AAC Accepts, published online ahead of print on 20 October 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.03979-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay

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El-Mowafi, S. A., Sineva, E., Alumasa, J. N., Nicoloff, H., Tomsho, J. W., Ades, S. E. and Keiler, K. C.* Department of Biochemistry & Molecular Biology The Pennsylvania State University

*correspondence: 401 Althouse Lab University Park, PA 16803 [email protected] (814)863-0787

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Abstract

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Gram-negative bacteria are formidable pathogens because their cell envelope

32

presents an adaptable barrier to environmental and host-mediated challenges. The stress

33

response pathway controlled by the alternative sigma factor σE is critical for maintenance

34

of the cell envelope. Because σE is required for virulence or viability in several Gram-

35

negative pathogens, it might be a useful target for antibiotic development. To determine

36

if small molecules can inhibit the σE pathway, and to permit high-throughput screening

37

for antibiotic lead compounds, a σE activity assay that is compatible with high-throughput

38

screening was developed and validated. The screen employs a biological assay with

39

positive readout. An E. coli strain was engineered to express yellow fluorescent protein

40

(YFP) under negative regulation by the σE pathway, such that inhibitors of the pathway

41

increase production of YFP. To validate the screen, the reporter strain was used to

42

identify σE pathway inhibitors from a library of cyclic peptides. Biochemical

43

characterization of one of the inhibitory cyclic peptides showed that it binds σE, inhibits

44

RNA polymerase holoenzyme formation, and inhibits σE-dependent transcription in vitro.

45

These results demonstrate that alternative sigma factors can be inhibited by small

46

molecules, and enable high-throughput screening for inhibitors of the σE pathway.

47 48

Introduction

49

Gram-negative bacteria are remarkably successful pathogens, and the increasing

50

prevalence of antibiotic resistance in these species presents a significant threat to human

51

health (1, 2). A major factor contributing to the success of these bacteria in virulence and

52

in evading antibiotic action is their ability to maintain the integrity of the outer

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53

compartment of the cell, the cell envelope, which consists of the outer membrane,

54

periplasmic space, peptidoglycan layer, and cytoplasmic membrane. The σE pathway is

55

one of the key regulatory systems used by Gram-negative bacteria to adapt to challenges

56

to the cell envelope encountered in the environment, including those presented by the

57

host during infection (3, 4).

58

Sigma factors, such as σE, are the subunits of RNA polymerase (RNAP)

59

responsible for promoter recognition and transcription initiation (5, 6). The majority of

60

transcription in the cell is directed by the housekeeping sigma factor, σ70. However, most

61

bacteria also possess a series of alternative sigma factors that are activated by particular

62

stresses and redirect RNAP to promoters for genes required to respond to the stress in

63

question (6, 7). σE is a member of the largest and most widespread group of alternative

64

sigma factors referred to as the group 4 or ECF (extracytoplasmic function) sigma factors

65

(8, 9). ECF sigma factors have been implicated in stress survival, virulence and antibiotic

66

resistance in many pathogens (3, 4, 10, 11).

67

rpoE, the gene encoding σE, is essential for viability of E. coli K12 and Yersinia

68

enterocolitica (12-16). rpoE is also likely required for viability in E. coli AIEC LF82

69

(associated with Crohn’s disease), Haemophilus ducreyi, and Bordetella pertussis,

70

because deletion mutants could not be obtained (17-19). Likewise, deletion mutants could

71

only be obtained under certain conditions and suppressors arose frequently in Vibrio

72

cholerae (20). In bacterial pathogens that do not require σE for viability, mutants lacking

73

σE are often attenuated for virulence. These bacteria include Salmonella enterica serovar

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Thyphimurium, E. coli UTI89, Pseudomonas aeroguinosa and Klebsiella pneumoniae

75

(21-24). In addition, the V. cholerae ∆rpoE strains were still highly attenuated for

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76

virulence despite the appearance of suppressor mutations that allowed growth in culture.

77

Given these phenotypes, the σE pathway presents a potential target for new antibacterials.

78

In Escherichia coli and related bacteria, the major role of the σE pathway in cell

79

envelope homeostasis is to control the integrity and composition of the outer membrane

80

by two major mechanisms. First, σE transcribes several sRNAs that act in conjunction

81

with the Hfq protein to silence gene expression of outer membrane porins and a major

82

cellular lipoprotein (25, 26). Second, σE transcribes genes encoding proteins required for

83

the folding and delivery of porins to the outer membrane, as well as genes required for

84

the export of lipopolysaccharide to the outer membrane (27). In this manner, σE ensures

85

proper porin production, controls the amount and identity of the porins produced, and

86

ensures proper LPS export to the outer membrane (27, 28).

87

The regulatory pathway controlling σE has been studied extensively in E. coli, and

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genes encoding the major players in the pathway are found in the genomes of other

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bacteria that have homologues of σE (8). σE activity is strongly inhibited by the anti-sigma

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factor RseA, an inner membrane protein (29, 30). RseA binds σE with high affinity and

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prevents σE from binding core RNAP (31). Stresses that disrupt the proper delivery of

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LPS and outer membrane porins to the outer membrane trigger proteolysis of RseA,

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freeing σE to interact with RNA polymerase and initiate transcription of genes required to

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combat the stress (32-34). A low basal level of degradation of RseA provides sufficient

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free σE to maintain viability of strains of E. coli that require σE activity (32, 35, 36).

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The bacterial cell envelope is a proven target for antibiotic action. Targeting the

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σE pathway presents a new approach to simultaneously disrupt several components of this

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compartment. Drugs that block the σE pathway would prevent the ability of the bacterium

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99

to ensure envelope integrity and to modulate the cell envelope during infection, resulting

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in cell death for pathogens in which σE is essential for viability, or reducing virulence of

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pathogens in which σE is important for causing disease. Currently, no inhibitors are

102

available that target any step in the σE pathway.

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To determine if the σE pathway can be inhibited by small molecules, an assay

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compatible with high-throughput screening was developed. The assays was used to

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identify inhibitors from libraries of cyclic peptides generated in E. coli using SICLOPPS

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(Split Intein Circular Ligation Of Proteins and PeptideS), a genetic system based on

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spontaneous protein splicing by inteins. SICLOPPS has been used to isolate inhibitors of

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several bacterial proteins including the ClpXP protease, Hfq, and Dam methyltransferase

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(37-39). One of the inhibitory cyclic peptides inhibited σE-dependent transcription by

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decreasing the affinity of σE and core RNAP, demonstrating that the assay is effective

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and that inhibitors of σE can be obtained.

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Materials and Methods

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Bacterial strains and growth conditions

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Bacterial strains used in this study are described in Table 1. Mutant alleles ydcQ,

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lacIq, and rseA were mobilized into the appropriate strains using P1 phage transduction,

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and the antibiotic resistance markers were removed using FLP recombinase (40, 41). E.

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coli strains were grown in LB at 30 °C with aeration unless otherwise noted. 100 μg/ml

119

ampicillin, 30 μg/ml kanamycin, 30 μg/ml chloramphenicol and 0.0002% arabinose were

120

added where appropriate.

121

Plasmid constructions

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122

Plasmids used in this study are described in Table 1 and oligonucleotide

123

sequences are listed in Table 2. To make prpoErybB, the rrnBT1T2 transcription

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terminator was amplified from pTrc99a using primers rrnbT1Ba and rrnBT2X. The rybB

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gene and its promoter were amplified from E. coli genomic DNA using primers rybBX

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and rybBSI. Both PCR products were digested with XbaI, ligated using T4 DNA ligase,

127

and the resulting rrnBT1T2-rybB product was amplified by PCR using primers rrnbT1Ba

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and rybBSI. The amplified DNA was digested with BamHI and SalI, and ligated into

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pLC245 cut with the same enzymes.

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For construction of pSRE, the rybB promoter was amplified from E. coli genomic

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DNA using primers rybBE and rybBB. The egfp gene was amplified from pEGFP-N2

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using primers egfpB and egfpP. Both PCR products were digested with BspHI, ligated,

133

and the resulting rybB-egfp product was amplified by PCR using primers rybBE and

134

egfpP, digested with EcoRI and PstI, and ligated into pSB4K5 cut with the same enzymes.

135

Site directed mutagenesis to generate the σE N80C/C165A variant was performed

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using the QuikChange mutagenesis kit (Stratagene) with primers rpoE_N80C_for and

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rpoE_N80C_rev, and primers rpoE_C165A_for and rpoE_C165A_rev according to the

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manufacturers instructions.

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Chemical synthesis of SI24

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The linear peptide was purchased from the Huck Institute of the Life Sciences

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Macromolecular Core Facility (PSU Hershey, PA). Head-to-tail chemical cyclization was

142

initiated

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dimethylaminopropyl)carbodiimide, and 66 mg 1-hydroxy-7-azabenzotriazole in 75 ml

144

dimethylformamide under argon. The reaction was incubated at room temperature for 12

by

dissolving

100

mg

linear

peptide,

70

mg

1-ethyl-3-(3’-

6

145

h and evaporated under reduced pressure to near dryness. The resulting residue was

146

triturated in diethyl ether to obtain a crude cyclized product. Purification was performed

147

on a preparative High Performance Liquid Chromatography system (Waters Corporation)

148

using a C18 reverse phase column (Varian Dynamax) with a combination of acetonitrile

149

and water, both containing 0.1% trifluoroacetic acid as the mobile phase. Excess solvent

150

was removed by lyophilization and the resulting solid was analyzed by electrospray-

151

ionization mass spectrometry (positive mode) to confirm its mass.

152

Screen for inhibitors of the σE pathway

153

The SGWX5 SICLOPPS library was constructed as previously described (42). E.

154

coli strain SEA6805 was transformed with the SICLOPPS plasmid library, and the

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resulting colonies were scraped, grown overnight, diluted 100-fold, and grown to OD600 =

156

0.2. Expression of rpoE and rybB was induced by addition of IPTG to 1 mM for 1 h,

157

ompC’-yfp expression was induced by addition of anhydrous tetracycline (AHT) to 100

158

ng/ml for 3 h, and cells were sorted by fluorescence activated cell sorting (FACS) using a

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Beckman Coulter Elite cell sorter equipped with Autoclone for detection of YFP

160

fluorescence intensity. Cells were selected based on YFP fluorescence intensity, and the

161

brightest 0.01% were selected and deposited on agar plates for clonal growth. Cells from

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each colony were grown as described above, imaged by epifluorescence microscopy, and

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the fluorescence intensity was measured using Simple PCI software (Compix, Inc.).

164

Plasmid DNA was prepared from selected clones and sequenced. Peptide sequences were

165

determined by conceptual translation of the DNA sequences.

166

Protein sequence analysis

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167

E. coli strain SAE020 was grown to OD600 = 0.6, and expression of SI24 was

168

induced by addition of arabinose to 2% for 5 h. Cells were harvested by centrifugation

169

and the cell pellet was resuspended in 50 ml chitin buffer (20 mM Tris-HCl (pH 7), 500

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mM NaCl, 1mM EDTA, 0.1% Tween 20, 1 mM PMSF). Cells were lysed by sonication

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and debris was removed by centrifugation. The clarified lysate was passed over a column

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equilibrated in chitin buffer and loaded with 1 ml chitin resin (New England BioLabs).

173

The column was washed with twenty volumes chitin buffer, 200 μl 3X SDS-PAGE buffer

174

(188 mM Tris-HCl (pH 6.8), 3% SDS, 30% glycerol, 0.01% bromophenol blue, 15% β-

175

mercaptoehanol) was added to the resin. The mixture was boiled, the protein was

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resolved on a 12% SDS polyacrylamide gel, and bands corresponding to SI24 were

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excised. The sequence was determined at the Proteomics and Mass Spectrometry Core

178

Facility at Pennsylvania State University, University Park. A Thermo LTQ Orbitrap

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Velos mass spectrometer with a Dionex Ultimate 3000 nano-LC system was used to

180

analyze chymotryptic peptides. The data was processed using Proteome Discoverer 1.3

181

(Thermo Scientific).

182

Fluorescent reporter and cell staining assays

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E. coli strains were grown with or without addition of arabinose and examined

184

under inducing and non-inducing conditions. For induced cultures, IPTG was added at

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OD600 = 0.2 to a final concentration of 1 mM, the cultures were grown for 1 h, AHT was

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added to 100 ng/ml, and cultures were grown for an additional 3 h. Cells were examined

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using epifluorescence microscopy. For propidium iodide staining, cultures were grown

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and induced as described above, and propidium iodide was added to 3 μg/ml. Cells were

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incubated with shaking for 30 min, harvested by centrifugation, resuspended in 50 μl LB,

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and examined using epifluorescence microscopy. All experiments were done in triplicate.

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For assays in microtiter plates, cultures of E. coli prpoErybB pompC’-yfp and E.

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coli pTrc99a pompC’-yfp strain were grown in flasks and IPTG was added at OD600 = 0.2

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to a final concentration of 1 mM. Following the addition of IPTG, 50 µl aliquots were

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transferred to wells in 384-well trays. Trays were incubated at 37 °C for 45 min shaking

195

at 300 rpm, AHT was added, and trays were incubated at 37 °C for an additional 3 h.

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Fluorescence was measured with excitation at 500 nm and emission at 540 nm, and the

197

fluorescence intensity was normalized to the OD600 of the well. Z’ values were calculated

198

as described for each day (43), and Z’ values for 3 different days were averaged. The E.

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coli pTrc99a pompC’-yfp strain was used as the positive control.

200

Western blotting

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E. coli strains were grown as described for protein sequence analysis, and

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equivalent OD600 units were harvested. Cells were lysed in SDS-PAGE buffer, separated

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on 15% SDS-polyacrylamide gels, and transferred to Hybond-P PVDF membrane (GE

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Healthcare). SICLOPPS proteins were detected using rabbit polyclonal antibodies raised

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against chitin binding domain (S6654S, New England Biolabs).

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Protein Purification and dye labeling

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N-terminally His-tagged wild-type σE and σEN80C/C165A proteins were purified as

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previously described and stored in storage buffer (20 mM Tris-HCl (pH 8.0), 150 mM

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NaCl, 1mM DTT, 15% glycerol) (44). Proteins were quantified by absorbance at 280 nm

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(ε= 14,770 M-1 cm-1). DTT levels were maintained at 1 mM throughout storage. Core

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RNAP and σ 70 were gifts from Katsuhiko Murakami (Penn State University) and purified

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as described (45).

213

Immediately before labeling N-terminally His-tagged wild-type σE and

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σEN80C/C165A, the storage buffer was removed by chromatography over a Bio-Gel P4

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desalting column (Bio-Rad) equilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 0.5

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mM EDTA and 10% glycerol. Proteins were then incubated on ice with 100 µM

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BODIPY-FL maleimide (Invitrogen/Molecular Probes) diluted from a 2.5 mM BODIPY-

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FL stock in acetone. Unreacted dye was removed using a Bio-Gel P4 desalting column

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equilibrated in 50 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM EDTA and 10% glycerol.

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The degree of dye labeling was determined by measuring the absorbance of the labeled

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protein at 505 nm (ε 505 = 79,000 M-1cm-1,ε 280 = 1,300 M-1cm-1) and was typically higher

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than 50%.

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Fluorescence anisotropy

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Anisotropy assays were performed in Greiner black 384-well microplates using a

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microplate reader (Infinite M1000, Tecan) at 30 °C, with excitation of 470 nm and

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emission of 514 nm. The G-factor was determined to be 1.13 using 1 nM fluorescein in

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0.01 M NaOH. Binding reactions (20 µl) containing 100 nM BODIPY-σEN80C/C165A and 0

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– 1 mM SI24 or 5 nM BODIPY-σEN80C/C165A and 0 – 1 µM RNAP in transcription buffer

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(50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 50 mM NaCl, 5% glycerol, 10 mM 2-

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mercaptoethanol, 0.01% NP40) were incubated at 30 °C for 30 min, and anisotropy

231

measurements were performed. The fractional occupancies were calculated as

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θ = (P - P0)/(Pmax – P0)

Eq. 1

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where P0 and P are polarization values before and after addition of ligands and Pmax is the

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polarization value at saturation. Binding constants were obtained by fitting of results to

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the equilibrium binding equation for a bimolecular association where E, S, and ES are the

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concentrations of the BODIPY-σEN80C/C165A, ligand (RNAP or SI24) and the bound

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complex, respectively, and KD is the equilibrium dissociation constant.

238 239

[E] + [S]0 + KD − {([E]0 + [S]0 + KD )2 − 4 ⋅ [E]0 ⋅ [S]0} 2 [ES] = 0

240

Competition assays were used to determine the affinity of unlabeled σE and σ70

1

2

Eq. 2 (46)

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for core RNAP and to measure the KI for inhibition of holoenzyme formation by SI24. In

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these assays, reaction mixtures (30 µl) contained 5 nM BODIPY-σEN80C/C165A, 75 nM core

243

RNAP and either 0 – 4 mM SI24 or 0 – 1 µM unlabeled sigma factor. BODIPY-

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σEN80C/C165A was incubated with competitors for 20 min at 30 °C, core RNAP was added

245

and the mixture incubated for 30 min, then fluorescence anisotropy was measured. IC50

246

values were calculated using the Hill equation (47), and equilibrium dissociation

247

constants (KI) were calculated from the IC50 as follows. KD refers to the affinity of

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BODIPY-σEN80C/C165A and RNAP.

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KI= IC50/(1+ [RNAP]/KD)

250 251

Eq. 3 (48)

In vitro transcription For multiple-round transcription reactions, σE holoenzyme was formed by

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incubating 0.25 µM core RNAP with 0.25 µM σE for 30 min at 30 °C in transcription

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buffer. Transcription reactions were initiated by adding the σE holoenzyme to the

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transcription template along with nucleotides and [α-32P]UTP. The transcription reaction

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mixtures (10 μl final volume) contained 25 nM E. coli RNAP core enzyme, 25 nM σE, 10 11

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nM transcription template containing positions –70 to +100 of the rybB gene, 200 μM

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ATP, 200 μM CTP, 200 μM GTP, and 20 μM [α-32P]UTP in transcription buffer.

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Transcription was allowed to proceed for 10 min at 30 °C and terminated by the addition

259

of TBE buffer with 8 M urea, 10 mM EDTA, 0.04% bromophenol blue, and 0.04%

260

xylene cyanol. Samples were heated 2 min at 95 °C, cooled 5 min on ice, and applied to

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6% polyacrylamide gel (19:1 acrylamide:bisacrylamide) with 7 M urea. Bands were

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visualized on a Typhoon 9410 (GE Healthcare) and quantified using ImageQuant

263

(Molecular Dynamics). To assess the effects of SI24, the cyclic peptide was incubated

264

with σE for 30 min at 30 °C before the addition of core RNAP, or was added after core

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RNAP. The 215 bp transcription template DNA fragment was generated by PCR of the

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rybB promoter from genomic DNA using primers RybB_EcoRI_for and RybB_XhoI_rev.

267

For single round transcription experiments, 0.25 µM σE was pre-incubated with 0.25

268

µM core RNAP 30 min at 30 °C to form the holoenzyme. Template DNA containing the

269

rybB promoter was added to σE holoenzyme and reaction mixes were incubated for 30

270

min at 30 °C to allow for open complex formation. A single round of transcription was

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initiated by the addition of NTPs and heparin. The transcription reactions (20 µl final

272

volume) contained 25 nM core RNAP, 25 nM σE, 10 nM transcription template, 200 μM

273

ATP, 200 μM CTP, 200 μM GTP, 20 μM [α-32P]UTP, and 25 μg/ml heparin in

274

transcription buffer. Following a 10 min incubation at 30 °C, reactions were terminated,

275

transcripts separated by polyacrylamide gel electrophoresis, and visualized as described

276

for the multiple-round transcription assays. The effects of the SI24 were measured by

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incubating the cyclic peptide with σE for 30 min at 30 °C before holoenzyme formation

278

or after holoenzyme formation, but before the addition of the DNA template to form open

12

279

complexes. In all cases, IC50 values were calculated using Hill equation (47), and KI

280

values were determined using eq. 3 (48). The effect of SI24 on transcription by σ70-

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RNAP was measured using the T7A1 promoter as the transcription template (49) (gift

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from Katsuhiko Murakami) in single round assays as described above for σE.

283 284

Results

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Screen for inhibitors of the σE pathway

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To identify inhibitors of the σE pathway, a cell-based assay with positive readout

287

for inhibition was developed. In this assay, σE transcribes the RybB sRNA, which is a

288

member of its regulon. A gene fusion between the region of ompC that is targeted by

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RybB and yfp allows σE pathway activity to be monitored using YFP fluorescence (Fig.

290

1A). In the absence of an inhibitor, σE transcribes rybB, and RybB-Hfq represses

291

production of OmpC’-YFP, resulting in dark cells. Conversely, if an inhibitor of the σE

292

pathway is present, OmpC’-YFP is produced and the cells are fluorescent. To ensure that

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sufficient quantities of each component are present to give an accurate readout of

294

pathway activity, the rpoE and rybB genes are expressed from a plasmid (prpoErypB).

295

On this plasmid, rpoE expression is controlled by an IPTG-inducible promoter and rybB

296

expression is controlled by its native σE-dependent promoter. ompC’-yfp is expressed

297

from a tetracycline-regulated promoter on a separate plasmid (pompC’-yfp).

298

The maximum fluorescence that can be produced from the reporter under

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screening conditions was determined by constructing an isogenic control strain that

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contains pompC’-yfp and the pTrc99a vector lacking rpoE and rybB. This strain mimics

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strong inhibition of the σE pathway. OmpC’-YFP fluorescence intensity in the control

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strain was 4-fold higher than in the strain with prpoErybB (Fig. 1B). The difference in

303

fluorescence intensity between the two strains indicates that the assay has the sensitivity

304

to detect inhibitors that reduce activity of the σE pathway.

305

Inhibitors of the σE pathway were identified from the SGWX5 library of cyclic

306

peptides produced using SICLOPPS (50). In this library, five codons in the cyclic peptide

307

gene (corresponding to the X amino acids) are randomized at the DNA level, and the

308

SGW sequence is constant to ensure efficient cyclization. Cyclic peptide production was

309

induced in the E. coli prpoErybB pompC’-yfp strain, and FACS was used to isolate cells

310

with the highest fluorescence intensity from a population of 2 x 106 cells. Cultures of the

311

selected clones were grown and OmpC’-YFP production was measured in the presence

312

and absence of cyclic peptide expression using epifluorescence microscopy. To ensure

313

that increased fluorescence was caused by the SICLOPPS plasmid and not mutations in

314

the reporter strain, plasmids encoding putative inhibitors were purified, retransformed

315

into E. coli prpoErybB pompC’-yfp, and the fluorescence assay was repeated. Four

316

plasmids caused >2-fold higher fluorescence than the negative control (E. coli prpoErybB

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pompC’-yfp with no cyclic peptide). The strongest inhibition was observed in cells

318

carrying pSI24. The fluorescence intensities of cells with pSI24 showed a bi-modal

319

distribution. 10% of cells had fluorescence intensity within 2 standard deviations of the

320

average for the negative control, and were scored “dark”. The remaining 90% of cells

321

with pSI24 had fluorescence intensity 300 ± 13% higher than the negative control. Based

322

on these data pSI24 was chosen for further characterization (Fig. 1B).

323

Lysine is inserted at the TAG codon of pSI24

14

324

Sequencing of the four plasmids with >2-fold increased fluorescence, including

325

SI24, showed that each contained a TAG codon in the randomized region of the sequence

326

(Table 3). The presence of a stop codon suggested that either inhibition of the σE pathway

327

was the result of an uncircularized, truncated SICLOPPS protein, or read-through of the

328

stop codon resulted in a complete SICLOPPS protein that could produce an inhibitory

329

cyclic peptide. The SICLOPPS plasmids encode a chitin-binding domain (CBD) at the C

330

terminus of the full-length SICLOPPS protein. The CDB is removed during the first step

331

of cyclization, generating the IN-CBD fragment (42, 50). Because the full-length

332

SICLOPPS protein and IN-CBD are different sizes (22 kD and 19 kD, respectively),

333

western blotting to detect the CBD can be used to measure expression of the full-length

334

protein and the ability of the protein to initiate cyclization. Western blotting of extracts

335

from a strain with pSI24 revealed that full-length protein and the IN-CDB were produced,

336

albeit at low levels (Fig. 2A). This result suggested that read-through did occur at the stop

337

codon. The 22 kD protein was purified using chitin-affinity chromatography and

338

identified by LC-MS-MS (Fig. 2). The mass spectrometry results confirmed that the full-

339

length SICLOPPS protein was present, and revealed that a lysine residue was inserted at

340

the TAG codon in the randomized sequence region. These data indicated that a cyclic

341

peptide of the sequence SGWLGSKP was produced at low levels in cells with pSI24.

342

Expression of cyclic SGWLGSKP is toxic

343

To determine if cyclic SGWLGSKP was responsible for σE pathway inhibition in

344

cells containing pSI24, the stop codon in the randomized region of pSI24 was changed to

345

a lysine codon to generate pSI24Am7K. When cyclic peptide expression was induced

346

from pSI24Am7K in E. coli prpoErybB pompC’-yfp, the lag phase following dilution of

15

347

the overnight culture was extended and even after growth resumed the cultures grew

348

more slowly than isogenic strains with pSI24 or no SICLOPPS plasmid (Fig. 3A).

349

Epifluorescence microscopy of samples taken after 7-8 hours of culture growth showed

350

that cultures with pSI24Am7K contained a small percentage of fluorescent cells with

351

typical E. coli size, but the majority of cells formed long filaments (Fig. 3B). To assess

352

whether expression of cyclic peptide from pSI24Am7K affected viability, cells were

353

stained by propidium iodide, a membrane-impermeable dye that is excluded from viable

354

cells. Propidium iodide was retained in most cells with pSI24Am7K, but not by cells

355

expressing an unrelated cyclic peptide, RI20, that does not cause filamentation or alter

356

growth (Fig. 3C). These data suggest that expression from pSI24Am7K is toxic, and the

357

toxicity is not due to the SICLOPPS reaction or to general properties of cyclic peptides.

358

The toxicity produced by expression from pSI24Am7K is consistent with the cyclic

359

peptide inhibiting the σE pathway, because σE activity is required for viability of E. coli

360

K12 strains.

361

The toxic effect of expression from pSI24Am7K suggested that pSI24 passed the

362

screen because the amber codon allowed low expression of a highly active cyclic peptide.

363

To determine if the lysine inserted at the amber codon is required for activity, a variant of

364

pSI24 with a glutamine codon at position 7 (pSI24Am7Q) was tested. Expression from

365

pSI24Am7Q in E. coli prpoErybB pompC’-yfp resulted in fluorescent cells and caused

366

slow growth and filamentation similar to the phenotype observed for cells with

367

pSI24Am7K (Fig. 3C). These results indicate that lysine at position 7 is not required for

368

SI24 activity, and suggest that pSI24 with an amber codon at position 7 was identified in

16

369

the screen because cyclic SGWLGSKP inhibits the σE pathway, but is toxic when

370

expressed at typical levels.

371

SI24 inhibits σE-dependent transcription

372

Because repression of the prpoErybB pompC’-yfp reporter requires both σE-

373

dependent transcription of rybB and Hfq-RybB-mediated repression of ompC’-yfp, SI24

374

could inhibit either step of the σE pathway. To test if SI24 inhibits RybB activity, the

375

prpoErybB plasmid was replaced with prybB, which carries the rybB gene under the

376

control a σ70-dependent IPTG-inducible promoter. In the E. coli prybB pompC’-yfp

377

strain, rybB expression and ompC’-yfp repression are not dependent on σE. When SI24

378

was expressed in E. coli prybB pompC’-yfp, there was not an increase in fluorescence:

379

75% of cells showed no increase in fluorescence intensity, and the remaining 25% were

380

≤1.2-fold brighter (Fig. 4A). These data suggested that SI24 does not inhibit Hfq-RybB

381

activity.

382

To determine if SI24 inhibits σE-dependent transcription, egfp was placed under

383

control of the σE-dependent PrybB promoter to make the pSRE reporter. Because basal

384

levels of σE activity are low in unstressed E. coli, a plasmid with rpoE under the control

385

of an IPTG-inducible promoter was transformed into the E. coli pSRE strain to increase

386

GFP production from PrybB. Overproduction of σE in this strain resulted in a 4-fold

387

increase in the average GFP fluorescence intensity. When SI24 expression and

388

overproduction of σE were induced in the same cells, the average GFP fluorescence

389

intensity decreased by 64% (Fig. 4B), indicating that SI24 inhibits σE-dependent

390

transcription.

391

SI24 inhibits transcription in vitro

17

392

The in vivo data suggest that SI24 inhibits σE-dependent transcription, either directly

393

or by altering some aspect of cellular physiology. To determine if SI24 acts directly on

394

σE-dependent transcription, cyclic SGWLGSKP was chemically synthesized and added

395

to multiple-round transcription reactions in vitro. In this assay, RNAP goes through

396

several rounds of holoenzyme formation, transcription initiation, elongation, and

397

termination, recapitulating the fundamental steps of transcription that occur in the cell.

398

When SI24 was incubated with σE before RNAP was added to form the holoenzyme,

399

SI24 inhibited transcription with an IC50 of 270 µM and a KI = 37 ± 17 µM (Fig. 5A).

400

When SI24 was added to σE after holoenzyme formation, a small concentration-

401

dependent effect was observed on transcription with an IC50 of 1.25 mM. (Fig. 5B).

402

These data indicate that SI24 is a transcription inhibitor and suggest that SI24 may act at

403

a step before holoenzyme formation

404

To confirm that SI24 inhibits σE-dependent transcription, similar experiments were

405

performed using a single-round transcription assay. In these experiments, heparin is

406

included in the reaction mixture to prevent re-binding of polymerase to DNA, thereby

407

limiting transcription to a single round. When SI24 was added to σE prior to addition of

408

core RNAP, transcription was inhibited in a concentration-dependent manner, with a KI =

409

62 ± 6 µM (Fig. 5A). However, when σE was incubated with core RNAP to form the

410

holoenzyme before SI24 was added, little inhibition was observed (Fig. 5B). These data

411

are consistent with the results from the multiple-round assays. The observations that SI24

412

is more potent when added before holoenzyme formation in multiple-round transcription

413

assay and that it is only active when added before holoenzyme formation in the single-

414

round transcription assay suggest that SI24 interferes with formation of an active σE-

18

415

RNAP holoenzyme. The multiple-round transcription assay involves reassociation of σE

416

and RNAP to form holoenzyme and initiate a new round of transcription following

417

termination providing an opportunity for SI24 to act whereas this recycling does not

418

occur in the single-round assay.

419

SI24 binds σE in vitro and inhibits holoenzyme formation

420

Because SI24 only inhibited transcription when it was pre-incubated with σE, the

421

simplest explanation for the mechanism of action of SI24 is that it binds σE or core

422

RNAP and disrupts holoenzyme formation. To test if SI24 directly binds σE, cyclic SI24

423

was used in fluorescence anisotropy experiments with BODIPY-labeled σEN80C/C165A. In

424

this σE variant, the naturally occurring cysteine was replaced with alanine and a cysteine

425

residue was introduced at position 80 in place of asparagine, so the protein could be

426

labeled at a surface exposed region that does not interfere with binding to core RNAP

427

(51). Incubation of BODIPY-σEN80C/C165A with increasing concentrations of SI24K altered

428

the anisotropy of BODIPY-σEN80C/C165A in a concentration-dependent manner. SI24 bound

429

BODIPY-σEN80C/C165A with a KD = 32 ± 8 μM (Fig. 6A). The KD was similar when the

430

anisotropy experiment was repeated using wild-type σE labeled with BOPIPY at cysteine

431

165, indicating that the position of the label did not influence peptide binding (not

432

shown).

433

Given that SI24 binds σE, we next examined whether SI24 binding could interfere

434

with σE holoenzyme formation. Fluorescence anisotropy was used to first determine the

435

binding affinity of σE and core RNAP, and then to determine whether SI24 alters the

436

binding affinity. Core RNAP bound to BODIPY-σEN80C/C165A with a KD = 8.7 ± 5 nM (Fig.

437

6B). Because the σE variant contains two mutations and a fluorescent label which might

19

438

affect binding to core RNAP, the binding affinity of wild-type σE and core RNAP was

439

determined using a competition assay in which increasing amounts of unlabeled wild-

440

type σE were added to the core RNAP BODIPY-σEN80C/C165A complex. The KI for wild-

441

type σE and core RNAP was 4 ± 1 nM, which is close to the KD measured with the

442

labeled σE variant. To ensure that the anisotropy assay was providing accurate

443

measurements of binding affinity, the binding constant for core RNAP binding with σ70,

444

the housekeeping sigma factor, was measured using the same competition assay with σ70

445

as the competitor. σ70 bound core RNAP with a KI = 3 ± 2 nM (Fig. 6C), in agreement

446

with published KD values (52).

447

Having established the assay, the ability of SI24 to disrupt the interaction between

448

core RNAP and BODIPY-σEN80C/C165A was tested. When BODIPY-σEN80C/C165A was pre-

449

incubated with SI24, SI24 decreased the affinity of BODIPY-σEN80C/C165A for core RNAP

450

in a concentration-dependent manner (Fig 6D). The KI for binding of SI24 and BODIPY-

451

σEN80C/C165A calculated from the inhibition data was 53 µM, similar to the KD value

452

obtained using the direct binding assay. The KI values for σE binding with core RNAP

453

calculated from the multi-round transcription and single-round transcription experiments

454

were similar to those measured from the anisotropy experiment, indicating that binding of

455

SI24 with σE could account for the observed inhibition.

456

E. coli sigma factors share some conserved regions, including those required to bind

457

core RNAP (7). To determine if SI24 is specific for σE or can inhibit binding of other

458

sigma factors with core RNAP, the effect of SI24 on σ70-dependent transcription was

459

assayed. When SI24 was incubated with σ70 before holoenzyme formation in a single-

460

round transcription experiment, some inhibition was observed. However, higher

20

461

concentrations of SI24 were needed to inhibit σ70-dependent transcription than σE-

462

dependent transcription; the KI for SI24 inhibition of σ70-dependent transcription was

463

>100 µM (Fig. 5C). Therefore, although SI24 can inhibit σ70-dependent transcription in

464

vitro, it preferentially inhibits σE.

465

In a wild-type E. coli strain, most σE in the cell is bound to the anti-sigma factor

466

RseA, so the concentration of free σE is low. If SI24 acts by inhibiting σE-dependent

467

transcription in vivo, increasing the free concentration of σE should counteract the effects

468

of SI24. To test this prediction, the active concentration of σE in the cell was increased by

469

deleting the gene encoding RseA. σE activity is 25-30 fold higher in a ∆rseA strain

470

compared to a wild-type strain (S. Ades unpublished observations, (29)). In the ∆rseA

471

strain, cyclic peptide expression from pSI24 did not affect fluorescence from E. coli

472

prpoErybB pompC’-yfp, and cyclic peptide expression from pSI24Am7K did not alter

473

the growth rate or morphology of the cells (Fig. 7). Western blotting demonstrated that

474

expression of the full-length SI24 SICLOPPS protein and the first cyclization product

475

from pSI24 and pSI24Am7K were the same in the wild-type and ∆rseA strains. These

476

results suggest that additional σE can overcome the inhibition by SI24, and are consistent

477

with SI24 acting by preventing σE binding with core RNAP.

478

SI24 is not an effective inhibitor when added to E. coli cultures

479

For SI24 to act as an antibiotic, it must be effective when added exogenously to

480

cultures of bacterial cells. However, when chemically synthesized SI24 was added to

481

cultures of E. coli prpoErybB pompC’-yfp, the peptide had no effect on growth or

482

OmpC’-YFP fluorescence. These data indicate that although SI24 is an effective inhibitor

483

of σE in vitro and when it is expressed inside the cell, either SI24 cannot cross the cell 21

484

envelope of E. coli or it does not accumulate to high enough concentrations in the

485

cytoplasm to block σE activity.

486

Validation of σE pathway inhibitor screen under HTS conditions

487

Screening for high YPF fluorescence in the E. coli prpoErybB pompC’-yfp reporter

488

strain successfully identified inhibitors of σE pathway using a FACS-based screen with a

489

library of compounds expressed in vivo. To determine if the reporter could be used to

490

screen compound libraries in high-throughput format, YFP fluorescence from E. coli

491

prpoErybB pompC’-yfp and E. coli pompC’-yfp was measured in 384-well microtiter

492

plates. The average fluorescence in E. coli pTrc99a pompC’-yfp, the positive control

493

strain mimicking full inhibition of σE activity, was 5-fold higher than that in E. coli

494

prpoErybB pompC’-yfp, with Z’ = 0.6 ± 0.05. These data indicate that the assay is

495

appropriate for high-throughput screening.

496 497 498 499

Discussion The results described here characterize a cell-based assay that can efficiently identify

500

inhibitors of the σE pathway. The SI24 cyclic peptide binds σE with a KD ≈ 32 µM,

501

inhibits binding of σE to RNAP, and inhibits transcription from σE-dependent promoters

502

in vitro and in vivo (Figs. 5 & 6). The affinity of σE for RNAP is 1000-fold higher than

503

for SI24, so high concentrations of SI24 (0.1–1 mM) are required to inhibit σE-dependent

504

transcription in vitro under equilibrium binding conditions (Fig. 5A). However, reporter

505

assays demonstrated that SI24 inhibits σE-dependent transcription in vivo even when it is

506

expressed from pSI24 and requires read-through of a stop codon for production (Fig. 1B).

507 508

What is responsible for the increased effectiveness of SI24 in vivo? Transcription of σE-dependent promoters in E. coli requires binding of free σE to free core RNAP, and the

22

509

reaction is likely to be controlled by kinetics and not thermodynamics. The binding

510

affinity of σE and RseA is 0.2 nM (data not shown) and there is ~2.5 fold more RseA than

511

σE in E. coli, so there is little free σE at equilibrium (53). However, RseA is an unstable

512

protein in E. coli. The half-life of RseA is ~8 min (54), so turnover of RseA will produce

513

free σE that can interact with core RNAP or SI24. The concentration of free core RNAP

514

available to bind to σE is limited by its engagement in transcription elongation and by

515

binding with σ70. The affinity of core RNAP for σ70 is 3 ± 2 nM (Fig. 6C), and the

516

concentration of σ70 in the cell is higher than the concentration of core RNAP (and

517

significantly higher that the concentration of free σE) (55). However, sigma factors are

518

released from core RNAP after transcription initiation (56). After transcription

519

termination, core RNAP can bind σE or another sigma factor, and it is likely to be kinetic

520

competition for sigma factor binding that controls the amount of σE-dependent

521

transcription. In this model, SI24 could inhibit σE-dependent transcription in vivo by

522

decreasing the rate of association of σE and core RNAP. Consistent with this kinetic

523

regulation model, SI24 did not inhibit transcription in vitro when it was added after

524

holoenzyme formation in the single-round transcription assay (Fig. 5B).

525

SI24 can inhibit transcription by σ70 in addition to σE, but with lower affinity.

526

Therefore, it is possible that the toxicity associated with expression from pSI24Am7K is

527

due in part to inhibition of σ70. However, there are several indications that SI24 acts by

528

inhibiting σE. First, increasing the amount of free σE in the cell by deleting rseA

529

eliminates the effects of pSI24Am7K, consistent with SI24 acting through σE. Second,

530

there is much more σ70 in E. coli than σE, and concentrations of cyclic peptide are

531

unlikely to reach a level at which they can fully inhibit σ70. Third, clear selectivity for σE

23

532

was seen when SI24 was expressed from the pSI24 plasmid. Transcription of ompC’-yfp

533

depends on σ70, but expression of ompC’-yfp increased in the presence of SI24 (Fig. 1B),

534

consistent with SI24 inhibiting σE more efficiently than σ70. Overall, the work presented

535

here demonstrates that inhibitors of σE-dependent transcription can be obtained using the

536

prpoErybB pompC’-yfp assay.

537

SI24 is not a lead compound for drug development because it does not enter E. coli

538

cells when added exogenously. However, elucidation of the binding interface between

539

SI24 and σE would reveal interactions that could be targeted for structure-based drug

540

design. Moreover, the assay used to identify SI24 is appropriate for high-throughput

541

formats, and could be used to identify inhibitors from libraries of molecules with good

542

pharmaceutical properties. Because repression of ompC’-yfp requires both σE activity and

543

Hfq-RybB activity, the assay could identify inhibitors of either pathway.

544

In this screen, plasmids from all the selected cells contained a stop codon. In pSI24,

545

this stop codon is read with low efficiency by tRNAlys , which is consistent with reported

546

data about UAG nonsense codon being misread by tRNAlysUUU (57). It is notable that the

547

read-through allowed identification of a potent inhibitor that was toxic when a sense

548

codon was used. The growth and morphological defects produced by pSI24Am7K were

549

severe enough that cells with the sense codon would have been unlikely to survive the

550

screening process. Plasmids with amber codons have also been selected in other screens

551

((37). In addition to tRNAlys, amber codons can be read at low efficiency by tRNAgln and

552

tRNAtyr. It is not yet known what peptides were produced by these other nonsense

553

plasmids, but stop codon read-through might provide a means to identify peptides and

554

plasmids produced by SICLOPPS or other mechanisms that are extremely potent.

24

555

Bacterial RNA polymerase has been explored as a target for drug development for

556

many years. The majority of polymerase inhibitors bind in or near the active site and

557

interfere with RNA synthesis (15, 16). Recent HTS and rational design approaches have

558

identified compounds that interfere with sigma-core interactions (58-60). However, these

559

compounds were designed to inhibit transcription by the housekeeping sigma factor.

560

Inhibitors of alternative sigma factors involved in infection and virulence, such as σE,

561

present a different approach to antibiotic development, because such compounds will

562

interfere with a pathogen’s ability to cause disease. One other inhibitor of an alternative

563

sigma factor, σB from Listeria monocytogenes has been reported (61). However, this

564

inhibitor does not bind to σB and instead appears to act by interfering with the signaling

565

pathway leading to activation of σB (62). SI24 is the first compound reported to bind to

566

an alternative sigma factor and interfere with transcription by that sigma factor. The assay

567

is currently being used in small molecule screening with the goal of identifying more

568

selective inhibitors of σE. In addition, the assay can be readily adapted to find inhibitors

569

of other alternative sigma factors by changing the sigma factor gene carried on the

570

plasmid and replacing the promoter for rybB with the appropriate promoter sequence.

571

This is the third published study in which a SICLOPPS library was used in

572

conjunction with FACS to validate antibiotic targets and screens (37, 38). We suggest

573

that this is a useful route to validate assays for high-throughput screening. The

574

SICLOPPS system provides a complex library that is very inexpensive to produce.

575

Likewise, FACS allows rapid isolation of positive clones at low cost. All assays that have

576

been developed for SICLOPPS/FACS have adapted easily to microtiter plate format for

25

577

HTS. In addition to validating the screening assay, the SICLOPPS library yields cyclic

578

peptides that can be used as controls for HTS.

579 580

Acknowledgments

581

Thanks to Katsuhiko Murakami for gifts of proteins and DNA, to Susan Margaree in the

582

Microscopy and Cytometry Facility of the Huck Institutes of the Life Sciences for

583

assistance with FACS, and to Dr. Tatiana Laremore in the Proteomics and Mass

584

Spectrometry Core Facility of the Huck Institutes of the Life Sciences for performing the

585

protein sequence analysis. Thanks also to Stephen Benkovic for advice and support. This

586

work was supported by NIH grant NS071542 to S.E.A. and K.C.K., NIH grant GM68720

587

to K.C.K. and The International Fulbright Science & Technology Award to S.A.E.

588

26

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Table 1: Strains and plasmids Name BW27786 SEA5036 SEA6805 SEA6809 SEA6833 SAE006 SAE008 SAE009 SAE018 SAE019 SAE020 SAE021 SAE028 SAE051 SAE052 SAE057 SAE173 SAE174 SAE197 SAE200 SAE248 pTrc99a pSB4K5 pEGFP-N2 pRybB pJV300 prpoErybB pLC245 pompC’-yfp pSRE pARCBD pSI5 pSI18 pSI19 pSI24 pSI24Am7K pSI24Am7Q pPER76 prpoEN80C/C165A

Description DE(araBAD) DE(rhaBAD) DE(araFGH) Φ(∆araEp PCP13-araE ∆rhaD-B) BL21 (DE3) slyD::kan pLysS pPER76 BW27786 prpoErybB pompC’-yfp BW27786 pTrc99a pompC’-yfp BW27786 ΔydcQ ΦλrpoHP3::lacZ BW27786 lacIq SAE006 pRybB pompC’-yfp SAE006 pJV300 pompC’-yfp SEA6805 pSI18 SEA6805 pSI19 SEA6805 pSI24 SEA6805 pSI24Am7Q SAE008 pSI24 SEA6833 pSRE pLC245 SEA6833 pSRE pTrc99a SAE051 pSI24 SEA6809 ΔrseA SAE020 ΔrseA SEA6805 pSI24Am7K SEA6805 pRI20 SAE197 ΔrseA Plasmid containing the Ptrc promoter, AmpR BioBrick vector, KanR Plasmid encoding egfp, KanR Same as pFM1-1 (pBRpLac encoding rybB, AmpR) sRNA control vector, AmpR pTrc99a encoding rpoE and rybB, AmpR pTrc99a encoding rpoE, AmpR pSB4K5 with ompC’-yfp, KanR pSB4K5 encoding egfp, KanR SICLOPPS system plasmid, CmR pARCBD encoding SGWWDAV*, CmR pARCBD encoding SGWSER*T, CmR pARCBD encoding SGWAD*CK, CmR pARCBD encoding SGWLGS*P, CmR pARCBD encoding SGWLGSKP, CmR pARCBD encoding SGWLGSQP, CmR pET15b encoding rpoE, AmpR pET15b encoding rpoE N80C/C165A, AmpR

Source (63) (44) This study This study This study (38) (38) (38) This study This study This study This study This study This study This study This study This study This study This study This study This study (64) (65) (66) (67) (68) This study (27) (38) This study (50) This study This study This study This study This study This study (69) This study

783 784 785 786 787

32

788

Table 2: Oligonucleotides Name rrnbT1Ba rrnBT2X rybBX rybBSI rybBE rybBB egfpB egfpP rpoE_N80C_for rpoE_N80C_rev rpoE_C165A_for rpoE_C165A_rev rybB_EcoRI_for rybB_XhoI_rev E24K_top E24K_bottom

789 790 791 792

799 800 801

Sequence TATTAGGATCCTCAGAAGTGAAACGCCGTAGCG TATTATCTAGATCAGGGTTATTGTCTCATGAGC ATAATTCTAGAAAACTGAAGTTGCCCTGAAAATG ATAATGTTCGACTAAGCCGCTATCGCGCGAGGAG ATAATGAATTCAAACTGAAGTTGCCCTGAAAATG TATTATCATGACTAACCTCCTGACATCAAAGAAAAGCAGTGGCAC TATTATCATGAGCAAGGGCGAGGAGCTGT ATAATCTGCAGTTACTTGTACAGCTCGTCCATG GTATCGGATTGCTGTATGTACAGCGAAAAATTA CC GGTAATTTTTCGCTGTACATACAGCAATCCGATAC CACCGTACCTACCGGAGCATCCATGATAG GGC GCCGCTATCATGGATGCTCCGGTAGGTACGGTG CATGGTATGGCCAGGATTAGG GAGGGTTGCAGGGTAGTAG GGGCGATCGCCCACAATTCCGGCTGGTTGGGCTCGAAGCCGTGC TTAAGCACGGCTTCGAGCCCAACCAGCCGGAATTGTGGGCGATCG CCCCAT

Table 3. Sequences of cyclic peptide inhibitors. 793 % fluorescent cells Name Sequence1 794 pSI24 SGWLGS*P 90 795 pSI5 SGWWDAV* 80 796 pSI18 SGWSER*T 82 797 pSI19 SGWAD*CK 85 798 1 Amino acid sequence of randomized region determined by conceptual translation of the plasmid DNA sequence; * indicates a stop codon.

33

802 803 804

Figure legends

805

representation of the σE pathway reporter. (Left) When the pathway is functional, σE

806

directs transcription of RybB, which binds an Hfq hexamer. Hfq-RybB represses

807

translation of ompC’-yfp mRNA and targets it for degradation, preventing production of

808

OmpC’-YFP. (Right) Inhibition of a step in the pathway, such as σE-dependent

809

transcription or RybB-Hfq activity, derepresses translation, resulting in OmpC’-YFP

810

production and fluorescent cells. B) pSI24 increases fluorescence in E. coli prpoErybB

811

pompC’-yfp. With no inhibitor, RybB represses ompC’-yfp expression, and E. coli

812

prpoErybB pompC’-yfp cells have low YFP fluorescence intensity (prpoErybB panel).

813

When SI24 is produced in E. coli prpoErybB pompC’-yfp (prpoErybB+pSI24 panel),

814

fluorescence intensity is similar to that observed in the positive control strain (No

815

prpoErybB panel).

Figure 1. Screen for cyclic peptide inhibitors of the σE pathway. A) Schematic

816 817

Figure 2. SI24 contains lysine at position 7. A) Schematic diagram of SICLOPPS

818

reaction. In the first step, the IN-CDB domain is spliced out to give a lariat intermediate,

819

and in the second step the cyclic peptide is released. B) Representative Western blot

820

using anti-CBD antibody showing the relative amounts of full-length protein and IN-CBD

821

produced from E. coli prpoErybB pompC’-yfp cells with no SICLOPPS plasmid, with

822

expression induced from pSI24, and with expression induced from an unrelated

823

SICLOPPS plasmid. C) Purified full-length SICLOPPS protein from pSI24 resolved by

824

SDS-PAGE. Lysate from cells expressing an unrelated SICLOPPS protein is shown for

825

comparison. D) MS/MS spectrum from one chymotryptic peptide of SI24 protein from

34

826

(C). The masses of the parent ion (495.9) and fragmentation ions are indicated in black,

827

with b+1 and y+1 assignments shown in gray.

828 829

Figure 3. Expression from pSI24Am7K is toxic. A) Representative growth curves of E.

830

coli prpoErybB pompC’-yfp pSI24 with (open circles) and without (filled circles)

831

induction, and E. coli prpoErybB pompC’-yfp pSI24Am7K with (open triangles) and

832

without (filled triangles) induction. B) Fluorescence micrographs of E. coli prpoErybB

833

pompC’-yfp and E. coli pompC’-yfp as in Fig. 1B. Expression from pSI24Am7K or

834

pSI24Am7Q in E. coli prpoErybB pompC’-yfp results in fluorescence and filamentous

835

growth. C) Propidium iodide staining of E. coli prpoErybB pompC’-yfp with

836

pSI24Am7K or an unrelated cyclic peptide, pRI20, with and without induction. Red color

837

indicates permeability of cells to propidium iodide. Samples were taken after 6-7 hours of

838

growth in parts B and C.

839 840

Figure 4. SI24 inhibits σE-dependent transcription. A) SI24 does not inhibit Hfq-RybB

841

activity. With no inhibitor, RybB represses ompC’-yfp expression, and E. coli prybB

842

pompC’-yfp cells have low YFP fluorescence intensity (prybB panel). When SI24 is

843

produced (prybB+pSI24 panel), fluorescence intensity is similar to that in a strain with no

844

inhibitor. Cells are fluorescent when RybB is not present (No prybB panel). B) SI24

845

inhibits production of GFP from a σE-dependent promoter in E. coli prpoE pSRE. With

846

no inhibitor, rpoE expression causes GFP production and fluorescent cells (prpoE panel).

847

When SI24 is produced (prpoE+pSI24 panel), fluorescence intensity is decreased. Cells

848

are not fluorescent in a strain with no RpoE expression (No prpoE panel).

35

849 850

Figure 5. SI24 inhibits transcription in vitro. A) Multiple-round and single round

851

transcription assays with SI24 incubated with σE before addition of core RNAP. B)

852

Multiple-round and single round transcription assays with SI24 after σE holoenzyme

853

formation. C) Single round transcription assays with SI24 incubated with σ70 before

854

addition of core RNAP. Representative gels and plots with averages from 2 independent

855

experiments with whiskers indicating standard deviation are shown for multiple-round

856

(filled circles) and single round (open circles) assays. Solid lines show fits to the Hill

857

equation.

858 859

Figure 6. SI24 binds σE and inhibits interaction with core RNAP. A) Fluorescence

860

anisotropy experiments using SI24 and BODIPY-σEN80C/C165A were used to determine the

861

dissociation constant for binding (Kd = 32 ± 8 μM). B) Fluorescence anisotropy

862

experiments using core RNAP and BODIPY-σEN80C/C165A were used to determine the

863

dissociation constant for binding (Kd = 8.7 ± 5 nM). Solid lines in panels A and B show

864

fits to Eq. 2. C) Competition binding experiments were used to determine the affinity of

865

core RNAP for σE and σ70. Pre-formed complexes with 5 nM BODIPY-σEN80C/C165A and

866

75 nM RNAP were challenged with unlabeled σE or σ70 competitor. D) Competition

867

binding experiments show SI24 inhibits holoenzyme formation. SI24 was added to

868

binding reactions containing 5 nM BODIPY-σEN80C/C165A and 75 nM core RNAP. Solid

869

lines in panels C and D show fits to the Hill equation.

870

36

871

Figure 7. Deletion of rseA suppresses the phenotypes caused by SI24 A) SI24 did not

872

increase fluorescence when expressed in E. coli prpoErybB pompC’-yfp ΔrseA. Results

873

of SI24 expression in E. coli prpoErybB pompC’-yfp (wt panels) as performed in Fig. 1B

874

are shown for comparison. B) Representative growth curves of E. coli prpoErybB

875

pompC’-yfp ΔrseA pSI24Am7K with (open triangles) or without (filled triangles)

876

induction. Growth curves for E. coli prpoErybB pompC’-yfp pSI24Am7K with (open

877

circles) and without (filled circles) induction as performed in Fig. 3A are shown for

878

comparison.

37