Characterization of interactions between LPS transport proteins of the Lpt system

Bowyer, A.; Baardsnes, Jason; Ajamian, Eunice; Zhang, Linhua; Cygler, Mirosław

doi:10.1016/j.bbrc.2010.12.121

Cited by 41 publications

(33 citation statements)

References 21 publications

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“…LPS can be transferred from LptC to its structural homolog LptA, but not vice versa, consistent with the expected directionality in the LPS transport pathway (18). LptA was shown to bind to LptC (42, 43) but not to LptE (42), which would be consistent with the idea that LptE is not the LPS recipient at the OM. A more likely recipient for LPS could be the N terminus of LptD because it shows sequence similarity to LptA (6).…”

Section: Discussionsupporting

confidence: 77%

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

Bos

Tommassen

2011

Journal of Biological Chemistry

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The biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPSproducing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all Gram-negative bacteria.Lipopolysaccharide (LPS) is a glycolipid, uniquely present at the cell surface of Gram-negative bacteria. It consists of a hydrophobic membrane anchor, called lipid A, which is embedded in the outer membrane (OM), 2 and an extracellular oligosaccharide core extended in some bacteria with a polysaccharide moiety, the O-antigen. The lipid A part can evoke strong, often toxic, innate immune responses; hence, it is also known as endotoxin. LPS is an essential component of the OM of most Gram-negative bacteria. Therefore, molecules involved in its biogenesis may represent attractive antimicrobial targets, as demonstrated by the successful development of antibiotics targeting LpxC, an enzyme involved LPS biogenesis (1), and LptD, an OM protein involved in LPS transport (2). However, much of the LPS biogenesis is not understood yet. The complete lipid A biosynthetic pathway, which takes place in the cytoplasm and at the inner leaflet of the inner membrane, has been elucidated in Escherichia coli and Salmonella. The ubiquitous presence of the lipid A biosynthetic enzymes in most other Gram-negative organisms suggests that this process is well conserved (3). The transport process of LPS from its site of synthesis to the cell surface is much less well understood (4 -7).The ATP-binding cassette transporter MsbA is thought to transport the lipid A-core moiety of newly synthesized LPS over the inner membrane (8, 9). At the OM, two proteins have been shown to be involved in the transport of LPS to the cell surface: the integral OM protein designated LptD (formerly Imp/OstA) (...

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Section: Discussionsupporting

confidence: 77%

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

Bos

Tommassen

2011

Journal of Biological Chemistry

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“…The current model of LPS transport posits that LptBFG, which constitute an ABC transporter, extract LPS from the IM (5,(7)(8)(9)(10)(11), and then the molecule is passed to the periplasmic domain of LptC (8,(10)(11)(12). Next, additional ATP hydrolysis by LptBFG facilitates passage of LPS from LptC to LptA in the periplasm (6,(11)(12)(13). Multiple LptA proteins polymerize in end-on-end fashion to span the periplasm, from the IM LptBCFG components to the OM LptDE complex (14)(15)(16).…”

mentioning

confidence: 99%

Dominant Negative lptE Mutation That Supports a Role for LptE as a Plug in the LptD Barrel

Grabowicz

Yeh

Silhavy

2013

Journal of Bacteriology

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Lipopolysaccharide (LPS) is the major outer leaflet constituent of the Gram-negative outer membrane (OM) bilayer. A bipartite protein complex of LptD and LptE assembles LPS into the OM. It has been established that LptE assists folding and assembly of its ␤-barrel partner LptD, yet reported biochemical evidence suggested additional LptE functions. Here, we isolated dominant negative lptE mutations, seeking to inform these functions. The lptE14 mutation increased OM permeability to erythromycin, even when the wild-type lptE gene was present. We show that the lptE14 mutation does not cause a defect in either LptD assembly or LPS export. A spontaneous IS1 insertion in secA suppressed lptE14 erythromycin sensitivity by removing the C-terminal SecB-binding domain of SecA. While this suppressor mutation broadly impeded SecB-dependent secretion of preproteins, we show that suppression was a direct and specific consequence of reduced LptD levels in the OM. We suggest that lptE14 causes poor plugging of the LptD ␤ barrel and that a reduction of ineffectively plugged LptD-LptE14 complexes in the OM decreases permeability to erythromycin. Hence, lptE14 supports a proposed plug-and-barrel LptE-LptD arrangement.

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“…Moreover, an intermolecular interaction that LptA can be co-purified with overexpressed LptC has already been established [17], and the two proteins can be stably associated when purified separately [12]. Recent studies showed that LptA interacts with the C-terminal part of LptC at the IM and the N-terminal part of LptD at the OM in vivo [13].…”

Section: Lpta Interacts With Both Im and Om Lpt Complexmentioning

confidence: 99%

“…However, its function is not well understood within the complex LptBFGC [5,[9][10][11]. LptA was proposed to act as a periplasmic chaperone for LPS transport across the periplasm and reported to interact with IM protein LptC [12,13] and OM protein LptD [13]. LptD and LptE form a stable complex at the OM which is proposed to serve as a translocon that facilitates the passage of LPS across the OM bilayer [14].…”

Section: Introductionmentioning

confidence: 99%

Characterization of lipopolysaccharide transport protein complex

Xiang

Wang

et al. 2013

Open Life Sciences

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Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro

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Characterization of interactions between LPS transport proteins of the Lpt system

Cited by 41 publications

References 21 publications

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

Dominant Negative lptE Mutation That Supports a Role for LptE as a Plug in the LptD Barrel

Characterization of lipopolysaccharide transport protein complex

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