ACS Infectious Diseases

Multidrug-resistant Staphylococcus aureus is a major global health threat, with the VraTSR three-component system playing a key role in sensing and conferring resistance to cell-wall active antibiotics, particularly vancomycin. VraTSR comprises the membrane histidine kinase VraS, the cytoplasmic response regulator VraR, and the uncharacterized membrane protein VraT, which regulate the cell wall stress stimulon. However, the molecular signals sensed by VraTSR remain unknown. To elucidate the activation mechanism of this regulatory system, we investigated interactions with {beta}-lactams and glycopeptides. Using a transcriptional reporter strain, we confirmed VraTSR activation by {beta}-lactams, glycopeptides, a vancomycin-derived photoprobe (VPP), and the previously unreported activators A47934 and moenomycin A. Photo-crosslinking assays with VPP and full-length VraS expressed in membranes revealed a direct interaction with vancomycin, which was further confirmed in purified VraS reconstituted in liposomes. VPP binding was concentration-dependent, saturable, and displaced by vancomycin. Saturation transfer difference (STD) Nuclear Magnetic Resonance (NMR) experiments confirmed vancomycin binding to VraS and demonstrated ampicillin interaction, highlighting the involvement of aryl protons from both antibiotics. These findings establish VraS as a receptor for vancomycin and ampicillin. In contrast, assays with membrane vesicles expressing only VraT or co-expressing VraS/VraT did not show covalent adduct formation between VraT and VPP. While VraTs exact role remains unclear, its participation in antibiotic sensing or signal transduction cannot yet be excluded. These results demonstrate that vancomycin and ampicillin directly activate VraS, providing critical insights into the activation of the cell wall stress stimulon and the mechanisms underlying antibiotic resistance. Disrupting VraTSR signaling is a promising strategy to combat multidrug resistance in S. aureus, and we provide invaluable in vitro platforms for identifying potential VraS inhibitors. Author SummaryMultidrug-resistant Staphylococcus aureus poses a major global health threat due to its resistance to cell-wall active antibiotics. Our study focuses on the VraTSR three-component system, a key regulator of the cell wall stress response in S. aureus, whose activation signals have remained unknown. We demonstrate that VraS, the membrane histidine kinase of the system, acts as a direct receptor for vancomycin and ampicillin--two structurally distinct antibiotics. These findings uncover the activation mechanism of VraTSR and position VraS as a central player in antibiotic sensing and resistance. By identifying VraS as a direct antibiotic receptor, we provide a promising target for developing inhibitors to disrupt VraTSR signaling and restore antibiotic efficacy. Additionally, the in vitro platforms we established enable the identification and testing of potential VraS inhibitors. This study highlights the importance of understanding bacterial stress-response pathways to combat antibiotic resistance, offering critical insights for developing new therapeutic strategies against multidrug-resistant S. aureus, a growing global health challenge.

Carbapenem resistant Klebsiella pneumoniae extreme drug resistance has warranted the use of colistin as a last-resort antibiotic but these isolates are now displaying increasing rates of colistin resistance. This is in large part due to the modifications made to the lipid A by the PhoPQ two component system. Host defense peptides (HDPs) are like colistin in that they are both positively charged and display amphipathic character, however are not impacted by lipid A modifications to the extent of colistin. To understand how HDPs can penetrate colistin resistant membranes we performed a deep mutational scanning analysis of protegrin-1 and revealed that amino acids mutations that resulted in alteration of peptide structure had more impact on antimicrobial activity than a reduction in charge. Probing single and double amino acid variants using membrane analysis and molecular modeling revealed the loss of antimicrobial activity correlated with decreased inner membrane leakage and pore modeling predicting decreased pore size,

Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobials mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpress to identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpress identified that all three analogues targeted the lipid IVA biosynthetic pathway in E. coli and Salmonella Typhimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues in E. coli when lpxD was overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in both E. coli and S. Typhimurium. LPS composition was a likely reason for differences between E. coli and S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpress has proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations. Data SummaryNucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13250. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Many commonly prescribed non-antibiotic medicines have off-target antimicrobial activity, yet their impact on antibiotic efficacy remains poorly understood. In this study, we investigated eight widely used UK prescription medicines and identified simvastatin, amlodipine, and fluoxetine as growth inhibitory towards methicillin-resistant Staphylococcus aureus (MRSA). These drugs disrupt bacterial membranes, with amlodipine and fluoxetine also triggering stress responses linked to cell wall and membrane damage. Further mechanistic analysis using transposon mutant screening revealed that simvastatin impairs cell wall synthesis by inhibiting the mevalonate pathway. Notably, checkerboard assays demonstrated antagonistic interactions: simvastatin reduced the efficacy of {beta}-lactams and vancomycin, amlodipine with vancomycin and daptomycin, and fluoxetine with vancomycin activity. Prolonged exposure to these drugs also accelerated resistance development to vancomycin and daptomycin. Together, these findings underscore the potential for commonly prescribed non-antibiotic medicines to undermine antibiotic therapy, warranting further study given the rising S. aureus treatment failures.

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected in standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and {beta}-lactamase assays) and the inner membrane (Escherichia coli {beta}-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analog library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

Neisseria gonorrhoeae is an obligate human pathogen and the etiological agent of the sexually transmitted infection, gonorrhoea. The rapid emergence of extensively antimicrobial-resistant strains, including those resistant to all frontline antibiotics, has led to N. gonorrhoeae being labelled a priority pathogen by the World Health Organization, highlighting the need for new antimicrobial treatments. Given its absence in humans, targeting de novo cysteine biosynthesis has been identified as a promising avenue for developing new antimicrobials against drug-resistant bacteria. The biosynthesis of cysteine is catalyzed by two enzymes; serine acetyltransferase (SAT/CysE) which catalyzes the first step and O-acetylserine sulfhydrylase (OASS/CysK) that catalyzes the second step incorporating sulfur to form L-cysteine. CysE is reported to be essential for bacterial survival in several bacterial pathogens including N. gonorrhoeae. Here, we have conducted virtual inhibitor screening of commercially available compound libraries against SAT from N. gonorrhoeae (NgSAT). We have identified a hit compound with an IC50 of 13.9 {micro}M and analyzed its interactions with the enzymes active site. This provides a platform for the identification and development of novel SAT inhibitors to combat drug-resistant bacterial pathogens.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has developed its own ways of evading defense mechanisms to counteract the lethal effects of reactive oxygen species (ROS) generated within the host macrophages during infection. The melH gene present in Mtb and Mycobacterium marinum (Mm) plays an important role to reduce ROS generated during infection. The melH gene encodes for an epoxide hydrolase. Bioinformatics data suggests that encoded enzyme utilizes lipid substrates for its function. Initially, we used a lipid fractionation approach coupled with liquid chromatography mass spectrometry (LC-MS) and treatment with active MelH enzyme to identify potential substrates for MelH. We found classes of mycolic acids, predominantly epoxy mycolic acids accumulate in the melH mutant and upon treatment with MelH are reduced in the lipid fraction. These results provide insight into how MelH encoded in the mel2 operon contributes to Mtb virulence and persistence and present further evidence for potential mechanisms of action if MelH is targeted for antitubercular drug discovery.

Nitroaromatic drugs are of critical importance for the treatment of trypanosome infections in Africa and the Americas. Fexinidazole recently joined benznidazole and nifurtimox in this family when it was approved as the first oral therapy against Human African trypanosomiasis (HAT). Nitroaromatic prodrugs are bioactivated by the trypanosome-specific type I nitroreductase (NTR) enzyme that renders the compounds trypanocidal. A caveat to the specificity of NTR activation is the potential for drug resistance and cross-resistance that can arise if NTR expression or functionality is altered through mutation. The outcomes of NTR bioactivation of nitroaromatic compounds is variable but can include the formation highly reactive open chain nitriles that can damage biomolecules including DNA. A proposed mechanism of action of nitroaromatic compounds is the formation of reactive oxygen species (ROS) resulting in the formation of trypanocidal levels of DNA damage. Fexinidazole made its way to clinical approval without a significant interrogation of its effects on trypanosome biology and a limited understanding of its mechanism of action. Early reports mentioned fexinidazole potentially affects DNA synthesis but without supporting data. In this study, we evaluated and compared the cytotoxic effects of nifurtimox, benznidazole, and fexinidazole on Trypanosoma brucei using in vitro analyses. Specifically, we sought to differentiate between the proposed effects of nitroaromatics on DNA damage and DNA synthesis. Toward this goal we generated a novel {gamma}H2A-based flow cytometry assay that reports DNA damage formation in conjunction with cell cycle progression. Here we report that fexinidazoles cytotoxic outcomes are distinct from the related drugs nifurtimox and benznidazole. Specifically, we show that fexinidazole treatment results in a pronounced defect in DNA synthesis that reduces the population of parasites in S phase. In contrast, treatment with nifurtimox and benznidazole appear accumulate DNA damage early in cell cycle and result in a defective G2 population. The findings presented here bring us closer to understanding the anti-trypanosomatid mechanisms of action of nitroaromatic compounds, which will promote improved drug design and help combat potential drug resistance in the future. Our findings also highlight DNA synthesis inhibition as a powerful anti-parasitic drug target.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 M. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the{Delta} lytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

Drug susceptibility profiles of NIAID Category A and B priority and emerging pathogens to standard of care drugs were assessed to determine susceptibility against clinical strains in addition to reference laboratory strains to establish a comparison resource for the performance of drug candidates, to define non-redundant minimal strain panels for individual species and the group of species for use in drug screening programs, and provide pharmacophore classification. Profiling of standard of care drugs against strains of each species revealed a broad spectrum of susceptibility among strains in each species with important differences between standard laboratory reference strains and strains sof clinical origin. Unbiased hierarchical clustering analyses of strain susceptibilities within each species group and strains from all the species identified subsets of non-redundant strains that are able to classify the susceptibility range for each species and able to classify all the species. This analysis established a reduced targeted set of Category A and B priority pathogen strains for testing the potency of drug candidates against each species and pan-species. This approach also discriminated pharmacophore classification for each species. This information can be applied to directed screening efforts to guide the selection of drug classes for derivatization and repurposing, and advance drug candidates with the greatest potential for efficacy against NIAID Category A and B priority and emerging pathogens.

MmpL3 is a promising new target for antitubercular drugs, but the microbiological properties of MmpL3 inhibitors are not fully understood. We compared the activity and mode of action of 11 structurally diverse compound series that target MmpL3. We confirmed activity was via MmpL3 using strains with differential expression of MmpL3. MmpL3 inhibitors had potent activity against replicating M. tuberculosis, with increased activity against intramacrophage bacilli and were rapidly bactericidal. MmpL3 inhibition induced cell wall stress concomitantly with a boost in ATP levels in M. tuberculosis. Mutation in MmpL3 conferred resistance to all series at different levels. Molecules did not negatively impact membrane potential, pH homeostasis or induce reactive oxygen species and were inactive against starved bacilli. Our study revealed common features related to the chemical inhibition of MmpL3, enabling the identification of off-target effects and highlighting the potential of such compounds as future drug candidates.

The global rise of antimicrobial resistance among the ESKAPE pathogens represents a major challenge to public health. Here, we report the broad-spectrum antibacterial activity of the synthetic antimicrobial and pore-forming peptide C14R against all six ESKAPE species. Using a radial diffusion assay and resazurin-based viability testing, C14R exhibited potent bactericidal effect with minimum inhibitory concentrations (MICs), defined as the lowest concentration of an antimicrobial agent that completely inhibits visible growth of planktonic microorganisms, ranging from 3.4 g/mL (Enterococcus faecium, vancomycin-resistant) to 45.2 g/mL (Klebsiella quasipneumoniae, ESBL). C14R also inhibited biofilm formation by Gram-positive pathogens, with minimum biofilm inhibitory concentrations (MBICs), referring to the minimal concentration required to prevent the development of biofilms, of 15.0 g/mL (Staphylococcus aureus, MRSA) and 22.0 g/mL (E. faecium, VRE), whereas Gram-negatives biofilms showed higher tolerance. Together, these findings demonstrate that C14R retains high activity against multidrug-resistant ESKAPE strains, highlighting its potential as a lead compound for the development of next-generation antimicrobial drugs to expand the portfolio of available antibiotics and brace health systems against emerging severe infections. Author summaryAntibiotic-resistant infections are a growing threat worldwide. A small group of hospital-associated bacteria is especially problematic because they often evade multiple drugs and cause hard-to-treat infections. In this study, we tested the designed antimicrobial peptide C14R as a novel and effective way to fight these bacteria. Peptides are short protein fragments with the ability to puncture and disrupt microbial membranes. We evaluated C14R against six hospital related priority species (so called ESKAPE pathogens) and measured its ability to stop growth and to limit biofilm formation. C14R killed every species we tested and reduced biofilm of two bacteria. Our findings identify C14R as a promising lead for new treatments, particularly for difficult infections and those involving biofilms.

Fluoroquinolone antibiotics, such as ciprofloxacin, are important broad-spectrum agents for a range of bacterial infections; however, fluoroquinolone usage is increasingly challenged by the emergence of resistance. Ciprofloxacin resistance mechanisms include mutations in the antibiotic target DNA gyrase, downregulation of porins required for bacterial cell penetration, and upregulation of efflux pumps to expel the antibiotic. However, new pathways driving bacterial tolerance to fluoroquinolones are still being discovered, suggesting that additional ciprofloxacin-binding proteins may exist in bacteria. In this study, we report the use of affinity-based protein profiling (AfBPP) with photo-crosslinking chemical probes to identify protein binding partners of ciprofloxacin in live E. coli cells. AfBPP identified novel ciprofloxacin binding proteins including YjdN and YbaA, whose molecular functions are as yet unannotated. Target engagement was validated using genetic knockout and biophysical binding assays, and key interactions identified in the ciprofloxacin binding site of YbaA. Collectively, this study demonstrates that additional and previously unreported biological interactions can exist for well-established antibiotics, and provides methodology to identify and interrogate these interactions in detail.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments into novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX(R), a broad-spectrum silver containing antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX(R) could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX(R) on several antibiotic classes. We found that the combination of AGXX(R) and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX(R)/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX(R)/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX(R)/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCEThe emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the silver containing antimicrobial AGXX(R) in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX(R) and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX(R) induces increased endogenous oxidative stress, membrane damage and iron sulfur cluster disruption. These findings emphasize AGXX(R)s potential as a route of antibiotic adjuvant development and shed light into potential targets to enhance aminoglycoside activity.

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacteriums complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.

Multidrug-resistant (MDR) Klebsiella pneumoniae, classified by the World Health Organization (WHO) as a critical priority pathogen, represents a global health thereat requiring novel antimicrobials urgently. Here we evaluated the in vitro antimicrobial activity of a novel iridium-based compound (OMKP-3), against MDR K. pneumoniae. OMKP-3 exhibited robust antimicrobial activity in M9 minimal media (MIC=6.25{micro}g/mL) and rapid bactericidal effect (MBC=12.5{micro}g/mL) against the tested MDR K. pneumoniae strains. OMKP-3 showed antibiofilm ability and was active against multiple MDR Gram-negative pathogens, including Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa and Serratia marcescens (MIC range:6.25-25{micro}g/mL). Importantly, OMKP-3 showed no cytotoxicity against mammalian cells after 24 hours of exposure. When combined with polymyxin B, OMKP-3 acted as an adjuvant, enhancing polymyxin B activity (FIC[≤]0.5). OMKP-3 was less prone to induce high-level resistance in MDR K. pneumoniae compared to ciprofloxacin, and supressed the growth of resistant bacteria at a low and non-cytotoxic concentration (4xMIC). K. pneumoniae strains harboring truncated Ompk35/36 porin genes exhibited higher OMKP-3 MICs, indicating that these porins may serve as an important entry pathway. Spectrometry analysis revealed that OMKP-3 was able to accumulate intracellularly (1.57{micro}g/mL), with minimal Resistance-Nodulation-Division (RND) efflux pump extrusion involvement. Furthermore, analysis of the resistant mutant, harboring a mutation in the outer membrane protein DegS, together with fluorescence microscopy, suggests that OMKP-3 induces membrane-associated damage. No cross-resistance between OMKP-3 and commonly used antibiotics was observed. Collectively, these findings identify OMKP-3 as a promising novel antimicrobial agent against MDR K. pneumoniae, likely acting through an unexplored bacterial target. ImportanceMultidrug-resistant (MDR) Klebsiella pneumoniae is a critical global health threat and is among the leading causes of hospital0hyphenorendash;associated mortality, largely due to the scarcity of effective therapeutic options. Alarmingly, the current antimicrobial pipeline fails to address this issue, relying largely on derivatives of existing scaffolds that offer only short-term clinical benefit due to rapid resistance emergence. Developing antibiotics against Gram-negative pathogens is particularly challenging because of their highly impermeable outer membrane and efficient efflux systems, limiting intracellular drug accumulation. Metal-based antimicrobials emerge as a promising alternative. Our findings showed that OMKP-3, an iridium complex, exhibits potent bactericidal activity against MDR K. pneumoniae without selecting for high-level resistance, suggesting the potential for sustained therapeutic efficacy. Additionally, it demonstrated to accumulate intracellularly with minimal efflux involvement. Together, these features position OMKP-3 as a valuable and underexplored novel antimicrobial strategy for addressing the escalating threat of MDR K. pneumoniae infections.

Antibiotic resistance is a critical public health issue, causing resistant bacterial strains to be increasingly difficult to control. Antivirulence therapies, which target bacterial virulence factors rather than kill bacteria, present a promising approach. Sortase enzymes, particularly SrtA, are crucial for Gram-positive bacterial virulence by anchoring surface proteins essential for bacterial adhesion and biofilm formation to the bacterial outer cell wall. This study evaluates the selectivity of the peptidomimetic inhibitor BzLPRDSar towards various Gram-positive bacteria. The BzLPRDSar significantly inhibited biofilm formation in multidrug-resistant S. aureus and S. epidermidis. Conversely, it showed variable and generally lower selectivity to Gram-positive species such as E. faecalis, B. cereus and S. agalactiae. The selectivity towards Staphylococcus species is attributed to conserved structural elements in the SrtA enzyme, particularly the {beta}7/{beta}8 loop region with a key tryptophan, likely facilitating strong binding interactions with the inhibitor. ImportanceThis study addresses the pressing issue of antibiotic resistance by exploring antivirulence therapy as an innovative alternative to conventional antibiotics, focusing on inhibiting bacterial virulence rather than bacterial growth. By evaluating the selectivity of the peptidomimetic inhibitor BzLPRDSar against various Gram-positive bacteria, the study highlights its potent selectivity in inhibiting biofilm formation in multidrug-resistant S. aureus and S. epidermidis. The findings underscore the potential of targeting conserved structural elements in bacterial Sortase enzymes, particularly in Staphylococcal species, to develop more selective and effective antivirulence therapies.

Although receptor-mediated mechanisms account for the therapeutic action of numerous FDA-approved drugs, emerging evidence suggests that many of these therapeutics have off-target antimicrobial activities. One example is fingolimod, an immunomodulator used to treat multiple sclerosis, that has been reported to have antimicrobial effects associated with membrane permeabilization. Yet the molecular mechanism by which fingolimod alters bacterial membranes remains unknown. As a cationic amphiphilic drug (CAD), fingolimod is comprised of both hydrophobic and positively charged regions that can enable membrane interactions. We show that fingolimod compromises membrane integrity in E. coli and P. aeruginosa, contributing to its antimicrobial activity. To determine how fingolimod disrupts membrane integrity, we used planar lipid bilayer electrophysiology with phospholipid compositions mimicking E. coli membranes. Using gramicidin A channels as molecular biosensors, we show that fingolimod alters both mechanical properties and surface charge of lipid bilayers at concentrations that have antimicrobial effects. At higher concentrations, fingolimod directly permeabilizes lipid bilayers, as revealed by conductance measurements and Bilayer Overtone Analysis. Molecular dynamics simulations correlate fingolimods preference for pore-favoring curvature with its strong interactions with lipids and trans-leaflet translocation. These findings establish a molecular mechanism for fingolimods off-target activity and provide a starting point for understanding how some CAD structures can drive membrane-specific effects that compromise bacterial physiology. ImportanceMany commonly prescribed drugs, beyond their primary action via receptor targets, modify cell membranes. A mechanistic understanding of how these drugs interact with bacterial membranes will have a significant impact on drug design and on the evaluation of potential side effects. Furthermore, the emerging need for new antimicrobial drugs has led to increased interest in drug repurposing. Elucidating the molecular mechanisms of these compounds interactions with bacterial membranes can ultimately provide critical insights into redesigning existing drugs as antimicrobials and into identifying unintended membrane-related effects that may contribute to their therapeutic or off-target effects.