Uropathogenic Escherichia coli remodels LPS to evade Caspase-11-dependent pyroptosis to establish intracellular bacterial communities

<title>Abstract</title> <p> Uropathogenic <italic>Escherichia coli</italic> (UPEC), a major urinary tract infection (UTI) pathogen, is the currently known bacterium capable of extensively replicate in host cytosol. UPEC rapidly and clonally replicates to approximately 10 ⁵ bacteria, establishing biofilm-like intracellular bacterial communities (IBCs) in superficial bladder epithelial cell (BEC) cytosol. Caspase-4/11-dependent noncanonical inflammasome is the major cytosolic immune surveillance monitoring gram-negative bacteria, <italic>via</italic> lipopolysaccharide (LPS) detection. However, how UPEC evades recognition by Caspase-4/11 to replicate extensively in the BEC cytosol is unknown. In this study, transcriptional profiling of UPEC during the IBC formation process found that the expression of <italic>lpxT</italic> , which encodes a phosphotransferase that adds an additional phosphate group at the 1-position of LPS lipid A, was upregulated. We showed that lack of this modification not only reduced initial IBC formation, but also reduced the reinfection of UPEC to form subsequent rounds of IBCs in acute infection, and also reduced the incidence to cause recurrent UTIs in mice. We confirmed that LpxT modifies LPS in UPEC, and that the expression of <italic>lpxT</italic> is highly induced in UPEC-infected mouse bladders, resulting in an increase in the percentage of 1-pyrophosphate lipid A. We showed that phosphate modification of lipid A reduces the interaction between LPS and Caspase-4 in BECs, and diminishes Caspase-4/11 activation both in BECs and in mouse bladders, leading to reduced host cell pyroptosis. Caspase-11 knockout mouse experiments confirmed that UPEC increases LPS lipid A modification by LpxT to reduce Caspase-11 activation in mouse bladder, thereby facilitating its IBC formation and virulence. </p>