Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability
The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential p...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2020.00909/full |
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| author | Elisabete C. C. M. Moura Tiago Baeta Alessandra Romanelli Cedric Laguri Alessandra M. Martorana Emanuela Erba Jean-Pierre Simorre Paola Sperandeo Alessandra Polissi |
| author_facet | Elisabete C. C. M. Moura Tiago Baeta Alessandra Romanelli Cedric Laguri Alessandra M. Martorana Emanuela Erba Jean-Pierre Simorre Paola Sperandeo Alessandra Polissi |
| author_sort | Elisabete C. C. M. Moura |
| collection | DOAJ |
| description | The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC–LptA and LptA–LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC–LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria. |
| first_indexed | 2024-12-12T00:01:48Z |
| format | Article |
| id | doaj.art-93112f27e305496cb7e920c8295fceac |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T00:01:48Z |
| publishDate | 2020-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-93112f27e305496cb7e920c8295fceac2022-12-22T00:45:12ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-05-011110.3389/fmicb.2020.00909532591Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA StabilityElisabete C. C. M. Moura0Tiago Baeta1Alessandra Romanelli2Cedric Laguri3Alessandra M. Martorana4Emanuela Erba5Jean-Pierre Simorre6Paola Sperandeo7Alessandra Polissi8Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, ItalyUniversité Grenoble Alpes, CNRS, CEA, IBS, Grenoble, FranceDipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Milan, ItalyUniversité Grenoble Alpes, CNRS, CEA, IBS, Grenoble, FranceDipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, ItalyDipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Milan, ItalyUniversité Grenoble Alpes, CNRS, CEA, IBS, Grenoble, FranceDipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, ItalyDipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, ItalyThe outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC–LptA and LptA–LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC–LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.https://www.frontiersin.org/article/10.3389/fmicb.2020.00909/fullbacterial cell walllipopolysaccharideLpt systemthanatinantimicrobial peptidesBACTH technique |
| spellingShingle | Elisabete C. C. M. Moura Tiago Baeta Alessandra Romanelli Cedric Laguri Alessandra M. Martorana Emanuela Erba Jean-Pierre Simorre Paola Sperandeo Alessandra Polissi Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability Frontiers in Microbiology bacterial cell wall lipopolysaccharide Lpt system thanatin antimicrobial peptides BACTH technique |
| title | Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability |
| title_full | Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability |
| title_fullStr | Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability |
| title_full_unstemmed | Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability |
| title_short | Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability |
| title_sort | thanatin impairs lipopolysaccharide transport complex assembly by targeting lptc lpta interaction and decreasing lpta stability |
| topic | bacterial cell wall lipopolysaccharide Lpt system thanatin antimicrobial peptides BACTH technique |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2020.00909/full |
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