Identification of novel genes including rpmF and yjjQ critical for Type II persister formation in Escherichia coli

Abstract

7Persister cells, which are characterized by inactive metabolism and tolerance to antibiotics or 1 8 stresses, pose a significant challenge to the treatment of many persistent infections. Although 1 9 3 1(ydcX), which are two well-known persister genes while the remaining 11 novel genes (gnsA, 3 2 gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, yncN, rpmF, yohJ, rbsD) have not been reported 3 3 previously. Persister levels of 7 constructed knockout mutants (ΔgnsA,Δ ybfA, Δ yjjQ, Δ yhdU, 3 4 Δ csgD, Δ yohJ andΔ rpmF) and 10 overexpression strains (gnsA, gnsB, ybfA, yjjQ, ymdF, 3 5 yhdU, csgD, rpmF, yohJ, rbsD) in E. coli uropathogenic strain UTI89 were determined upon 3 6 treatment with different cidal antibiotics (ampicillin, levofloxacin and gentamicin). 7Additionally, ranking of these overlapping genes according to their impact on persister levels 3 8 2 were also performed. Two genes (rpmF encoding 50S ribosomal subunit protein L32, and 3 9 yjjQ encoding a putative LuxR-type transcription factor) showed the most obvious phenotype 4 0 on persister levels in both knockout and overexpression studies, which suggests they are 4 1 broad and key factors for persister formation. While previous studies cannot distinguish if a 4 2 given persister gene is involved in persister formation or persister survival, our findings 4 3 clearly identify novel persister forming genes and pathways involving a ribosome protein and 4 4 a LuxR type transcription factor during the bona fide persister formation process and may 4 5 7 1 involved in type II persister formation. Fifty-three and thirty-two genes were found to have 7 2 significantly different expression in S2 and S3 compared with S1, respectively. Specially, 13 7 3 genes overlap in both comparison groups. Apart from 4 genes (gnsB, ymdF, yncN, ybfA) with 7 4 uncharacteristic functions and 2 genes (ssrA, ydcX) with definite effect on persistence (Li et 7 5 al., 2013; Islam et al., 2015), the remaining 7 overlapping genes were mapped to ribosomal 7 6 protein (rpmF), regulator of phosphatidylethanolamine synthesis (gnsA), membrane protein 7 7(yohJ, yhdU), DNA-binding transcriptional activator (csgD), transcription factor (yjjQ) and 7 8 cytoplasmic sugar-binding protein (rbsD). We confirm two genes (rpmF and yjjQ) play 7 9 crucial roles in persister formation under multiple antibiotic treatment and stress conditions. 8 0 Together, our data provide a dynamic profile of genes involved in type II persister formation 8 1 3 which leads to novel insight on bona fide mechanisms of persister formation. 8 2 Methods 8 3 Bacteria and culture conditions 8 4 E. coli K12 strain W3110 from glycerol stocks at -80 °C were 1:1000 diluted in Luria-Bertani 8 5(LB) broth (10 g Bacto-tryptone, 5 g yeast extract, and 10 g NaCl/liter) without antibiotics 8 6 and incubated at 37 °C for 16 hours. For transcriptomics, the overnight culture was diluted 8 7 1:10 5 inoculated in 3.5 liters, 2 liters and 1 liter fresh LB medium for each time point (3 hrs, 4 8 8 hrs and 5 hrs) to satisfy the quantity requirement fo...

Proteomic Analyses of Morphological Variants ofBorrelia burgdorferiShed New Light on Persistence Mechanisms: Implications for Pathogenesis, Diagnosis and Treatment

“…Meanwhile, we also found while some ribosomal proteins were downregulated, some others were up-regulated in both stationary phase forms (Figure 2, Table 1). In a recent study, rpmF encoding a ribosomal protein was also identified as a persister gene in E. coli [47], which supports the possibility that certain ribosomal proteins may play a role in persister formation or survival in Borrelia SP and MC forms. In future studies we will confirm whether these proteins or genes are involved in persistence to antibiotics and multiple stresses.…”

Prediction of Streptococcus uberis clinical mastitis treatment success in dairy herds by means of mass spectrometry and machine-learning

“…RpmC was previously shown to bind puromycin, an aminonucleoside antibiotic, in both E. coli and Bacillus stearothermophilus 33 . Also, rpmF was previously shown to be involved in membrane lipid synthesis 34 and persister cell formation which may help bacteria to survive antibiotic treatment 35 . RpmF depleted E. coli mutants were shown to increase susceptibility against ampicillin, gentamicin, levofloxacin, metronidazole, rifampicin and sulfamethoxazole in different studies 35 – 37 .…”

“…Also, rpmF was previously shown to be involved in membrane lipid synthesis 34 and persister cell formation which may help bacteria to survive antibiotic treatment 35 . RpmF depleted E. coli mutants were shown to increase susceptibility against ampicillin, gentamicin, levofloxacin, metronidazole, rifampicin and sulfamethoxazole in different studies 35 – 37 . RpsU was shown to be related to motility and biofilm formation in Bacillus subtilis 38 , stress resistance in Listeria monocytogenes 39 , metronidazole resistance in Helicobacter pylori 40 , and daptomycin and vancomycin susceptibility in Staphylococcus aureus 41 , 42 .…”

Transcriptomic determinants of the response of ST-111 Pseudomonas aeruginosa AG1 to ciprofloxacin identified by a top-down systems biology approach

“…The rpmF gene encodes for the 50S ribosomal subunit protein L32, which is responsible for protein synthesis and membrane lipid synthesis 101 . It is also involved in multidrug tolerance by modulating biofilm formation and persister cell induction 102 .…”