Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

Structural determination of carbohydrates is mostly performed by liquid-state NMR, and it is a demanding task because the NMR signals of these biomolecules explore a rather narrow range of chemical shifts, with the result that the resonances of each monosaccharide unit heavily overlap with those of others, thus muddling their punctual identification. However, the full attribution of the NMR chemical shifts brings great advantages: it discloses the nature of the constituents, the way they are interconnected, in some cases their absolute configuration, and it paves the way to other and more sophisticated analyses. The purpose of this review is to provide a practical guide into this challenging subject. It will drive through the strategy used to assign the NMR data, pinpointing the core information disclosed from each NMR experiment, and suggesting useful tricks for their interpretation, along with other resources pivotal during the study of these biomolecules.

Summary: One common mechanism of resistance against antimicrobial peptides in Gram‐negative bacteria is the addition of 4‐amino‐4‐deoxy‐l‐arabinose (l‐Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l‐Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l‐Ara4N synthesis and transfer to the LPS. The absence of l‐Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi‐protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l‐Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.