FIG. 4.

(A and B) Entry and replication in J774A.1 macrophages of the F. tularensis LVS wild type and ΔpurMCD, FTL_0741::HimarFT, and attenuated mutants with insertions in loci encoding hypothetical proteins. The experiments were performed using a 24-well format with an MOI of 100 in triplicate. The results are expressed as mean log CFU ml⁻¹; the error bars indicate standard deviations. Mutants found to be significantly impaired in entry at 2 h or in replication at 24 h are indicated by asterisks (P < 0.05). (C) Quantitation of cytotoxicity by measuring LDH release into the supernatant compared to an uninfected monolayer (maximum lysis) at 48 h. LDH release was monitored alongside assessment for entry and replication defects as described above for panels A and B. Experiments were performed in triplicate, The bars indicate mean percentages of cytotoxicity, and the error bars indicate standard deviations. Mutants found to have a significant reduction in the percentage of cytotoxicity at 48 h are indicated by asterisks (P < 0.05).