Modification of structures of lipooligosaccharides (LOS) represents one prevalent mechanism by which Gramnegative bacteria can become resistant to key antibiotics. Owing to the significant complexity of LOS, the structural characterization of these amphipathic lipids has largely focused on elucidation of the lipid A substructures. Analysis of intact LOS enables detection of core oligosaccharide modifications and gives insight into the heterogeneity that results from combinations of lipid A and oligosaccharide substructures. Top-down analysis of intact LOS also provides the opportunity to determine unknown oligosaccharide structures, which is particularly advantageous in the context of glycoconjugate vaccine development. Advances in mass spectrometry technologies, including the development of MSn capabilities and alternative ion activation techniques, have made top-down analysis an indispensable tool for structural characterization of complex biomolecules. Here we combine online chromatographic separations with MS3 utilizing ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD). HCD generally provides information about the presence of labile modifications via neutral loss fragments in addition to the saccharide linkage arrangement, whereas UVPD gives more detailed insight about saccharide branching and the positions of nonstoichiometric modifications. This integrated approach was used to characterize LOS from Acinetobacter baumannii 1205 and 5075. Notably, MS3 analysis of A. baumannii 1205, an antibiotic-resistant strain, confirmed phosphoethanolamine and hexosamine modification of the lipid A substructure and further enabled derivation of a core oligosaccharide structure.

The defining feature of the Gram-negative cell envelope is the presence of two cellular membranes, with the specialized glycolipid lipopolysaccharide (LPS) exclusively found on the surface of the outer membrane. The surface layer of LPS contributes to the stringent permeability properties of the outer membrane, which is particularly resistant to permeation of many toxic compounds, including antibiotics. As a common surface antigen, LPS is recognized by host immune cells, which mount defences to clear pathogenic bacteria. To alter properties of the outer membrane or evade the host immune response, Gram-negative bacteria chemically modify LPS in a wide variety of ways. Here, we review key features and physiological consequences of LPS biogenesis and modifications.

Outer membrane biogenesis is a complex process for Gram-negative bacteria as the components are synthesized in the cytoplasm or at the inner membrane and then transported to the outer membrane. Stress pathways monitor and respond to problems encountered in assembling the outer membrane. The two-component system CpxAR was recently reported to be a stress pathway for transport of lipoproteins to the outer membrane, but it was unclear how this stress is sensed. May et al. [K.L. May, K.M. Lehman, A.M.Mitchell, and M. Grabowicz, mBio 10(3): e00618-19, 2019, https://doi.org/10.1128/mBio.00618-19] determined that an outer membrane lipoprotein, NlpE, is the sensor for lipoprotein biogenesis stress. The group demonstrated that CpxAR is activated by the N-terminal domain of NlpE when the lipoprotein accumulates at the inner membrane. Further, this work resolved a previously debated role for NlpE in sensing copper stress; copper was shown to inhibit acylation of lipoproteins, preventing them from being transported to the outer membrane.