Infection of H. vermiformis by L. pneumophila strains. Wells containing Hartmannella amoebae were infected with approximately 5 × 103 CFU of wild-type 130b (●), pilD mutant NU243 (□), and pNPPC-hydrolase mutants NU247 (◊) and NU253 (▵). Bacterial CFU per well were quantitated at 0, 24, 48, and 72 h after inoculation. Each datum point represents the mean and standard deviations for three wells. Significant differences in recovery between 130b and its mutant derivatives were evident at 48 and 72 h (P < 0.005, Student's t test). These differences were observed in two additional experiments (data not shown).