The SaeRS two-component system likely mediates the major dopamine-dependent regulation of virulence in Staphylococcus aureus ATCC 25,923

Investigating a TREM2-TLR2 axis mediating Staphylococcus aureus infection 2329158

Staphylococcus aureus can express a large arsenal of virulence factors, readily acquire antibiotic resistance, and survive harsh environmental conditions through biofilm formation. These traits facilitate evasion of host immunity and antibiotic killing, leading to chronic infections. A better understanding of pathogenicity and immune evasion are critical for more effective treatments. In a mouse model of S. aureus biofilm infection, we uncovered a critical role for triggering receptor expressed on myeloid cells-2 (TREM2) in exacerbating infection severity via anti-inflammatory responses to pathogen (PAMPs) and damage-associated molecular patterns (DAMPs). Interestingly, the role for TREM2 was specific to females, suggesting a role for estrogens. Further studies with bone marrow-derived macrophages showed that TREM2 loss enhanced surface expression of Toll-like receptor 2 (TLR2), a key immune receptor that senses bacterial lipoprotein and lipoteichoic acid for proinflammatory activation. Cross-regulation between TREM2, TLR2, and estrogens in the context of infection has not been described. Mouse background (8-10 week old C57BL/6) and S. aureus (USA300 LAC-13C) strains were identical for all studies. Craniotomy infection was induced by inoculating bone flaps with 103 CFU. In vitro studies used primary bone marrow-derived macrophages. Flow cytometry (surface markers, metabolism, ROS/RNS), Cytometric Bead Array (mediator production), and Western blot (protein quantification) were also used. Macrophages with individual knockouts of TREM2 and TLR2, and a double knockout of TREM2/TLR2, showed cross-regulatory effects from these receptors and 17β-estradiol on inflammatory surface markers, mediator production, metabolism, and reactive oxygen/nitrogen species production. TREM2, TLR2, and estrogen appear to have an interconnected role in driving immune responses, which could be exploited for moderating pro- vs anti-inflammatory immune activation during S. aureus infection. Preliminary work was supported by NIH F32NS126302 to Lee Korshoj and R01NS107369 and R01AI169788 to Tammy Kielian. Current work is supported by startup funds from the University of Nebraska Medical Center to Lee Korshoj. Microbial, Parasitic, and Fungal Immunology (MPF)

Two-Component System as Drug Target: Is It a Bandwagon or a Breakthrough?

MDR ESKAPE pathogens are the leading cause of hospital-acquired infections (HAIs) that resist most antibiotics and form biofilms. Biofilm formation is dependent on the two-component system (TCS), which regulates virulence traits including adhesion to host tissues, evasion of innate immunity, the synthesis of exopolysaccharides, and antibiotic resistance. TCS sense environmental stimuli such as pH, osmotic pressure and antimicrobial peptides. They regulate gene expression to promote bacterial survival and persistence during infection. TCS represents a contemporary and novel pathway for the advancement of targeted, adjuvant-based options to fend off infections caused by ESKAPE pathogens. Targeting TCS may improve drug penetration and reduce resistance pressure, although challenges remain due to their structural conservation, regulatory complexity, and mutation-driven resistance. However, there are several limitations in targeting TCS for drug development like TCS not always being essential for bacterial viability, structural homologies between TCS are not sufficient for broad-spectrum inhibitors, sometimes TCS can be involved in complex and essential regulatory networks and can evolve resistance mechanisms by mutations. To further investigate this paradox, we implemented comprehensive computational and phylogenetic analyses of selected ESKAPE TCS. The findings from this investigation form the basis for our in-depth review of the significance and challenges of TCS as emerging drug targets.

Staphylococcus aureus biofilms: molecular mechanisms, resistance determinants, and emerging therapeutic strategies

The alternative sigma factor SigH modulates biofilm formation and stress tolerance in a raw milk-derived Staphylococcus aureus.

Staphylococcus aureus is an important raw milk contaminant that can persist in dairy environments through biofilm formation and adaptation to environmental stresses. Although the alternative sigma factor SigH has been described in S. aureus, its role in raw milk-associated strains remains poorly understood. In this study, we investigated the function of SigH in the raw milk-derived S. aureus strain RMSA24 in biofilm formation, stress tolerance and antibiotic susceptibility. Deletion of sigH did not affect bacterial growth under routine culture conditions but significantly reduced biofilm formation. In contrast, the sigH mutant exhibited enhanced tolerance to osmotic, acid, and heat stresses. Loss of sigH also reduced susceptibility to the glycopeptide antibiotics vancomycin and teicoplanin and was accompanied by pronounced cell wall thickening. Transcriptomic analysis further supported these phenotypes by revealing differential expression of genes associated with biofilm formation, stress tolerance, and cell wall homeostasis. Overall, these findings indicate that SigH contributes to the regulation of persistence-associated phenotypes in a raw milk-derived S. aureus strain and provide new insights into the regulatory mechanisms that may influence survival of this pathogen in dairy-related environments.

Disruption of ytrE attenuates methicillin-resistant Staphylococcus aureus virulence and compromises membrane integrity and oxidative stress resistance

6‐Aminoflavone Inhibits Planktonic Growth, Disrupts Biofilm Architecture, and Regulates Quorum Sensing in Klebsiella pneumoniae

Antibiotic resistance has emerged as a major global health challenge, particularly in biofilm‐forming pathogens that exhibit enhanced tolerance to antimicrobial therapies. K. pneumoniae, a multidrug‐resistant pathogen, is a leading cause of hospital‐acquired infections, including pneumonia, septicemia, and device‐associated infections. Its robust biofilm‐forming capacity facilitates immune evasion, restricts antibiotic penetration, and contributes to recurrent infections. Therefore, the identification of agents capable of targeting both planktonic bacterial growth and biofilm architecture is of considerable therapeutic importance. In the present study, the antibacterial, antibiofilm, and antivirulence potential of 6‐Aminoflavone was investigated against K. pneumoniae. Antibacterial activity was evaluated using growth inhibition assays, time–kill kinetics, and clonogenic survival analysis. Mechanistic investigations revealed significant intracellular ROS accumulation, increased oxidative stress susceptibility, and induction of apoptosis‐like cell death, suggesting ROS‐mediated antibacterial activity. In addition to suppressing bacterial proliferation, 6‐Aminoflavone exhibited promising antibiofilm efficacy, inhibiting biofilm formation by 86.23% and eradicating 84.77% of established biofilms. These effects were associated with a substantial reduction in cell surface hydrophobicity (∼45.39%), and EPS levels were reduced to ∼2.33% as compared to untreated controls, indicating severe destabilization of the biofilm matrix. Furthermore, eDNA, a key structural component of the biofilm scaffold, exhibited a maximum ∼2.13‐fold reduction following treatment. Confocal microscopy confirmed marked disruption and collapse of biofilm architecture. Additionally, quorum sensing (QS)–regulated virulence factors, including urease (∼51.92%), protease (∼55.18%), and lipase (∼44.90%), were significantly attenuated. These findings demonstrate that 6‐Aminoflavone exerts antimicrobial activity by inducing oxidative stress–mediated apoptosis‐like bacterial death, disrupting biofilm structural components, and suppressing QS‐regulated virulence in K. pneumoniae.