RESUMEN
There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity, yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realize this potential. Here, we have adapted our recently developed in vitro assay for Sec activityâbased on the split NanoLuc luciferaseâto work at scale and in living cells. A simple counterscreen allows compounds that specifically target Sec to be distinguished from those with other effects on cellular function. As proof of principle, we have applied this assay to a library of 5000 compounds and identified a handful of moderately effective in vivo inhibitors of Sec. Although these hits are unlikely to be potent enough to use as a basis for drug development, they demonstrate the efficacy of the screen. We therefore anticipate that the methods presented here will be scalable to larger compound libraries, in the ultimate quest for Sec inhibitors with clinically relevant properties.
Asunto(s)
Antibacterianos , Ensayos Analíticos de Alto Rendimiento , Antibacterianos/farmacología , Antibacterianos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Luminiscencia , Mediciones Luminiscentes/métodos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Canales de Translocación SEC/antagonistas & inhibidores , Canales de Translocación SEC/metabolismoRESUMEN
Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.