Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling

Non-typeable Haemophilus influenzae (NTHi) invades host cells by bidding, o f the platelet-activating factor (PAF) receptor via lipooligosaccharide (LO S) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infec tion on host cell signalling and its role in NTHi invasion was examined. Th e infection of human bronchial epithelial cells with NTHi 2019 increased cy tosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was I nhibited by pretreatment with the cell-permeant intracellular Ca2+ chelator BAPTA-AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosp hate (IP) levels were also increased after infection with NTH! 2019 (P < 0. 001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulat ed IP production in a dose-dependent manner. NTH! 2019 invasion was inhibit ed by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitor s wortmannin and LY294002. The less invasive strain NTHi 7502 also initiate d IP production, but was unaffected by PAF receptor antagonist or PTX. Thes e data demonstrate that the binding of the PAF receptor by NTHi initiates r eceptor coupling to a PTX-sensitive heterotrimeric G protein complex, resul ting in a multifactorial host cell signal cascade and bacterial invasion. M oreover, the data suggest that NTHi strains initiate cell signalling and in vade by different mechanisms, and that invasion mediated by PAF receptor ac tivation is more efficient than macropinocytosis.