Dataset Information

Channel formation by CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

ABSTRACT: It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M(w) and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a beta-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

SUBMITTER: Siroy A

PROVIDER: S-EPMC1315959 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Channel formation by CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

Siroy Axel A Molle Virginie V Lemaître-Guillier Christelle C Vallenet David D Pestel-Caron Martine M Cozzone Alain J AJ Jouenne Thierry T Dé Emmanuelle E

Antimicrobial agents and chemotherapy 20051201 12

It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M(w) and pI). W ...[more]

PMID: 16304148

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Project description:β-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. Carbapenem β-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections, but treatment failure is increasingly prevalent. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate tolerance to high-level meropenem treatment, form spheroplasts upon exposure to the antibiotic, and revert to normal growth after antibiotic removal. Using transcriptomics and genetic screens, we show that several genes associated with outer membrane integrity maintenance and efflux promote tolerance, likely by limiting entry into the periplasm. Genes associated with peptidoglycan homeostasis in the periplasm and cytoplasm also answered our screen, and their disruption compromised cell envelope barrier function. Finally, we defined the enzymatic activity of the tolerance determinants penicillin-binding protein 7 (PBP7) and ElsL (a cytoplasmic ld-carboxypeptidase). These data show that outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to A. baumannii meropenem tolerance. IMPORTANCE Carbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting the urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote the acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well understood. Here, we characterized the role of peptidoglycan recycling in outer membrane integrity maintenance and meropenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlight physiological processes that could be targeted to improve antimicrobial treatment.

Project description:BackgroundWith an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant Acinetobacter baumannii infections, colistin resistance is emerging.MethodsPatients with infection or colonization due to colistin-resistant A. baumannii were identified at a hospital system in Pennsylvania. Clinical data were collected from electronic medical records. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. To investigate the mechanism of colistin resistance, lipid A was subjected to matrix-assisted laser desorption/ionization mass spectrometry.ResultsTwenty patients with colistin-resistant A. baumannii were identified. Ventilator-associated pneumonia was the most common type of infection. Nineteen patients had received intravenous and/or inhaled CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection prior to identification of colistin-resistant isolates. The 30-day all-cause mortality rate was 30%. The treatment regimen for colistin-resistant A. baumannii infection associated with the lowest mortality rate was a combination of CMS, a carbapenem, and ampicillin-sulbactam. The colistin-susceptible and -resistant isolates from the same patients were highly related by PFGE, but isolates from different patients were not, suggesting evolution of resistance during CMS therapy. By MLST, all isolates belonged to the international clone II, the lineage that is epidemic worldwide. Phosphoethanolamine modification of lipid A was present in all colistin-resistant A. baumannii isolates.ConclusionsColistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection. Lipid A modification by the addition of phosphoethanolamine accounted for colistin resistance. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.

Dataset Information

Channel formation by CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

Publications

Channel formation by CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

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