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

Abstract

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) (...

Ralstonia solanacearum RSc0411 (lptC) is a determinant for full virulence and has a strain-specific novel function in the T3SS activity

“…We found that this gene cluster is largely conserved among Gram-negative bacteria in the gene composition and order and homology of protein sequences, indicating common functions of these genes. A recent report showed that the Neisseria meningitidis LptC orthologue is involved in LPS transport and the disruption of N. meningitidis lptC and other Lpt genes reduced cellular LPS (Bos & Tommassen, 2011). However, the specific effect of Lpt disruption on LPS biosynthesis was not clear.…”

Involvement of Neisseria meningitidis Lipoprotein GNA2091 in the Assembly of a Subset of Outer Membrane Proteins

“…1C). Increased protein levels in spent medium can also be diagnostic for periplasmic leakage, a process observed in mutants deficient in LPS biosynthesis or transport (22) or in strains depleted for essential Bam complex components such as ComL, the BamD homolog in N. meningitidis (11). The extracellular protein profile of the ⌬GNA2091 mutant showed bands characteristic of both a ComL-depleted strain and a ⌬rmpM mutant (Fig.…”

“…Hence, we first considered the possibility that GNA2091 is involved in LPS transport. To address this possibility, we analyzed cellular LPS levels because defects in LPS transport in N. meningitidis lead to a feedback inhibition on LPS synthesis (1,22). SDS-PAGE analysis, however, revealed similar LPS levels in parent and mutant cells ( Fig.…”

Colonize, evade, flourish

“…The expected loci for the two remaining periplasmic Lpt components (LptC and LptE) encode proteins sharing minor domain characteristics with their E. coli counterparts. 13 Interestingly, the proteins encoded in these H. pylori loci are found across Epsilonproteobacteria, suggesting that this class of bacteria may contain unique conserved functional equivalents to E. coli LptC and LptE proteins. 13 Finally, H. pylori does contain a strong homolog of LptD, which delivers LPS to the outer leaflet of the outer membrane, completing the transport process.…”

“…13 Interestingly, the proteins encoded in these H. pylori loci are found across Epsilonproteobacteria, suggesting that this class of bacteria may contain unique conserved functional equivalents to E. coli LptC and LptE proteins. 13 Finally, H. pylori does contain a strong homolog of LptD, which delivers LPS to the outer leaflet of the outer membrane, completing the transport process. All in all, H. pylori seems to possess most of the machinery that E. coli uses to transport LPS, though genetic loci, sequence, and structure of several proteins are not perfectly conserved.…”