Llobet Brossa, Enrique et al. ; 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-hydroxyacylmodified 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 multidrugresistant 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. ; This work was funded by Sigrid Juselius Foundation (T.A.S.); the Tor, Joe, and Pentti Borg’s Foundation (T.A.S.); and Medicinska Understödsföreningen Liv och Hälsa (T.A.S. and K.M.D.). This work was also supported by a Spanish Ministry of Economy and Competitiveness Grant (Biomedicine Programme, SAF2012-39841), Marie Curie Career Integration Grant U-KARE (PCIG13-GA-2013-618162), and Queen’s University Belfast start-up funds (to J.A.B.). Centro de Investigación Biomédica en Red Enfermedades Respiratorias is an initiative from Instituto de Salud Carlos III. ; Peer Reviewed