EvaluatingaroAgene essentiality and EPSP synthase vulnerability inMycobacterium smegmatisunder different nutritional conditions

Abstract

26The epidemiological importance of bacteria from the genus Mycobacterium is 27 indisputable and the necessity to find new molecules that can inhibit their growth 28 is urgent. The shikimate pathway, required for the synthesis of important 29 metabolites in bacteria, represents a target for inhibitors of Mycobacterium 30 tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate 31 synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In 32 this study, we combined gene knockout, gene knockdown and kinetic assays to 33 evaluate aroA gene essentiality and the vulnerability of its protein product, 34 EPSPS synthase from Mycobacterium smegmatis (MsEPSPS), under different 35 nutritional conditions. We demonstrate by an allelic exchange-based gene 36 knockout approach the essentiality of MsEPSPS under rich and poor nutritional 37 conditions. By performing gene complementation experiments with wild-type 38 (WT) and point mutant versions of aroA gene, together with kinetic assays using 39 WT and mutant recombinant proteins, we show that aroA gene essentiality 40 depends on MsEPSPS activity. To evaluate MsEPSPS vulnerability, we 41 performed gene knockdown experiments using the Clustered Regularly 42 Interspaced Short Palindromic Repeats interference (CRISPRi) system. The 43 experiments were performed in both rich and defined (poor) media, using three 44 different repression forces for aroA gene. We only observed growth impairment 45 when bacteria were grown in defined medium without supplementation of 46 aromatic amino acids, thereby indicating that MsEPSPS vulnerability depends on 47 catalyzes the sixth step of the shikimate pathway, the aroA-encoded 5-51 enolpyruvylshikimate-3-phosphate synthase from Mycobacterium smegmatis 52 (MsEPSPS). Combining gene knockout experiments and kinetic assays, we 53 established a causal link between aroA gene essentiality and the biological 54 function of EPSPS protein, which we advocate is an indispensable step for target 55 validation. Moreover, we characterized MsEPSPS vulnerability under different 56 nutritional conditions and found it is a vulnerable target only when M. smegmatis 57 is grown under poor nutritional conditions without supplementation with aromatic 58 amino acids. Based on our findings, we suggest that gene essentiality information 59 should be obtained from gene knockout experiments and not knockdown 60 approaches, as even low levels of a protein after gene silencing can lead to a 61 different growth phenotype when compared to that under its complete absence, 62 as was the case with aroA and MsEPSPS in our study. 63 64 Keywords 65 Gene silencing, Chorismate, Essentiality, Vulnerability, CRISPRi, Molecular 66 genetics. 67 68 69Human tuberculosis (TB) is an important infectious disease that continues 71 to be a public health threat worldwide. Despite the joint global efforts to lower TB 72 burden, which resulted in a 6.3% and 11% cumulative decline in incidence and 73 mortality, respectively, between 2015 and 2018, the End TB...