Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex

Mizutani, Masanori; Mukaiyama, Keita; Xiao, Jing; Mori, Makiko; Satou, Rika; Narita, Shin‐ichiro; Okuda, Suguru; Tokuda, Harukuni

doi:10.1016/j.febslet.2012.11.009

Cited by 29 publications

(30 citation statements)

References 27 publications

(53 reference statements)

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“…Cross-linking studies suggest that despite their structural similarity, LolC and LolE are functionally different, which is consistent with both proteins being essential for growth (9). These studies show that LolC interacts with LolA and that LolE interacts with the lipoprotein to mediate lipoprotein transfer from the inner membrane to LolA (39). Both LolC and LolE are predicted to have 4 transmembrane-spanning (TMS) regions, with a large periplasmic loop between TMS1 and TMS2 and a smaller periplasmic loop between TMS3 and TMS4 (38).…”

Section: Discussionsupporting

confidence: 60%

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

et al. 2015

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cIn Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.T he most distinguishing feature of Gram-negative bacteria is their cell envelope, which is comprised of both an inner and an outer membrane bilayer. The outer membrane has a unique composition of lipoproteins, ␤-barrel proteins, lipopolysaccharides, and phospholipids. Lipoproteins, membrane proteins that are covalently modified with lipids, are involved in a variety of integral cellular functions, such as the synthesis and maintenance of the cell surface and the transport of substrates (reviewed in reference 1). Lipoproteins are synthesized as precursors in the cytoplasm. Upon transit across the inner membrane by either the Sec or Tat machinery, the export signal peptide is cleaved, and attached to its amino terminus is a lipid moiety. This lipid serves as a membrane anchor for the lipoprotein. Some lipoproteins remain in the outer leaflet of the inner membrane, while others must cross the hydrophilic periplasmic space to the outer membrane. This sorting of lipoproteins to the outer membrane is achieved by the Lol system, which consists of five proteins (Fig. 1) (reviewed in reference 1). In this process, lipoproteins destined for the outer membrane are released from the inner membrane by the LolCDE complex, an inner membrane ABC transporter. LolCDE transfers the lipoproteins to LolA, a diffusible periplasmic chaperone (2, 3). LolA then transfers the lipoprotein to LolB, the outer membrane lipoprotein receptor, which incorporates these lipoproteins into the inner leaflet of the outer membrane (1, 4). This is in contrast to Grampositive bacteria, which have a single membrane bilayer; therefore, localization of lipoproteins to the cell surface requires only export through the cytoplasmic membrane and acylation (5).Infections c...

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Section: Discussionsupporting

confidence: 60%

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

et al. 2015

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“…In E. coli, the key to sorting to the OM is recognition of the mature lipoproteins by the LolCDE system, with subsequent transfer to the LolA and LolB proteins (48). It was recently shown that deletion of lnt is possible in E. coli if the lolCDE genes are overexpressed, but only in the absence of Braun's lipoprotein (49).…”

Section: Discussionmentioning

confidence: 99%

Revisiting the Gram-Negative Lipoprotein Paradigm

et al. 2015

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The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS ⌬lnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the ⌬lnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. IMPORTANCEThis paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria.

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“…In a first step, LolE captures an OM lipoprotein from the inner membrane [73]. The precise mechanism remains to be determined, but it involves LolE’s periplasmic loop, which may fold into a hydrophobic domain similar to the hydrophobic cavities found in LolA/B.…”

Section: Rules and Pathways Of Lipoprotein Sorting Within The Perimentioning

confidence: 99%

“…At the same time, LolC procures an empty LolA carrier molecule [74]. In a second step, ATP binding to LolD weakens the hydrophobic interaction of LolE with the OM lipoprotein [73]. In the third and final step at the IM, ATP hydrolysis leads to the transfer of the lipoprotein to LolA and the release of the lipoprotein:LolA complex into the periplasm, where it is captured at the OM by LolB [74].…”

Section: Rules and Pathways Of Lipoprotein Sorting Within The Perimentioning

confidence: 99%

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

Zückert

2014

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

178

193

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Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation, likely through interaction with a periplasmic holding chaperone, which delivers the proteins to an outer membrane lipoprotein flippase.

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Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex

Cited by 29 publications

References 27 publications

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

Revisiting the Gram-Negative Lipoprotein Paradigm

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

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