Regulation of the Staphylococcus aureus stress response pathway by copper ions.

The transcriptional regulator CasR controls mycobacterial antioxidant defense and biofilm formation via multiple direct targets.

AIMS The antioxidant defense system of Mycobacterium tuberculosis is critical for pathogenicity and persistence within macrophages, yet the regulatory networks remain poorly understood. This study aims to elucidate the molecular mechanism by which the transcription factor CasR regulates antioxidant defense in mycobacteria through delineation of the regulatory axis linking CasR activity, target gene expression, and the antioxidant phenotype. METHODS AND RESULTS Using Mycobacterium smegmatis as a model organism, we demonstrate that overexpression of CasR renders the bacteria significantly susceptible to hydrogen peroxide. Electrophoretic Mobility Shift Assay (EMSA) and β-galactosidase reporter analyses reveal that CasR directly binds and represses the promoter of cyp144, an uncharacterized cytochrome P450-encoding gene. Deletion of casRMsmreduces biofilm formation, consistent with the expected derepression of cyp144Msm, a gene that negatively regulates both biofilm and oxidative stress tolerance. EMSA and β-galactosidase activity assays also demonstrate that CasR negatively regulates antioxidant gene katGI, suggesting that CasR exerts a broader, global regulatory role within the mycobacterial antioxidant defense network. Furthermore, we identify isoleucine 18 as a critical residue for the DNA-binding and regulatory function of CasR. CONCLUSION This study establishes CasR as a pleiotropic transcriptional regulator that directly controls multiple antioxidant genes, including cyp144 and katGI, in mycobacteria. We report a previously unrecognized role for a cytochrome P450 family member in suppressing bacterial antioxidant capacity, as cyp144 overexpression reduces biofilm formation. These findings provide a valuable reference for further investigation into mycobacterial antioxidant mechanisms and identify CasR and Cyp144 as potential targets for the development of anti-tuberculosis drugs.

The oxidative stress regulator, PerR, is required for staphyloferrin B-mediated iron acquisition in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a globally prevalent gram-positive pathogen that can cause numerous types of infection. Due to host nutritional immunity and iron (Fe) sequestration, S. aureus experiences Fe limitation during infection. To overcome this, S. aureus expresses an arsenal of Fe acquisition systems whose expression is coordinated through the Fe-binding transcriptional regulator, Fur. Here, from a screen to identify S. aureus mutants defective for Fe-restricted growth, we identified several with mutations in perR, encoding a transcriptional regulator involved in resistance to oxidative stress. RNA-seq identified that the most downregulated genes in a perR mutant growing in Fe-restriction are those from the sbn operon that encodes staphyloferrin B biosynthesis. In agreement, perR mutants grew poorly in Fe-deficient media due to deficient staphyloferrin B production. In a subcutaneous model of S. aureus skin infection, S. aureus perR caused significantly smaller lesions, consistent with our finding that this mutant had decreased alpha-hemolysin expression during Fe-restricted growth. These findings are consistent with the hypothesis that PerR acts to fine-tune access to Fe ostensibly to avoid Fe-dependent toxicity. The importance of the PerR function to S. aureus was further highlighted by examination of over 8,000 human bloodstream isolates of S. aureus, showing that the PerR sequence was highly conserved. Together, these findings demonstrate the importance of PerR to S. aureus in providing an additional level of regulation of Fe homeostasis beyond Fur-dependent Fe sensing. IMPORTANCE Staphylococcus aureus is a notoriously antibiotic-resistant human pathogen and has the potential to cause a myriad of potentially life-threatening infections. Understanding the intricacies of S. aureus pathogenesis in the host will underpin the development of novel therapeutic approaches that target bacterial virulence mechanisms rather than essential cellular processes. The significance of our research lies in identifying how S. aureus regulates Fe acquisition in response to environmental signals, providing further insight into nutrient sensing and acquisition at the host–pathogen interface to facilitate the development of therapeutics that may target this process. Staphylococcus aureus is a notoriously antibiotic-resistant human pathogen and has the potential to cause a myriad of potentially life-threatening infections. Understanding the intricacies of S. aureus pathogenesis in the host will underpin the development of novel therapeutic approaches that target bacterial virulence mechanisms rather than essential cellular processes. The significance of our research lies in identifying how S. aureus regulates Fe acquisition in response to environmental signals, providing further insight into nutrient sensing and acquisition at the host–pathogen interface to facilitate the development of therapeutics that may target this process.

is required for an effective

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

Transcriptional analysis revealed that compared to neutral conditions, osmotic stress-related genes were significantly upregulated in the ΔliaS strain under acidic conditions, indicating that LiaS modulates acid tolerance through transcriptional regulation.

HsfA-driven gene transcription regulates fungal morphogenesis and pathogenicity

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.

Oxidative stress induces E. coli aryl polyene expression, sensitizing the bacterial stress response and modulating the redox environment of innate immune cells

ABSTRACT Aryl polyenes (APEs) are specialized polyunsaturated outer membrane lipids that protect their producers from oxidative stress and contribute to biofilm formation. APEs are produced by an abundant biosynthetic gene cluster (BGC) family conserved across Gram-negative bacterial clades. The APE biosynthesis pathway involves 11 different enzymes and culminates in the attachment of APEs to an anchor molecule in the Gram-negative outer membrane. Unlike most other small molecule BGCs, the APE BGC does not contain a dedicated regulatory gene that controls the production of its metabolically costly compounds. Building from our prior observations of APEs’ role in acute oxidative stress protection, we use a uropathogenic Escherichia coli (UPEC) strain to show that APE expression conveys a potential competitive advantage characterized by increased early-stage growth, sensitization of the bacterial oxidative stress response, and dampening of the redox stress of innate immune cells after in vitro infection. Our data indicate that APEs could act as a UPEC fitness factor, and in future work, we aim to study their contribution to overall bacterial pathogenicity and survival, as well as how APEs could facilitate the transition from an oxygen-poor environment, such as the gut, to the oxygen-rich environment of the urinary tract. IMPORTANCE Bacterial pathogens use various mechanisms to achieve a competitive advantage under harsh conditions, such as during interactions with their host. We studied the function of aryl polyenes (APEs), specialized polyunsaturated fatty acids in the outer membrane, in the context of a uropathogenic E. coli strain. APE expression is induced by an oxidative environment and contributes to early-stage growth and sensitization of the oxidative stress response. Furthermore, APE-expressing E. coli dampen the intracellular oxidative milieu of target host phagocytes. These findings suggest a role for APEs as a fitness factor and create opportunities to study their in vivo function and explore them as a potential drug target. Bacterial pathogens use various mechanisms to achieve a competitive advantage under harsh conditions, such as during interactions with their host. We studied the function of aryl polyenes (APEs), specialized polyunsaturated fatty acids in the outer membrane, in the context of a uropathogenic E. coli strain. APE expression is induced by an oxidative environment and contributes to early-stage growth and sensitization of the oxidative stress response. Furthermore, APE-expressing E. coli dampen the intracellular oxidative milieu of target host phagocytes. These findings suggest a role for APEs as a fitness factor and create opportunities to study their in vivo function and explore them as a potential drug target.