Klebsiella pneumoniae is an important cause of multidrug‐resistant infections worldwide. Recent studies highlight the emergence of multidrug‐resistant K. pneumoniae strains which show resistance to colistin, a last‐line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB‐associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K. pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ‐governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc3Man9GlcNAc2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc3Man9GlcNAc2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man7GlcNAc2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 °C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447−450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.

SummaryKlebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia.