Figure 3.

Validation of attenuation by genetic transduction and complementation. (A) Neutrophils were co-incubated with media, JE2 or S. aureus transductants at MOI 10 for 3 h, followed by flow cytometry. Viable neutrophil counts were generated as previously described. JE2 + 2D is illustrated by dotted line and attenuated strains to be taken forward indicated by arrows (n = 3). (B) Neutrophils were co-incubated with media, JE2, transductants, or complements (indicated by ⁺) at MOI 10 for 3 h. JE2 and lspA transductant was also incubated in the presence empty pKasbar plasmid (pKB) (n = 6). (C) Chemical complementation of purB mutant was performed by addition of both inosine and adenine (I/A) to solid BHI agar during bacterial growth or RPMI media during neutrophil infection (indicated by single ⁺) or both (indicated by double ⁺⁺) at a final concentration of 0.02 mg/ml. The absence of I/A in RPMI or BHI is indicated by (⁻). Neutrophils were incubated with S. aureus for 3 h at MOI 10 and viable neutrophils enumerated (n = 3). Data expressed at mean ± SEM and analyzed by ANOVA with Bonferoni post-test ^*p < 0.05, ^**p < 0.01. Comparisons were between JE2 and transductant (A) or as indicated (B,C).