Global regulation and virulence mediated by the histidine-responsive local transcription factor HutC in Pseudomonas aeruginosa

TL;DR

This study shows that the HutC protein in Pseudomonas aeruginosa regulates virulence by controlling gene expression in response to histidine and urocanate.

Contribution

The study reveals that HutC uses low-affinity DNA binding to globally regulate virulence traits in Pseudomonas aeruginosa.

Findings

HutC binds to promoters of hut operons and regulates histidine and urocanate utilization.

HutC also regulates virulence-related genes like arr and affects biofilm formation and pyoverdine production.

Deletion of hutC reduces virulence in a C. elegans infection model.

Abstract

Host-derived nutritional substrates fuel infection in invading bacteria, yet their potential as signaling molecules for host perception remains largely unexplored. Here, we report the functional characterization of HutC, a transcriptional repressor of hut genes for the utilization of histidine and its derivative, urocanate, in the human pathogenic bacterium Pseudomonas aeruginosa PAO1. Using electrophoretic mobility shift assay (EMSA) and DNase I footprinting combined with site-directed mutagenesis, we demonstrate that HutC specifically binds with high affinity to the promoters of the two hut operons. This analysis led to the identification of a noncanonical HutC-binding site in the hutF promoter of a non-pathogenic Pseudomonas strain, which is absent in P. aeruginosa. A genome-wide search of the PAO1 genome using a probability matrix of the canonical HutC-binding motif identified 172 candidate sites, many associated with bacterial pathogenesis. Their predicted low-affinity binding was experimentally validated by EMSA for six selected targets, including the aminoglycoside response regulator (arr). Deletion of hutC resulted in increased tobramycin-induced biofilm formation and impaired production of pyoverdine, an iron-scavenging siderophore. Moreover, the hutC mutant exhibited altered motility and significantly reduced virulence in the Caenorhabditis elegans infection model. Finally, transcriptome sequencing of three genetically distinct hutC mutants provided further support for the HutC-mediated global regulation. Together, these findings highlight the functional significance of low-affinity DNA binding by this transcription factor and support the hypothesis that HutC mediates P. aeruginosa virulence, with histidine and urocanate as effectors. Thus, HutC may represent a potential therapeutic target within the bacterial host-perception system.

Pseudomonas aeruginosa is a metabolically versatile environmental pathogen whose virulence relies on coordinated expression of catabolic genes, particularly the histidine utilization (hut) operon. Disruption of the hut operon reduces virulence, but the underlying mechanism remains rudimentary. Here, we genetically characterized the histidine-responsive transcriptional factor HutC in P. aeruginosa PAO1, alongside HutC in the non-pathogenic strain Pseudomonas fluorescens SBW25. Two important features emerged. First, HutC recognizes two distinct DNA-binding motifs with little sequence similarity; notably, a noncanonical-binding site was identified in the hutF promoter of SBW25 but was absent in PAO1. Second, HutC exhibits low-affinity binding to genes beyond histidine catabolism and contributes to the expression of multiple virulence traits. These findings identify HutC as a local regulator linking histidine catabolism with virulence and as a unique prokaryotic model for studying how noncanonical transcriptional factor-DNA interactions achieve binding specificity, a phenomenon so far investigated only in eukaryotes.

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