Histidinol dehydrogenase (HisD): a critical regulator of Staphylococcus aureus virulence and a promising target for antivirulence therapy.

UNLABELLED: Staphylococcus aureus is a pathogen responsible for diverse severe infections. The global spread of methicillin-resistant S. aureus (MRSA) necessitates innovative therapeutic approaches beyond traditional antibiotics. S. aureus virulence mechanisms remain a critical concern. Targeting histidinol dehydrogenase (HisD), a key enzyme in histidine biosynthesis, presents a novel anti-virulence strategy. We used the derivative strains of Newman strains (ΔhisD, ΔhisD::pRAB-hisD, and WT::pRAB-hisD) and clinical strains to study the role of HisD in the pathogenicity of S. aureus. HisD inhibition by pixantrone was further evaluated. The absence of hisD significantly reduced hemolytic activity and biofilm formation, accompanied by the downregulation of virulence genes (hla, coa, hlgA-C, lukE/S/F, and NWMN_1873) and the saeR/S two-component system (P < 0.05). The expression of biofilm-inhibiting proteases was elevated, notably Aur and ScpA. Murine challenge revealed that ΔhisD exhibited 5.6-fold higher LD50 (4.17 × 109 CFU/mL vs. WT 7.42 × 108 CFU/mL) and reduced organ colonization (P < 0.05). Through structure-based virtual screening and SPR affinity verification, we discovered pixantrone-a nitrogenated anthraquinone-as a potent HisD inhibitor binding via four hydrogen bonds and two salt bridges. Pixantrone dose-dependently (25-200 μM) suppressed virulence phenotypes in vitro, achieving hemolysis inhibition, virulence gene inhibition, and biofilm reduction. In vivo, pixantrone (30 mg/kg) decreased serum CRP, IL-6, and TNF-α levels while diminishing abscess sizes and splenic bacterial loads (P < 0.05 vs. untreated). These findings establish HisD as a pivotal virulence regulator in S. aureus through saeR/S-mediated pathways. Pixantrone demonstrates potent anti-virulence efficacy, positioning HisD inhibition as a promising therapeutic strategy against S. aureus infections. This study provides foundational insights for developing HisD-targeted agents to combat antibiotic-resistant staphylococcal pathogens.

IMPORTANCE: The increase in drug-resistant Staphylococcus aureus (MRSA) demands therapies that block virulence without promoting resistance. We identify histidinol dehydrogenase (HisD), a histidine-synthesis enzyme, as a key controller of S. aureus pathogenicity. Disrupting HisD genetically or with pixantrone-a newly identified inhibitor-reduces bacterial toxicity, biofilm formation, and virulence gene activity while improving survival and reducing organ damage in infected mice. Pixantrone's dose-dependent suppression of infection severity and inflammation positions it as a therapeutic candidate. Unlike traditional antibiotics, this strategy disarms bacteria rather than killing them, reducing resistance risks. By uncovering HisD's role in connecting metabolism to virulence through the saeR/S system, we reveal a druggable target for fighting multidrug-resistant infections. This work addresses the urgent need for innovative solutions to the global antibiotic resistance crisis, paving the way for therapies that outsmart evolving superbugs.