Transcriptomics and functional analysis reveal impaired motility, biofilm formation, invasion and intramacrophage survival of pimt gene-deleted strain of Salmonella Typhimurium.

The genotoxic stress-inducible gene GEI1(Orf19.3109) is a repressor of virulence in Candida albicans

ABSTRACT Transcriptional reprogramming under genotoxic stress is essential for the survival and pathogenesis of Candida albicans. While many DNA damage response (DDR) genes are well-characterized, the functions of pathogen-specific, stress-inducible genes remain poorly understood. Here, we characterized GEI1 (Orf19.3109), a gene significantly induced by genotoxic agents and oxidative stress, which lacks a clear ortholog in Saccharomyces cerevisiae. Interestingly, although GEI1 expression is triggered by DNA damage stress, its deletion does not impair genotoxin tolerance. Instead, the gei1Δ mutant shows increased resistance to oxidative stress, characterized by reduced intracellular reactive oxygen species (ROS) accumulation. Furthermore, loss of GEI1 leads to hyper-secretion of extracellular aspartyl proteases (Saps) and results in a hypervirulence phenotype in the Galleria mellonella infection model. Transcriptomic profiling and CRISPR-interference (CRISPRi) assays revealed that loss of GEI1 function triggers the hyperactivation of antioxidant defenses and protease secretion. We demonstrate that the hypervirulence of the gei1Δ mutant is primarily driven by the transcriptional derepression of SAP1, SAP3, SAP5 and the putative alcohol dehydrogenase ADH4. Repression of these downstream effectors in the gei1Δ background reduced protease secretion and restored virulence to wild-type levels. Overall, our findings uncouple transcriptional induction from conventional DNA repair functions and reveal a novel regulatory checkpoint where GEI1 links genotoxic stress signaling to the suppression of key pathogenic traits, highlighting a complex strategy by which C. albicans balances stress adaptation and virulence control.

Molecular mechanism underlying YibT-mediated RpoS regulation of virulence variation in Salmonella typhimurium.

Salmonella typhimurium, a Gram-negative pathogen widely distributed in the environment, poses a serious threat to public health by contaminating food and causing foodborne diseases. Our preliminary study found that under thymol stress, the expression of YibT, a poorly characterized factor in salmonella, was significantly reduced, and deletion of the yibT gene markedly impaired biofilm formation. However, the biological role of yibT in salmonella pathogenesis remains unclear. In this study, the λ-Red homologous recombination system and pET28a vector were used to construct the single deletion mutant STΔrpoS, the double deletion mutant STΔrpoSΔyibT, complementation strains, and an overexpression strain. Promoter reporter vectors were constructed using pKP302. Growth characteristics, biofilm formation, adhesion, invasion, virulence, and pathogenicity were evaluated. The regulatory relationship between rpoS and yibT was examined by β-galactosidase assays. Results showed that deletion of rpoS and yibT led to slow growth, reduced biofilm formation, and decreased flagella and surface appendages under transmission electron microscopy. qRT-PCRshowed that there is a negative feedback loop between yibT and rpoS, where RpoS positively regulates yibT transcription and YibT negatively regulates RpoS expression. Preliminary detection of β-galactosidase confirms that yibT may be located downstream of rpoS. Cellular infection models demonstrated that deletion of yibT and rpoS significantly reduced salmonella adhesion, invasion, and intracellular survival. In mouse infection experiments, bacterial loads in the liver, spleen, and ileum were markedly decreased in mutant-infected mice, with further reduction under quercetin intervention. Flow cytometry analysis of T-cell subsets suggested attenuated immune modulation by the mutant strains, indicating reduced pathogenicity. Collectively, yibT contributes to regulate adhesion and invasion of S. typhimurium, and its deletion attenuates bacterial virulence and host pathogenicity. YibT may participate in the virulence regulatory network by interacting with the transcriptional regulator RpoS. This study provides a theoretical basis for elucidating the molecular mechanism by which YibT modulates salmonella pathogenicity.

Pathogen-enriched transcriptional regulators modulate biofilm formation and virulence in Escherichia coli O157:H7.

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.

The two-component system histidine kinase LiaS contributes to the stress resistance and virulence of zoonotic Listeria monocytogenes

ABSTRACT LiaS is a histidine kinase receptor in two-component systems that senses environmental stress signals. This study investigated the roles of LiaS in Listeria monocytogenes stress resistance and virulence. Phenotypic assays showed that deletion of liaS (ΔliaS) resulted in significantly impaired growth under acidic, alkaline, and osmotic stresses, as well as reduced survival under strong acidic conditions. These defects were partially restored in the complemented strain (CΔliaS). The growth defects were also observed in the ΔliaS under Cu2+ or H2O2 exposure. Furthermore, the ΔliaS strain exhibited impaired invasion and intracellular migration in host cells, along with attenuated colonization in the liver and spleen at 24 h post-infection. Bacterial loads in the spleen of infected mice remained lower at 48 h. The mortality was also delayed in ΔliaS‑infected mice. Transcriptional analysis revealed that compared to neutral conditions, osmotic stress-related genes (except opucB and gbuC) were significantly upregulated in the ΔliaS strain under acidic conditions, indicating that LiaS modulates acid tolerance through transcriptional regulation. Collectively, LiaS is essential for the adaptation of Listeria monocytogenes to acidic, osmotic, and oxidative stresses. Its absence attenuates host cell invasion, intracellular motility, and organ-specific colonization, highlighting its dual role in environmental resilience and pathogenicity. These findings provide a theoretical basis for understanding host–pathogen interactions and offer new strategies against antimicrobial-resistant pathogens.

Transcriptomic Insights into Casein-Driven Adaptive Evolution of Burkholderia thailandensis: Implications for Biofilm Formation and Antibiotic Susceptibility.

How nutrient stress shapes bacterial evolution and the associated fitness trade-offs remains a central question in microbiology. Using Burkholderia thailandensis as a surrogate for the pathogen B. pseudomallei, we performed adaptive laboratory evolution with casein as the sole carbon source. Over 30 days of serial passaging, the population shifted towards protease deficiency, with mutants constituting 54.94% by the endpoint. These evolved strains exhibited pleiotropic virulence attenuation-including reduced rhamnolipid production, motility, auto-aggregation, and biofilm formation-alongside increased susceptibility to imipenem and chloramphenicol. RNA-seq analysis of evolved strain E3101 revealed 2,836 differentially expressed genes, with significant downregulation of quorum sensing (AHL synthesis), rhamnosyltransferases, flagellar assembly, and biofilm regulatory pathways. Our findings demonstrate that casein-driven evolution selects for social 'cheaters' that conserve energy by downregulating costly virulence determinants, revealing a fundamental virulence-fitness trade-off. The coordinated transcriptional repression of biofilm and antibiotic resistance pathways provides a mechanistic framework for understanding bacterial adaptation strategies and potential therapeutic vulnerabilities in Burkholderia.

Stage V sporulation protein G as a global regulator of biofilm formation and virulence in Staphylococcus species: molecular mechanisms and therapeutic potential