The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

Garam Choi,Kyung Ku Jang,Jong Gyu Lim,Zee-Won Lee,Hanhyeok Im,Sang Ho Choi

doi:10.1074/jbc.ra120.012724

Garam Choi, Kyung Ku Jang + Show 4 more

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https://doi.org/10.1074/jbc.ra120.012724

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Abstract

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter P vvhBA , which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of P vvhBA , whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the P vvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of P vvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

Highlights

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner
4 The abbreviations used are: CRP, cAMP receptor protein; H-NS, histone-like nucleoid-structuring protein; M9G, M9 minimal medium supplemented with 0.4% (w/v) glucose; qRT-PCR, quantitative RT-PCR; DMEM, Dulbecco’s modified Eagle’s medium; L-NMMA, L-NG-monomethyl arginine citrate; DEA NONOate, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium1,2-diolate; DP, 2,2Ј-dipyridyl; electrophoretic mobility shift assays (EMSAs), electrophoretic mobility shift assay; 6-FAM, 6-carboxyfluorescein; Fur, ferric uptake regulator; LBS, Luria-Bertani medium supplemented with 2% (w/v) NaCl; RPKM, reads per kilobase of transcript per million mapped sequence reads
Expression of vvhBA is induced upon exposure to murine blood and macrophages In an effort to identify virulence genes significantly induced in V. vulnificus upon invasion of the host bloodstream, transcriptomes of the bacteria exposed to murine blood or M9 minimal medium supplemented with 0.4% (w/v) glucose (M9G; negative control) were analyzed by RNA-seq

Summary

Introduction

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

Results

Conclusion