Scanning Mutagenesis.

The mutations were introduced by PCR mutagenesis (primers are listed in SI Appendix, Table S2) into the pilV gene inserted into the pKH37 plasmid. Plasmids were used to transform the PilV-deficient N. meningitidis 2C4.3 strain (ΔV). To examine the impact of the alanine substitutions, complemented ΔV mutants were first tested for their ability to assemble T4P and to incorporate PilV into the pili by shearing the pili of the bacterial surface, concentrating them using AS, and assessing PilE and PilV levels by SDS-PAGE and immunoblotting. Next, the piliated alanine-substituted mutants were tested for their ability to adhere to hCMEC/D3 endothelial cells. Mutations in the PilV sequence affecting PilV incorporation into pilus filament or T4P stability were excluded from our study.