Characterization of the Pseudomonas aeruginosa Lol System as a Lipoprotein Sorting Mechanism

Tanaka, Shinya; Narita, Shin‐ichiro; Tokuda, Harukuni

doi:10.1074/jbc.m611840200

Cited by 27 publications

(19 citation statements)

References 29 publications

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“…aeruginosa encodes homologs to each of the lolABCDE genes, and the LolCDE complex from P. aeruginosa recently was shown to release lipoproteins to the LolA chaperone (21). Reconstituted proteoliposomes with the LolCDE from P. aeruginosa did not release to LolA lipoproteins that contained the Lys ϩ3 Ser ϩ4 inner membrane retention signal.…”

Section: Discussionmentioning

confidence: 99%

Novel Inner Membrane Retention Signals inPseudomonas aeruginosaLipoproteins

2008

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The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFP OmlA -ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFP OmlA -ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions ؉2, ؉3, and ؉4 in CSFP OmlA -ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp ؉2 and Lys ؉3 Ser ؉4 function as inner membrane retention signals and identified four novel inner membrane retention signals:, and CQ ؉2 G ؉3 S ؉4 . These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370 CGDD -ChFP and PA3262 CDSQ -ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

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Section: Discussionmentioning

confidence: 99%

Novel Inner Membrane Retention Signals inPseudomonas aeruginosaLipoproteins

2008

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“…While the molecular mechanisms by which lipoproteins are targeted to the outer membrane have been extensively studied in E. coli, less is known about the Lol system in other gammaproteobacteria. For example, some work has been done to profile the proteins from P. aeruginosa, which showed that the five P. aeruginosa Lol proteins are responsible for the sorting of lipoproteins to the outer membrane, as in the case of E. coli lipoproteins (45). However, it was also shown that there are differences between E. coli and P. aeruginosa in the lipoprotein-sorting signals that dictate retention in the inner membrane or transfer to the outer membrane (45,46).…”

Section: Discussionmentioning

confidence: 99%

“…For example, some work has been done to profile the proteins from P. aeruginosa, which showed that the five P. aeruginosa Lol proteins are responsible for the sorting of lipoproteins to the outer membrane, as in the case of E. coli lipoproteins (45). However, it was also shown that there are differences between E. coli and P. aeruginosa in the lipoprotein-sorting signals that dictate retention in the inner membrane or transfer to the outer membrane (45,46). It is possible that the differences between the E. coli and P. aeruginosa Lol systems are significant enough that the pyridineimidazoles are not active against the P. aeruginosa LolCDE complex.…”

Section: Discussionmentioning

confidence: 99%

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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“…It is also unclear why Asp at position 2 can function as an inner membrane signal but is not used for many inner membrane lipoproteins in P. aeruginosa. 66) Finally, many lipoproteins still have no known function even in E. coli. Since lipoproteins are involved in various envelope activities, their structures and functions must be clarified in order to understand how bacterial envelopes are formed and maintained.…”

Section: Problems To Be Solvedmentioning

confidence: 99%