Teasing apart the evolution of lipoprotein trafficking in gram-negative bacteria reveals a bifunctional LolA

Smith, Hannah C.; May, Kerrie L.; Grabowicz, Marcin

doi:10.1073/pnas.2218473120

Cited by 7 publications

(4 citation statements)

References 50 publications

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“…Careful analysis is needed to pinpoint subtle deficiencies for subsequent investigations of lolCDE at native levels. Second, our findings are another indication that current biochemical assays of lipoprotein trafficking (reported by us and others) appear to have a low dynamic range and are highly sensitive to disruption ( 52 , 53 ). The trafficking system in vivo is remarkably robust by comparison.…”

Section: Discussionsupporting

confidence: 55%

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Lehman,

May,

Marotta

et al. 2024

mBio

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The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli , the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli . Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo , lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE. IMPORTANCE Resistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface—the outer membrane (OM)—is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery. In vitro evidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via “Hook” and “Pad” regions. We show that this model does not reflect lipoprotein trafficking in vivo . Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE.

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Section: Discussionsupporting

confidence: 55%

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Lehman,

May,

Marotta

et al. 2024

mBio

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“…Careful analysis is needed to pinpoint subtle deficiencies for subsequent investigations of lolCDE at native levels. Second, our findings are another indication that current biochemical assays of lipoprotein trafficking (reported by us and others) appear to have a low dynamic range and are highly sensitive to disruption (51, 52). The trafficking system in vivo is remarkably robust by comparison.…”

Section: Discussionsupporting

confidence: 55%

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Lehman,

May,

Marotta

et al. 2023

Preprint

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The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. InEscherichia coli, the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loopin vivousingE. coli. Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein traffickingin vivo, lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Ourin vivodata redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.IMPORTANCEResistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface— the outer membrane (OM)—is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery.In vitroevidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via “Hook” and “Pad” regions. We show that this model does not reflect lipoprotein traffickingin vivo. Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE.

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“…This would also be in line with phylogenomic evidence suggesting that many diderm bacteria have TamB but not TamA orthologs ( 60 ). Thus, either the other OM partner(s) of AsmA-like proteins have yet to be identified or, alternatively, some AsmA-like proteins could have intrinsic insertase activity, as it has been recently demonstrated for lipoprotein insertion by LolA in Gram-negative bacteria lacking the OM component LolB of the Lol system ( 61 ). Regarding the pathway(s) and mechanism(s) responsible for GPL extraction from the IM, if AsmA-like proteins require a dedicated IM protein machinery for GPL extraction, this should be essential for growth.…”

Section: Discussionmentioning

confidence: 99%

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa

Sposato,

Mercolino,

Torrini

et al. 2024

mSphere

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The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral β-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli , the opportunistic human pathogen Pseudomonas aeruginosa . Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA -like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa , including a novel AsmA-like protein (PA4735) that is not present in E. coli . Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM. IMPORTANCE Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli , thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

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Teasing apart the evolution of lipoprotein trafficking in gram-negative bacteria reveals a bifunctional LolA

Cited by 7 publications

References 50 publications

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa

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