Lipopolysaccharide (LPS), also known as endotoxin due to its severe pathophysiological effects in infected subjects, is an essential component of the outer membrane (OM) of most Gram-negative bacteria. LPS is synthesized in the bacterial inner membrane, a process that is now well understood. In contrast, the mechanism of its transport to the outer leaflet of the OM has remained enigmatic. We demonstrate here that the OM protein, known as increased membrane permeability (Imp) or organic solvent tolerance protein, is involved in this process. An Imp-deficient mutant of Neisseria meningitidis was viable and produced severely reduced amounts of LPS. The limited amount of LPS that was still produced was not accessible to LPS-modifying enzymes expressed in the OM or added to the extracellular medium. We conclude therefore that Imp mediates the transport of LPS to the cell surface. The role of Imp in LPS biogenesis and its high conservation among Gram-negative bacteria make it an excellent target for the development of novel antibacterial compounds.

Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.