Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides

Ghilarov, Dmitry; Inaba-Inoue, Satomi; Stępień, Piotr; Qu, Feng; Michalczyk, Elizabeth; Pakosz, Zuzanna; Nomura, Norimichi; Ogasawara, Satoshi; Walker, Graham C.; Rebuffat, Sylvie; Iwata, So; Heddle, Jonathan G.; Beis, Konstantinos

doi:10.1126/sciadv.abj5363

Cited by 32 publications

(50 citation statements)

References 54 publications

(62 reference statements)

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“…The expression of BacA Sm and all its tested homologs (SbmA, BacA Ba , BclA) reverted the PHZ-susceptible phenotype in PHZ-resistant double mutants, which is consistent with the promiscuous peptide uptake by these transporters [10][11][12][13][14]25,28]. While there is no in vitro system to study transport by YejABEF, we have previously developed a system to monitor P a g e | 11 of 36 substrate transport by SLiPTs including BacA Sm and SbmA Ec [10]. We used this liposome transport assay to provide direct evidence of the ability of BacA Sm to internalize PHZ.…”

Section: In Vitro Transport Assay Shows the Internalization Of Phz Vi...supporting

confidence: 74%

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Travin

Vigouroux

Inaba-Inoue

et al. 2022

Preprint

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Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides.SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

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Section: In Vitro Transport Assay Shows the Internalization Of Phz Vi...supporting

confidence: 74%

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Travin

Vigouroux

Inaba-Inoue

et al. 2022

Preprint

Self Cite

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“…Pyranine has been used to monitor the proton-pumping activity of different types of rhodopsins: sensory rhodopsin-I and II [129,130], xR [131], ChR [132], aR [133], pR [134], and xanthorhodopsin [135,136]. Recently, Ghilarov et al revealed the molecular mechanism of the membrane protein SbmA [137]. The proton-pumping activity of the protein reconstituted into liposomes was measured by pyranine fluorescence.…”

Section: Membrane-impermeable Dye-based Assaysmentioning

confidence: 99%

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

2021

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Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

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“…In the case of E. coli SbmA-mediated transport of microcin J25, it is thought that SbmA makes specific contacts with the peptide and is powered by a proton gradient (Ghilarov et al, 2021). Unlike SbmA, which possesses the transmembrane but not nucleotide-binding domain of ABC transporters (Ghilarov et al, 2021; Runti et al, 2013), B. cepacia YddA is predicted to have a C-terminal ATP-binding domain. As such, YddA may use a transport mechanism distinct from SbmA, another intriguing question for future studies.…”

Section: Discussionmentioning

confidence: 99%

“…Although SbmA homologs have been repeatedly associated with lasso peptide transport, how they interact with their cargo is poorly understood. In the case of E. coli SbmA-mediated transport of microcin J25, it is thought that SbmA makes specific contacts with the peptide and is powered by a proton gradient (Ghilarov et al, 2021). Unlike SbmA, which possesses the transmembrane but not nucleotide-binding domain of ABC transporters (Ghilarov et al, 2021; Runti et al, 2013), B. cepacia YddA is predicted to have a C-terminal ATP-binding domain.…”

Section: Discussionmentioning

confidence: 99%

The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry

Thokkadam

Leach

et al. 2022

Preprint

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New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodin’s spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.

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Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides

Cited by 32 publications

References 54 publications

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry

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