandMsEPSPS in our study.