Fig 5.

L. pneumophila ΔsdhA is severely attenuated in infection of G. mellonella larvae. (A) G. mellonella larvae were infected with a 1:1 ratio of WT:ΔsdhA or WT:ΔsdhA plus p4HA-SdhA, hemolymph was plated, and CFU were counted at 24 h p.i. to calculate the competitive index. The WT strain significantly outcompeted the ΔsdhA strain (CI, 0.002); fitness of the mutant could be restored by expression of 4HA-SdhA (CI, 0.88). (B) Hemolymph from larvae infected with WT, ΔdotA, ΔsdhA, or ΔsdhA plus p4HA-SdhA was extracted at 0, 5, 18, and 24 h p.i., and the CFU/0.1 g of extracted hemolymph were enumerated. The ΔsdhA strain did not replicate; however, it persisted over 24 h, unlike the ΔdotA strain, which was cleared by 18 h p.i. Results are means of at least three independent experiments ± standard deviations. (C) Larvae were infected with WT, ΔdotA, ΔsdhA, or ΔsdhA plus p4HA-SdhA L. pneumophila, and mortality was monitored for 72 h. Infection with WT resulted in 100% mortality within 24 h p.i., while the ΔsdhA strain only killed 40% of infected larvae by 72 h p.i.