Figure 5

Knock-down of BTH_s39 inhibits B. thailandensis growth and attenuates host response. (A) The efficiency of BTH_s39 transcript reduction was evaluated by Northern blot analysis of RNA isolated from wild type Bt CDC272 (WT) and from a bacterial clone expressing anti-sense to BTH_s39 transcript (as-s39) grown in LB containing 10% serum. 10 μg total RNA was loaded per sample and 5S rRNA was used as a reference control. (B) Growth curves of Bt CDC272 (WT) and the as-s39 knock-down strain cultured in RPMI containing 10% serum. Bacterial culture densities (OD₆₀₀) were measured at 2h intervals. The average values of four independent culture samples per bacterial strain are depicted. (C) The virulence of wild type Bt CDC272 (WT) and the as-s39 knock-down strain was evaluated by infection of THP-1 cells at MOI 10 for two hours, followed by addition of media containing 250 μg/ml kanamycin. The percentage (%) of dead cells was determined 24 hrs post-infection by comparison of total ATP in Burkholderia-infected versus uninfected THP-1 cells. The average and standard deviation values were obtained from three independent experiments. The ‘*’ denotes statistical significance (p < 0.05) in % dead cells between the as-s39 knock-down strain compared to WT. (D) The effect of BTH_s39 downregulation on the ability of Bt CDC272 to elicit an immune response was measured by RT-PCR. THP-1 cells were collected 5 h post infection with Bt CDC272 (WT) and the as-s39 knock-down strain at MOI 10. VCAM1 and IL-8 transcript levels were determined by Taqman qPCR using total RNA. The RNA levels are presented as fold change versus untreated THP1 control samples. Data is shown from three independent infection experiments performed in duplicate. The ‘*’ denotes statistical significance (p < 0.05) in IL-8 and VCAM1 expression between the WT and as-s39 knock-down strains.