Identification of small regulatory RNAs involved in persister formation

Abstract

12Small regulatory RNA (srRNA) is widely distributed in three kingdoms of life and 13 fulfills functions in many aspects of cellular life, but their role in bacterial persistence 14 remains unknown. In this study, we comprehensively interrogated the expression 15 levels of the known srRNAs on three critical time points, stage 1 (S1) where no 16 persisters are formed, stage 2 (S2) where persisters are beginning to appear, and stage 17 3 (S3) where persister numbers increase significantly. Three upregulated srRNAs 18 (OmrB, an outer member associated srRNA; RdlB, a swarming motility and curli 19 expression regulator; McaS, a flagellar motility and biofilm formation regulator) 20 overlapping in S2/S1and S3/S1, together with the other four upregulated srRNAs 21 (MicF, a ribosome binding inhibitor; MicL, an outer membrane associated srRNA; 22 RybB, a cell envelope stress regulator; RydB, regulator of a global regulator RpoS) in 23 S2/S1 are of special interest. By constructing deletion mutants and overexpression 24 strains in uropathogenic E. coli strain UTI89, we tested their persister-formation 25 capabilities in log phase and stationary phase cultures exposed to antibiotics 26 (gentamicin, cefotaxime and levofloxacin) and stresses (heat, hyperosmosis, H2O2, 27 and acid). The results of the deletion mutant studies showed that all the seven 28 identified sRNAs have varying effects on persister formation with different antibiotics 29 or stresses. Moreover, we found all the deletion mutants of these srRNAs have 30 reduced biofilm formation. Additionally, except the McaS and the RydB 31 overexpression strains, all of the srRNAs overexpression strains demonstrated 32increased persister-formation in antibiotic and stress persister assays, confirming the 33 role of these srRNAs in persistence. Together, we identified seven srRNAs (OmrB, 34 RdlB, McaS, MicF, MicL, RybB, and RydB) that are involved in type II persister 35 formation for the first time. These findings provide convincing evidence for a new 36 level of rapid persistence regulation via srRNA and furnish novel therapeutic targets 37 for intervention. 38 biofilm 40 Introduction 41 Persisters have drawn wide attention since they can be identified in almost every 42 bacterial species, even in fungi and eukaryotic human cancer cells [1][2][3][4][5]. They refer to 43 a small subpopulation of dormant cells that can survive lethal antibiotics or stresses 44 and regain susceptibilities when regrowing in fresh medium [6][7][8]. Persisters account 45 for the recalcitrance of treatment of many persistent bacterial infections and can 46 facilitate the emergence of antibiotic resistant bacteria [7][8][9][10][11]. Consequently, persisters 47 pose great challenges for effective treatment of many bacterial infections 48 Persisters are divided into, type I and type II persisters [12]. Type I persisters stem 49 from the stationary phase, while type II persisters are induced by triggering 50 environmental signals as the cultures grow older from log phase to...