Biology subjects

Chan, D. C. K.

Publications and source records attributed to Chan, D. C. K..

7 recordsLinked to original sources

bioRxiv2026

Vacidobactin A: An anti-Pseudomonas aeruginosa siderophore

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a significant clinical challenge due to its poorly permeable outer membrane, efflux systems, biofilm formation, and rapid acquisition of resistance genes. The lack of new treatments for P. aeruginosa infections underscores the necessity for innovative therapeutic solutions. Iron uptake is essential for bacterial survival, making it a promising target for the development of new antimicrobials. Iron-chelating siderophores are vital for bacterial iron acquisition, important agents for direct antimicrobial action, adjuvants to enhance the effectiveness of currently available antibiotics, and components of prodrugs that facilitate the transport of covalently linked antibiotics into the cell. Here, we report the anti-pseudomonal activity of vacidobactin A, a siderophore produced by the soil bacterium Variovorax paradoxus, identified through a screen of natural product extracts targeting a clinical MDR strain of P. aeruginosa. Vacidobactin A inhibits P. aeruginosa growth by limiting iron availability, particularly in strains that do not produce pyoverdine, their native siderophore. Expression of a TonB-dependent transporter sourced from the vacidobactin producer in a P. aeruginosa pyoverdine and pyochelin-null mutant restored its ability to acquire iron and grow in the presence of vacidobactin. Additionally, vacidobactin A synergized with thiostrepton, which hijacks pyoverdine receptors to enter the cell and inhibit protein synthesis. This study supports the therapeutic potential of targeting P. aeruginosa iron acquisition pathways and leveraging siderophores as adjuvants to enhance the efficacy of existing antimicrobials. These findings, along with recent advancements in siderophore-based research and combination therapies, offer innovative strategies to combat antibiotic-resistant infections. IMPORTANCEMultidrug-resistant Pseudomonas aeruginosa is a critical priority pathogen for which new therapeutic strategies are urgently needed. Iron acquisition is essential for P. aeruginosa survival and virulence, yet remains underexploited as a drug target. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. We further show that vacidobactin A enhances the activity of thiostrepton, an antibiotic that exploits siderophore uptake pathways. These findings highlight iron competition as a source of anti-pseudomonal agents and support the development of siderophore-based therapeutics and adjuvant strategies. Targeting iron acquisition networks offers a mechanistically distinct approach to combat antibiotic resistance and expands the repertoire of vulnerabilities that can be leveraged against this highly drug-resistant pathogen.

microbiology↗

bioRxiv2023

Nutrient Limitation Sensitizes Pseudomonas aeruginosa to Vancomycin

Traditional antibacterial screens rely on growing bacteria in nutrient-replete conditions which are not representative of the natural environment or sites of infection. Instead, screening in more physiologically relevant conditions may reveal novel activity for existing antibiotics. Here, we screened a panel of antibiotics reported to lack activity against the opportunistic Gram-negative bacterium, Pseudomonas aeruginosa, under low-nutrient conditions, and discovered that the glycopeptide vancomycin inhibited growth of P. aeruginosa at low micromolar concentrations through its canonical mechanism of action, disruption of peptidoglycan cross-linking. Spontaneous vancomycin-resistant mutants had activating mutations in the sensor kinase of the two-component CpxSR system, which induced cross-resistance to almost all classes of y-lactams, including the siderophore antibiotic cefiderocol. Other mutations that conferred vancomycin resistance mapped to WapR, an -1,3-rhamnosyltransferase involved in lipopolysaccharide core biosynthesis. A WapR P164T mutant had a modified LPS profile compared to wild type that was accompanied by increased susceptibility to select bacteriophages. We conclude that screening in nutrient-limited conditions can reveal novel activity for existing antibiotics and lead to discovery of new and impactful resistance mechanisms. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/536232v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1119ebeorg.highwire.dtl.DTLVardef@1ccd6c0org.highwire.dtl.DTLVardef@126f3deorg.highwire.dtl.DTLVardef@54a9c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

A single extracellular loop of FoxA controls ligand specificity, uptake, and signaling in Pseudomonas aeruginosa

The outer membrane of Gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic, thiocillin, exploits the nocardamine (ferrioxamine E) xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 [A] resolution the first crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which has implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa FpvB is a high-affinity transporter for xenosiderophores ferrichrome and ferrioxamine B

Iron is essential for many biological functions in bacteria but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer-membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intercellular targets - including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a common binding site in an aromatic pocket formed by the plug and barrel domains, but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach.

microbiology↗

bioRxiv2020

Thiocillin and Micrococcin Exploit the Ferrioxamine Receptor of Pseudomonas aeruginosa for Uptake

Thiopeptides are a class of Gram-positive antibiotics that inhibit protein synthesis. They have been underutilized as therapeutics due to solubility issues, poor bioavailability, and lack of activity against Gram-negative pathogens. We discovered recently that a member of this family, thiostrepton, has activity against Pseudomonas aeruginosa and Acinetobacter baumannii under iron-limiting conditions. Thiostrepton uses pyoverdine siderophore receptors to cross the outer membrane, and combining thiostrepton with an iron chelator yielded remarkable synergy, significantly reducing the minimal inhibitory concentration. These results led to the hypothesis that other thiopeptides could also inhibit growth by using siderophore receptors to gain access to the cell. Here, we screened six thiopeptides for synergy with the iron chelator deferasirox against P. aeruginosa and a mutant lacking the pyoverdine receptors FpvA and FpvB. Our findings suggest that thiopeptides such as thiocillin cross the outer membrane using FoxA, the ferrioxamine siderophore receptor. Other structurally related thiopeptides did not inhibit growth of P. aeruginosa, but had greater potency against methicillin-resistant Staphylococcus aureus than thiostrepton and related thiopeptides. These results suggest that thiopeptide structures have evolved with considerations for target affinity and entry into cells.

microbiology↗

bioRxiv2019

Forging new antibiotic combinations under iron-limiting conditions

Pseudomonas aeruginosa is a multidrug-resistant nosocomial pathogen. We showed previously that thiostrepton (TS), a gram-positive thiopeptide antibiotic, was imported via pyoverdine receptors and synergized with iron chelator deferasirox (DSX) to inhibit the growth of P. aeruginosa and Acinetobacter baumannii clinical isolates. A small number of P. aeruginosa and A. baumannii isolates were resistant to the combination, prompting us to search for other compounds that could synergize with TS against those strains. From literature surveys we selected 14 compounds reported to have iron-chelating activity, plus one iron analogue, and tested them for synergy with TS. Doxycycline (DOXY), ciclopirox olamine (CO), tropolone (TRO), clioquinol (CLI), and gallium nitrate (GN) synergized with TS. Individual compounds were bacteriostatic but the combinations were bactericidal. Our spectrophotometric data and chrome azurol S agar assay confirmed that the chelators potentate TS activity through iron sequestration rather than through their innate antimicrobial activities. A triple combination of TS + DSX + DOXY had the most potent activity against P. aeruginosa and A. baumannii isolates. One P. aeruginosa clinical isolate was resistant to the triple combination, but susceptible to a triple combination containing higher concentrations of CLI, CO, or DOXY. All A. baumannii isolates were susceptible to the triple combinations. Our data reveal a diverse set of compounds with dual activity as antibacterial agents and TS adjuvants, allowing combinations to be tailored for resistant clinical isolates.

microbiology↗

bioRxiv2019

Thiostrepton hijacks pyoverdine receptors to inhibit growth of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic, thiostrepton (TS) - considered inactive against Gram-negative bacteria - stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at sub-inhibitory concentrations, suggesting that TS was active against P. aeruginosa. Supporting this observation, TS inhibited growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. Expression of Tsr - a 23S rRNA-modifying methyltransferase - in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. Deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by FDA-approved iron chelators deferiprone and deferasirox. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. Our data show that the biofilm stimulation phenotype can reveal cryptic sub-inhibitory antibiotic activity, and that TS has activity against select multidrug resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

microbiology↗