Biology subjects

Velkov, T.

Publications and source records attributed to Velkov, T..

8 recordsLinked to original sources

bioRxiv2026

The structure-interaction model of polymyxin lipopeptides with human oligopeptide transporter 2

Multidrug-resistant (MDR) Gram-negative bacteria pose a critical global health threat, while polymyxins remain a last-line therapy. However, their clinical use is limited by nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal cells and contributes to their nephrotoxicity, but the molecular basis of their interaction remains unclear. Here, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical, and cell biology approaches. Bioinformatic modelling predicted an outward-facing hPepT2 structure and a potential transport pathway, with polymyxins interacting at the lateral opening, particularly E214, D215, D317, D342, and E622. Transporter mutagenesis and molecular analyses confirmed that D215 is critical for polymyxin binding, while other residues influence transporter turnover and/or expression. We subsequently synthesised polymyxin analogues with modifications at Dab1, Dab3, Dab5, and Dab9 of polymyxins, which reduced interactions with hPepT2. Notably, alanine substitution at Dab3 reduced nephrotoxicity in mice while retaining antibacterial activity. Overall, this proof-of-concept study demonstrates that the hPepT2-polymyxin SIR model provides a viable strategy for developing novel, safer lipopeptide antibiotics.

biochemistry↗

bioRxiv2025

Bioactive Cationic Lipidated Oligomers (CLOs) as Antimicrobial Materials: Metabolomic Insights into MRSA Membrane Disruption

The escalating incidence of antimicrobial resistance in Staphylococcus aureus, particularly the methicillin-resistant strain (MRSA), necessitates the development of novel therapeutic strategies. Cationic lipidated oligomers (CLOs) have emerged as promising membrane-active antimicrobial agents; however, their mechanisms of action remain insufficiently understood. In this study, untargeted metabolomics was employed to systematically profile the temporal metabolic perturbations induced by two structurally distinct CLOs, C12-o-DMEN-10 and C12-o-BEDA-10, in MRSA across four defined time points (0.25, 0.5, 1, and 3 hours). These CLOs previously demonstrated differential antibacterial activity, as evidenced by dose-dependent propidium iodide (PI) uptake and growth inhibition assays. Metabolomic analysis revealed pronounced and sustained disruptions in bacterial membrane lipid metabolism, including significant depletion of phosphatidylglycerols ([≥] -2.5 log2FC, p < 0.05), alongside elevated levels of phosphatidylethanolamines, lysophospholipids, and fatty acid-derived metabolites indicative of membrane destabilization and lipid remodelling. Although both CLOs affected overlapping metabolic pathways, they differed in the extent and temporal dynamics of their effects. These findings provide mechanistic insights into CLO-mediated antibacterial activity and highlight the value of metabolomics in elucidating both direct and downstream cellular responses, which extend beyond the scope of conventional membrane integrity assays, such as PI fluorescence.

systems biology↗

bioRxiv2025

Characterisation of SpoT-disruption induced metabolic shift in bacteria

SpoT, a pivotal stringent response regulatory enzyme, serves a dual function of both synthesizing and hydrolyzing the stress alarmones ppGpp and pppGpp, collectively known as (p)ppGpp. These alarmones act as global regulators, governing bacterial metabolic and physiological adaptation to diverse environmental stresses. Our previous investigation revealed that multidrug-resistant Acinetobacter baumannii notably upregulates SpoT in response to polymyxin treatment. Intriguingly, disrupting spoT gene enhances polymyxin killing. To comprehend SpoT metabolic regulatory function in mediating polymyxin tolerance, we conducted untargeted metabolomics, comparing metabolic perturbations upon polymyxin B (PMB) treatment between a spoT-disrupted A. baumannii mutant and its wild-type counterpart. Depletion of guanine-based purines GTP and GDP in the PMB-treated spoT-disrupted mutant suggests impaired (p)ppGpp hydrolysis, potentially leading to lethal accumulation of these molecules. PMB also induced more pronounced depletion of carbon sources (i.e., phosphoenolpyruvate, succinate, coenzyme A), energy metabolites (i.e., NADH biosynthesis pathway, ADP), amino acids, and the antioxidative system (i.e., glutathione, gamma-L-glutamyl-L-cysteine, (R)-S-lactoylglutathione) in the spoT-disrupted mutant. Interestingly, a distinctive time-dependent perturbation of fatty acyls was also observed following polymyxin treatment, with multiple fatty acyl conjugates significantly elevated in the spoT-disrupted mutant at 1 hour. However, this situation reversed at 4 hours, with more elevated fatty acyl groups in the wild-type compared to the spoT-disrupted mutant, indicating greater and more rapid PMB-induced membrane disruption in the spoT-disrupted mutant. Collectively, our findings suggest a potential role for SpoT in intricately coordinating A. baumannii energy expenditure and metabolite repertoire to ensure optimal functionality in stress tolerance and repair machineries (e.g., glutathione system, fatty acid regulation) following polymyxin treatment. IMPORTANCEPolymyxins, the last-resort antibiotics for multidrug-resistant Gram-negative bacteria, face increasing challenges due to growing tolerance. To tackle Acinetobacter baumannii polymyxin tolerance, we turned to SpoT, a (p)ppGpp synthetase/hydrolase protein. Our study focused on spoT-disrupted mutant, which exhibited heightened polymyxin antibacterial killing, yet the underlying mechanisms remained elusive. Through metabolomics, we unraveled key insights. Enhanced polymyxin killing in spoT-disrupted mutant likely resulted from lethal accumulation of stress alarmones (p)ppGpp, perturbations in carbon and energy metabolism, thiol-based antioxidant system depletion, and disrupted lipidic membrane repair. These findings illuminate the intricate metabolic signaling networks driving polymyxin tolerance in A. baumannii. By untangling pathogen-directed stress tolerance mechanisms, we deepen our understanding and identify potential therapeutic targets. Our discoveries offer new avenues to combat polymyxin resistance, paving the way for more effective treatments against superbugs. Through continued exploration, we can harness this knowledge to develop innovative strategies and overcome the challenges posed by polymyxin tolerance.

microbiology↗

bioRxiv2025

Single-cell transcriptomic dynamics exposes hidden survival trajectory under antibiotic treatment

Heterogeneity plays a major role in bacterial resistance to antibiotic treatment while the mechanism at single-cell level is largely unknown. Here, we employed a robust integration of bulk and bacterial single-cell RNA-seq (scRNA-seq) to uncover how individual cells of Acinetobacter baumannii reorganise the heterogenous transcriptome in response to antibiotic. Using polymyxin as a representative, bulk RNA-seq showed canonical envelope- and efflux-centred responses but obscured underlying heterogeneity. Single-cell profiling resolved these averages into discrete subpopulations whose abundances shifted with concentration and time. Specifically, in early time an envelope-stress programme predominated survival at the low concentration, whereas an outer-membrane repair/efflux programme dominated survival at the high concentration. These patterns revealed structured and time-resolved heterogeneity, highlighting an ingenious bacterial stress responsive strategy. Through trajectory inference, we further revealed a concentration-dependent bifurcation of cell fates: low-concentration treated cells detoured through a transient stress state and rejoined growth, whereas high-concentration treated survivors diverted into a slow-growing, tolerant branch. Perturbing marker genes from these programmes altered fitness eventually and rapidly increased permeability, depolarisation and reactive-oxygen burden, linking state to survival capability. Collectively, we map a dynamic, concentration-structured landscape of antibiotic responses at the single-cell level, revealing diverse survival trajectories that are obscured in conventional population-averaged analyses. Our developed single-cell based framework revealed tolerant bacterial cells with rewiring of the transcriptional landscape, causing emergence of antibiotic resistance. Importantly, these findings urge precision antimicrobial therapy in patients to minimise emergence of antibiotic resistance.

microbiology↗

bioRxiv2024

Providing insight into the mechanism of action of Cationic Lipidated Oligomers (CLOs) using metabolomics

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides, and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3 and 6 h following treatment with C12-o-(BG-D)-10 (48 {micro}g/mL i.e., 3x MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step towards its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points i.e., 1, and 3 h. Central carbon metabolism, and biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways. ImportanceAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

microbiology↗

bioRxiv2024

Transcriptomic interplay between Acinetobacter baumannii, human macrophage and polymyxin

Optimization of antibiotic therapy has been hindered by our dearth of understanding on the mechanism of the host-pathogen-drug interactions. Here, we employed dual RNA-sequencing to examine transcriptomic perturbations in response to polymyxin B in a co-culture infection model of Acinetobacter baumannii and human macrophages. Our findings revealed that polymyxin B treatment induced significant transcriptomic response in macrophage-interacting A. baumannii, exacerbating bacterial oxidative stress, disrupting metal homeostasis, affecting osmoadaptation, triggering stringent stress response, and influencing pathogenic factors. Moreover, infected macrophages adapt heme catabolism, coagulation cascade, and hypoxia-inducible signaling to confront bacterial invasion. Disrupting rcnB, ompW, and traR/dksA genes in A. baumannii impairs metal homeostasis, osmotic stress defense and stringent responses, thereby enhancing antibacterial killing by polymyxin. These findings shed light on the global stress adaptations at the network level during host-pathogen-drug interactions, revealing promising therapeutic targets for further investigation. IMPORTANCEIn the context of the development of bacterial resistance during the course of antibiotic therapy, the role of macrophages in shaping bacterial response to antibiotic killing remains enigmatic. Herein we employed dual RNA-sequencing and an in vitro tripartite model to delve into the unexplored transcriptional networks of the Acinetobacter baumannii-macrophage-polymyxin axis. Our findings uncovered the potential synergy between macrophages and polymyxin B which appear to act in co-operation to disrupt multiple stress tolerance mechanisms in A. baumannii. Notably, we discovered the critical roles of bacterial nickel/cobalt homeostasis (rcnB family), osmotic stress defense (ompW family), and stringent response regulator (traR/dksA C4-type zinc finger) in tolerating the last-line antibiotic polymyxin B. Our findings may lead to potential targets for the development of novel therapeutics against the problematic pathogen A. baumannii.

microbiology↗

bioRxiv2022

Diversified repertoire of phage defenses in Klebsiella pneumoniae: Bi-directional steering effects impacting antibiotic susceptibility

Bacteriophage (phage) therapy is rising as a promising anti-infective option to combat antimicrobial resistance; however, its clinical utilization is severely hindered by the potential emergence of phage resistance. Fortunately, certain phage resistance mechanisms can restore bacterial antibiotic susceptibility, making the combination of phages with antibiotics a potential strategic approach. Here, we demonstrated that phage resistance can also lead to increased antibiotic resistance and provided mechanistic insights into bacterial phage defense mechanisms. We discovered a repertoire of phage resistance mechanisms in Klebsiella pneumoniae, including the disruption of phage binding site (fhuA::Tn and tonB::Tn), extension of phage latent period (mnmE::Tn and rpoN::Tn) and increased mutation frequency (mutS::Tn and mutL::Tn). Different from the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we revealed a bidirectional steering effect on the bacterial antibiotic susceptibility. Specifically, it was uncovered that, while rpoN::Tn became more susceptible to colistin, mutS::Tn and mutL::Tn caused increased resistance to rifampicin and colistin. Our findings highlight the diversified strategies utilized by K. pneumoniae to overcome phage infection and the parallel effect on the antibiotic susceptibility. Mechanism-guided phage steering represents a rational strategy that should be incorporated into phage therapy to better inform clinical decisions.

microbiology↗

bioRxiv2020

Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

developmental biology↗