Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

Runti, Giulia; Ruiz, Maria del Carmen Lopez; Stoilova, Tatiana; Hussain, Rohanah; Jennions, Matthew; Choudhury, Hassanul G.; Benincasa, Monica; Gennaro, Renato; Beis, Konstantinos; Scocchi, Marco

doi:10.1128/jb.00818-13

Cited by 86 publications

(111 citation statements)

References 38 publications

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“…Our results show that the sbmA gene can be efficiently expressed in P. aeruginosa, resulting in functional transport, and confirm that the SbmA protein is a stand-alone transporter that does not need other subunits to translocate proline-rich peptides across the membrane (29).…”

Section: Mode Of Action Of Bac7(1-35) Against P Aeruginosamentioning

confidence: 56%

“…The mechanism of action of PR-AMPs, such as Bac7 and other proline-rich peptides, has previously been well depicted in E. coli and Salmonella enterica serovar Typhimurium (22,28). PR-AMPs cross the plasma membrane, exploiting the inner membrane protein SbmA (23,29), and enter the cytoplasm, where they inhibit vital functions such as protein synthesis (12). Bac7(1-35) and other PR-AMPs have also shown membranolytic activity in E. coli and S. Typhimurium, but only at much higher concentrations than their MIC values (16-to 64-fold); however, lytic effects due to PR-AMPs occur over longer times than those observed by most AMPs with lytic activity (30).…”

Section: Mode Of Action Of Bac7(1-35) Against P Aeruginosamentioning

confidence: 99%

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The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Runti

Benincasa

Giuffrida

et al. 2017

Antimicrob Agents Chemother

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Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa. By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli. The results evidenced a new mechanism of action for PRAMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

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Section: Mode Of Action Of Bac7(1-35) Against P Aeruginosamentioning

confidence: 56%

Section: Mode Of Action Of Bac7(1-35) Against P Aeruginosamentioning

confidence: 99%

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Runti

Benincasa

Giuffrida

et al. 2017

Antimicrob Agents Chemother

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“…Since the genes are under the same operon, production and export from the cell are very efficient and toxic levels of MccJ25 are prevented from building up. Inside the target cell (Runti et al , 2013; Mathavan et al , 2014), MccJ25 inhibits the bacterial RNA polymerase (Semenova et al , 2005). …”

Section: Introductionmentioning

confidence: 99%

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

et al. 2017

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Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.

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“…Initial bioinformatic analyses of the SbmA amino acid sequence allowed classification of the protein as a transmembrane domain (TMD), part of an ABC (ATP-binding cassette) transporter that would require a yetunidentified nucleotide binding domain (NBD) to be fully functional (5). Nevertheless, Runti et al, in the accompanying article (17), did not find any putative NBD partner in Escherichia coli for SbmA and showed that SbmA-mediated transport of Bac-7 requires the transmembrane electrochemical proton gradient and that it is independent of ATP hydrolysis.…”

mentioning

confidence: 99%

“…This observation is in agreement with an SbmA model having 8 TM domains. In addition, Runti et al (17) by means of a two-hybrid assay showed that SbmA forms homodimers and provided indirect evidence that both C and N termini of SbmA are orientated to the cytoplasm.…”

mentioning

confidence: 99%

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

Corbalán

Runti

Adler

et al. 2013

Journal of Bacteriology

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g SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

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Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

Abstract: SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization

Cited by 86 publications

References 38 publications

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

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