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 ␤-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.

Horizontal Gene Transfer and Assortative Recombination within the Acinetobacter baumannii Clinical Population Provide Genetic Diversity at the Single carO Gene, Encoding a Major Outer Membrane Protein Channel

“…3C). In this context, other authors (46) reported previously, by using a recombinant CarO protein from A. baumannii ATCC 19606 T (CarO variant I) (Fig. 1A) reconstituted in lipid bilayers, features compatible with unspecific monomeric channels with slight selectivity for cations, similarly to Pseudomonas OprF or E. coli OmpA.…”

Acinetobacter baumannii : Emergence of a Successful Pathogen

“…␤-Lactam resistance, including carbapenem resistance, has also been ascribed to nonenzymatic mechanisms, including changes in outer membrane proteins (OMPs) (47,108,119,165,209,336,380,446,510,511), multidrug efflux pumps (232,236,347), and alterations in the affinity or expression of penicillin-binding proteins (165,188,406,510). Relative to other gram-negative pathogens, very little is known about the outer membrane porins of A. baumannii.…”

“…Relative to other gram-negative pathogens, very little is known about the outer membrane porins of A. baumannii. Recently, the loss of a 29-kDa protein, also known as CarO, was shown to be associated with imipenem and meropenem resistance (336,380,511). This protein belongs to a novel family of OMPs found only in members of the Moraxellaceae family of the class Gammaproteobacteria (380).…”

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

“…In addition to the aforementioned enzymes, porins contribute to carbapenem resistance, the major ones being CarO and OprD (166)(167)(168)(169)(170)(171). These porins constitute channels for influx of carbapenems and can contribute to resistance whereby a reduction in carO transcription results in downregulation of the CarO porin system and thus a decrease in carbapenem entry.…”