Restriction of BCAA biosynthesis promotes Lm virulence.

(A) qRT-PCR analysis of hly, actA and prfA transcription levels in WT Lm and indicated mutants grown in LBMM. mRNA levels are represented as relative quantity (RQ) and normalized to rpoD. The data represent 4 biological replicates (N = 4). Error bars indicate standard deviation. Asterisks represent P-values (* = P<0.05, ** = P<0.01, *** = P<0.001, n.s. = non-significant), calculated using Student’s t-test. (B) Δrli60 and codY-R61A/Δrli60 bacteria are attenuated for virulence compared to WT Lm bacteria during in-vivo competitive infection of mice. Bacterial loads in the spleens and livers of C57BL/6 mice after 2 days of infection with a 1:1 ratio of WT Lm and mutant bacteria (either Δrli60 or codY-R61A/Δrli60). pPL2 containing a Kanamycin (Km) or Spectinomycin (Spec) resistance cassette were used to identify each bacterial strain. The experiment was done reciprocally, switching the antibiotic cassete between the two strains. Colony forming units (CFU) of mutant bacteria in each organ are presented as relative to the CFU of WT bacteria and are significantly different from CFU (WT/Mutant) = 1, calculated using Student’s t-test (* = p<0.05). Mutant Km/WT Spec CFU is not significantly different from the reciprocal Mutant Spec/WT Km CFU.