Browsing by Author "Velkov, T"

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    Arginine catabolism is essential to polymyxin dependence in Acinetobacter baumannii
    (Elsevier, 2024-07) Han, ML; Alsaadi, Y; Zhao, JX; Zhu, Y; Lu, J; Jiang, X; Ma, W; Patil, NA; Dunstan, RA; Le Brun, AP; Wickremasinghe, H; Hu, X; Wu, Y; Yu, HH; Wang, J; Barlow, CK; Bergen, PJ; Shen, HH; Lithgow, T; Creek, DJ; Velkov, T; Li, J
    Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii. ª 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC licence
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    An in vitro model to investigate the interactions between antimicrobial peptides and the outer membrane of gram-negative pathogens
    (Australian Institute of Nuclear Science and Engineering, 2016-11-29) Han, ML; Shen, HH; Zhu, Y; Le Brun, AP; Holt, SA; Roberts, K; Song, JN; Cooper, MA; Moskowitz, SM; Velkov, T; Li, J
    Increasing antibiotic resistance in Gram-negative bacteria led to polymyxins as the last therapy. Polymyxins present their antimicrobial activity through an initial electronical interaction with lipid A in the outer membrane (OM) of GNB, and the most common mechanism of polymyxin resistance is through modifications of lipid A with positively charged groups, such as 4-amino-L-arabinose (L-Aar4N) or phosphoethanolamine (pEtN). However, it is notable that Gram-negative bacteria employ a combination of charge-charge repulsion mechanism and the modification to fatty acyl chains of lipid A to obtain high-level polymyxin resistance. Hence, we designed hydrophobic polymyxin-related lipopeptides in order to overcome modified lipid A to insert into the outer membrane of Gram-negative bacteria. In this study, we employed neutron reflectometry (NR) study to investigate the interactions between lipid A and polymyxins. Lipid A was extracted from polymyxin-susceptible and -resistant pseudomonas aeruginosa strains, and analysed using ESI-MS in the negative ion mode. The asymmetric lipid A: deuterated DPPC bilayers were deposited on SiO2 surfaces by combined Langmuir-Blodgett and Langmuir-Schaefer disposition methods, and characterised by neutron reflectometer. Our results showed L-Ara4N modified lipid A was observed in polymyxin-resistant PAKpmrB6 strain, but not in the wild-type PAK strain. The NR data obtained from unmodified lipid A: DPPC bilayer was fitted into a five-layer model. Whereas, a six-layer model containing an extra outer headgroup was established for L-Ara4N modified lipid A: d-DPPC bilayer. Our results showed a dense of PMB (volume fraction of >20%) bound to the surface of both unmodified and modified lipid A: DPPC bilayers. While it is notable that the significant changes in NR profiles obtained from H2O contrast indicated about 15.8% and 6.1% of PMB penetrated into the wild-type lipid A headgroup and fatty acyl chains, respectively, but without penetration into L-Ara4N-lipid A: d-DPPC bilayer. However, the employment of octpeptin A3 induced higher hydrophobic interactions with L-Ara4N-lipid A: d-DPPC bilayer. Our study provides an in vitro model to investigate the interactions of polymyxins with OM bilayers in GNB, and confirmed that lipid A modification with L-Ara4N was certainly to reduce the penetration of PMB into bacterial membranes. Remarkably, the higher binding affinity between octapeptin A3 and L-Ara4N modified lipid A indicated its potential to be the new generation antibiotics for the therapy of infections caused by multi-drug resistant Gram negative bacteria.
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    Polymyxin-induced lipid A deacylation in pseudomonas aeruginosa perturbs polymyxin penetration and confers high-level resistance
    (ACS Publications, 2017-11-28) Han, ML; Velkov, T; Zhu, Y; Roberts, KD; Le Brun, AP; Chow, SH; Gutu, AD; Moskowitz, SM; Shen, HH; Li, J
    Polymyxins are last-line antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is increasingly reported, leaving a total lack of therapies. Using lipidomics and transcriptomics, we discovered that polymyxin B induced lipid A deacylation viapagL in both polymyxin-resistant and -susceptible Pseudomonas aeruginosa. Our results demonstrated that the deacylation of lipid A is an “innate immunity” response to polymyxins and a key compensatory mechanism to the aminoarabinose modification to confer high-level polymyxin resistance in P. aeruginosa. Furthermore, cutting-edge neutron reflectometry studies revealed that an assembled outer membrane (OM) with the less hydrophobic penta-acylated lipid A decreased polymyxin B penetration, compared to the hexa-acylated form. Polymyxin analogues with enhanced hydrophobicity displayed superior penetration into the tail regions of the penta-acylated lipid A OM. Our findings reveal a previously undiscovered mechanism of polymyxin resistance, wherein polymyxin-induced lipid A remodeling affects the OM packing and hydrophobicity, perturbs polymyxin penetration, and thereby confers high-level resistance. © 2017 American Chemical Society