Jul 2026· International Journal of Biological Macromolecules· Vol 379, pp.
153822
· 0 citations· 61 references
Medicine
TL;DR
This study characterizes a set of pathogen-enriched transcriptional regulatory proteins involved in biofilm formation in enterohemorrhagic E. coli and provides new insights into the macromolecular regulatory mechanisms underlying biofilm formation and virulence in pathogenic E. coli.
Abstract
Pathogenic Escherichia coli O157:H7 encodes numerous lineage-enriched transcriptional regulatory proteins that are absent or poorly conserved in non-pathogenic strains, yet their functions in transcriptional programming and biofilm-associated virulence remain largely uncharacterized. Here, we identified eight previously unreported biofilm-associated regulatory proteins involved in biofilm formation in enterohemorrhagic E. coli (EHEC) O157:H7 via comparative genomic analysis. Conserved domain analysis revealed that these regulators belong to diverse DNA-binding protein families, including LysR-, GntR-, LuxR-, AraC/XylS-, Cro/CI-, and Ogr/Delta-like families. Among these, two regulators (ECs_2620 and ECs_4457) were confirmed as direct DNA-binding transcriptional regulators through in vitro and in vivo DNA-binding assays. Functional analyses showed that overexpression of these regulators enhanced biofilm formation, whereas gene deletion impaired biofilm development and reduced bacterial virulence in both THP-1 cell and mouse infection models. Transcriptomic analysis revealed that these regulators primarily modulate genes involved in bacterial chemotaxis and flagellar assembly, representing a regulatory framework distinct from the classical c-di-GMP-centered regulatory paradigm described in non-pathogenic E. coli. Furthermore, we identified that the specific DNA-binding motifs of two regulators with direct in vitro DNA-binding activity. Evolutionary analysis showed that these regulators are highly conserved among pathogenic E. coli lineages, including EHEC, enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC), but are absent or truncated in non-pathogenic E. coli K-12 strains. Collectively, this study characterizes a set of pathogen-enriched transcriptional regulatory proteins and provides new insights into the macromolecular regulatory mechanisms underlying biofilm formation and virulence in pathogenic E. coli.
YgeP plays a central role in APEC pathogenesis by balancing two infection strategies: motility-mediated dissemination and colonization, and biofilm-mediated attachment and tolerance, and negatively regulates key virulence traits during the early stages of infection.
Weiyang Su, Zhe Li, Siqi Feng et al.· Veterinary Microbiology· 0 citations
ABSTRACT Aspergillus flavus, a ubiquitous filamentous fungus, severely compromises global food safety and public health by producing carcinogenic aflatoxins. Heat shock factor 1 (HSF1) orchestrates stress responses in eukaryotes, yet the functional role and regulatory mechanisms of its homolog, HsfA, in A. flavus remain elusive. Here, we demonstrate that A. flavus encodes two hsfA copies, whose knockdown or expression of a dominant-negative variant abrogates spore germination, a prerequisite for fungal development and colonization. Through integrated reverse genetics, ChIP-qPCR, electrophoretic mobility shift assay, and transcriptional profiling, we identify six non-chaperone targets – brlA, fksP, flbC, sntB, velB, and vosA – directly regulated by HsfA via binding to conserved heat shock elements (HSEs) in their promoters. Ectopic expression of each target partially restores germination, developmental progression, and pathogenicity in HsfA-deficient strains, confirming HsfA’s central role in driving these processes via transcriptional activation. Structural divergence between the A. flavus HsfA DNA-binding domain and human HSF1 explains the ineffectiveness of three HSF1 inhibitors against the fungus. Our findings establish HsfA as a pivotal regulator of A. flavus virulence and uncover a novel HSF regulatory pathway, highlighting HsfA as a promising target for mitigating aflatoxin contamination.
X. Nie, Guolong Zhu, Bei Qin et al.· Virulence· 0 citations
Biofilm formation is a fundamental survival strategy that enables bacterial persistence across diverse environments and hosts. While the mrk gene cluster encoding type 3 fimbriae is a well-established determinant of biofilm formation in Klebsiella pneumoniae, its presence and functional significance in Salmonella enterica remains poorly defined. In this study, we identified a plasmid-encoded mrkABCDF gene cluster carried on a highly conjugative IncX1 plasmid in a clinical Salmonella Thompson isolate. Using CRISPR/Cas9-mediated knockout of the mrk-containing Tn6011 transposon, plasmid curing, targeted gene deletion, and genetic complementation, we systematically dissected the contribution of mrkABCDF genes to biofilm development and associated phenotypes. Loss of the mrk gene cluster resulted in a profound reduction in biofilm biomass and a concomitant increase in bacterial motility. Type 3 fimbriae were detected exclusively on the surface of mrk-positive cells, confirming their structural role in surface attachment. The mrk operon was strongly expressed from an IS1-associated promoter, bypassing canonical MrkH-dependent regulation. Functionally, mrk expression enhanced bacterial tolerance to desiccation and oxidative stresses, and reduced susceptibility to macrophage phagocytosis. In vivo, mrk-positive strains exhibited enhanced gastrointestinal colonization and tissue invasion. Notably, carbapenems exhibited exceptional efficacy in inhibiting mrk-mediated biofilm formation, indicating their superior potential for treating biofilm-associated infections. Our findings demonstrate that plasmid-encoded mrkABCDF genes can act as key architectural and functional determinants of biofilm formation in Salmonella enterica. The horizontal dissemination of mrk-carrying IncX1 plasmids may promote the emergence of biofilm-adapted Salmonella lineages with enhanced environmental persistence and host colonization potential.
Ke Liu, Liya Feng, Lu Ouyang et al.· Biofilm· 0 citations
The Type VI secretion system (T6SS) is a key nanoweapon in Gram-negative bacteria that mediates microbial competition and pathogenesis via toxic effector delivery. Three functionally distinct T6SS clusters (H1-H3) are known in Pseudomonas aeruginosa, yet the broader evolutionary diversity and regulatory networks of T6SS in this pathogen remain poorly defined. Here, we identify Sfa4, a transcriptional regulator linked to a fourth T6SS (H4-T6SS) in clinical isolate LYSZa7. Sfa4 directly binds amrZ and H4-T6SS cluster to activate their transcription. AmrZ, in turn, directly regulates all four T6SS clusters. We further show that c-di-GMP receptor FleQ directly binds the promoters of all four T6SS clusters, revealing a direct regulatory link between c-di-GMP signaling and T6SS transcription. This regulation, together with Sfa4-mediated elevation of intracellular c-di-GMP levels, coordinately enhances H4-T6SS activity, biofilm formation, and virulence in A549 alveolar epithelial cells and Galleria mellonella models. Phylogenetic analysis shows Sfa4 homologs are present in Gram-negative bacteria, implying a potential T6SS-regulatory function. Collectively, our findings shed light on regulatory cascades and provide a mechanistic basis for understanding how clinically acquired T6SS clusters may be integrated into existing virulence networks.
Yizhou Zhang, T. Ye, Jie Deng et al.· International Journal of Bio...· 0 citations
Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain, and ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation.
Martina Janoušková, Yu-Ching Su, Sandra Jonsson et al.· Journal of Infectious Diseas...· 0 citations
This study provides a theoretical basis for elucidating the molecular mechanism by which YibT modulates salmonella pathogenicity and shows that yibT contributes to regulate adhesion and invasion of S. typhimurium, and its deletion attenuates bacterial virulence and host pathogenicity.
Bingbing Wang, Min Yue, Jianjun Chen et al.· Microbial Pathogenesis· 0 citations
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