Small molecule can be utilized to restore the effectiveness of existing major classes of antibiotics against antibiotic-resistant bacteria. In this study, it is demonstrated that celastrol, a natural compound, can modify the bacterial cell wall and subsequently render bacteria more suceptible to β-lactam antibiotics. It is shown that celastrol leads to incomplete cell wall crosslinking by modulating levels of c-di-AMP, a secondary messenger, in methicillin-resistant Staphylococcus aureus (MRSA). This mechanism enables celastrol to act as a potentiator, effectively rendering MRSA susceptible to a range of penicillins and cephalosporins. Restoration of in vivo susceptibility of MRSA to methicillin is also demonstrated using a sepsis animal model by co-administering methicillin along with celastrol at a much lower amount than that of methicillin. The results suggest a novel approach for developing potentiators for major classes of antibiotics by exploring molecules that re-program metabolic pathways to reverse β-lactam-resistant strains to susceptible strains.

Infections caused by Staphylococcus aureus, notably methicillin-resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti-persister activity through mutual reinforcement of their membrane-disrupting activities. Crucially, the "triple" combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane-active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti-persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments.

Small molecule can be utilized to restore the effectiveness of existing major classes of antibiotics against antibiotic-resistant bacteria. In this study, it is demonstrated that celastrol, a natural compound, can modify the bacterial cell wall and subsequently render bacteria more suceptible to β-lactam antibiotics. It is shown that celastrol leads to incomplete cell wall crosslinking by modulating levels of c-di-AMP, a secondary messenger, in methicillin-resistant Staphylococcus aureus (MRSA). This mechanism enables celastrol to act as a potentiator, effectively rendering MRSA susceptible to a range of penicillins and cephalosporins. Restoration of in vivo susceptibility of MRSA to methicillin is also demonstrated using a sepsis animal model by co-administering methicillin along with celastrol at a much lower amount than that of methicillin. The results suggest a novel approach for developing potentiators for major classes of antibiotics by exploring molecules that re-program metabolic pathways to reverse β-lactam-resistant strains to susceptible strains.

Infections caused by Staphylococcus aureus, notably methicillin-resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti-persister activity through mutual reinforcement of their membrane-disrupting activities. Crucially, the "triple" combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane-active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti-persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments.

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1α (HIF-1α) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1α was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1α protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1α and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1α and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1α and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.

Bacterial persisters are dormant phenotypic variants that are tolerant to antibiotics, contributing to treatment failure and the emergence of antimicrobial resistance. Although the formation of persisters has been extensively studied in regards to bacterial infections and treatment, such as antibiotic exposure or intracellular survival within macrophages, the role of environmental stressors in persister formation remains largely unexplored. In this study, we investigate the role of environmental heavy metals, specifically arsenic (As), cadmium (Cd), and mercury (Hg), in promoting persister cell formation in Staphylococcus aureus and Escherichia coli. Log-phase cultures were exposed to heavy metals (5 mM As, 1.25 mM Cd, 4 µM Hg for S. aureus; 12.5 mM As, 2 mM Cd, and 15 µM Hg for E. coli) for 0.5 h to induce persister cells. We observed that exposure to these metals induced persister cell formation, confirmed by intracellular ATP levels through microscopy and luciferase assays, as well as by reactive oxygen species (ROS) levels using carboxy-H2DCFDA. Short-term heavy metal exposure strongly depleted intracellular ATP while generating ROS. Moreover, we observed enhanced expression of genes involved in the SOS response, including recA, umuC, dinB, rexA, rexB, sulA, rpoS, and soxR, as measured by qPCR. This response was likely induced by elevated ROS levels following heavy metal exposure. Furthermore, we demonstrate that heavy metal-induced bacterial persisters exhibited a substantially increased emergence of antibiotic resistance, as shown by ciprofloxacin resistance developing in the presence of heavy metals. Therefore, our results clearly demonstrate that heavy metals can induce persister cells by depleting cellular ATP and generating ROS, and these bacterial responses to heavy metals substantially contribute to antibiotic resistance. These findings highlight the intricate relationship between environmental heavy metals, bacterial persister formation, and antibiotic resistance, emphasizing the need for a "One Health" strategy to address the growing antibiotic resistance crisis.

Infections associated with bacterial persisters are challenging to cure because they can evade antibiotics and regrow, often resulting in relapse. Current antibiotics are not optimized to target persisters, highlighting the urgent need for new therapeutics. Here, we report that bakuchiol, a plant-derived natural product, exhibits anti-persister and adjuvant properties. Bakuchiol eradicates persisters formed by the gram-positive bacterium <i>Staphylococcus aureus</i> at 8 μg/mL and, in combination with 1 μg/mL colistin, completely eliminates persisters formed by the gram-negative bacterium <i>Acinetobacter baumannii</i>. Mechanistic analyses revealed that bakuchiol selectively disrupted bacterial membrane phospholipids while sparing mammalian membranes and exhibited low cytotoxicity. In <i>Acinetobacter baumannii</i> persisters, bakuchiol likely damages phospholipid patches in the outer membrane, causing nominal lethality but facilitating membrane permeabilization. This activity synergizes with colistin, which targets the lipooligosaccharide layer, resulting in the mutual reinforcement of their bactericidal effects. These findings highlight the potential of dual glycolipid-phospholipid targeting as a strategy to combat gram-negative persisters and highlight natural products as valuable sources for anti-persister therapeutics with membrane selectivity.

The rapid rise of methicillin-resistant Staphylococcus aureus (MRSA) and its ability to form antibiotic-tolerant persisters pose a major challenge to antimicrobial therapy. These metabolically dormant cells survive antibiotic exposure without genetic resistance, driving chronic and relapsing infections. Here, we characterize RS17053-a selective α1A-adrenoceptor antagonist- as a membrane-active compound with potent activity against both antibiotic-resistant and antibiotic-tolerant S. aureus. RS17053 disrupts bacterial phospholipid bilayers, causing membrane permeabilization, intracellular leakage, accumulation of reactive oxygen species, and subsequent cell death, while exhibiting minimal cytotoxicity toward mammalian cells. The compound shows no detectable resistance after prolonged exposure and synergistically enhances aminoglycoside potency by promoting drug uptake into MRSA persisters. In a Caenorhabditis elegans infection model, RS17053 protects hosts from lethal MRSA challenge. Collectively, these findings support RS17053 as an antimicrobial lead compound suitable for repurposing as both a direct-acting antimicrobial and an adjuvant to aminoglycosides for treating persistent MRSA infections.