The effect of different Burkholderia pseudomallei isolates of varying levels of virulence on toll-like-receptor expression

The purpose of this investigation was to ascertain the degree of toll-like-receptor (TLR) activation by Burkholderia pseudomallei isolates with varying levels of virulence 2 h post infection. Standard antibiotic protection assays were performed on RAW 264.7 macrophages and peritoneal exudate cells (PEC) challenged with B. pseudomallei. Real-time PCR (RT-PCR) was performed to determine TLR2, TLR4, TLR5 and TLR9 expression. Internalization and killing of bacteria were determined 2h post infection. ELISAs were performed to determine the levels of TNF-α from cultured supernatants. Nitrate levels were determined by Griess assays. Up to 2h post infection, B. pseudomallei failed to significantly increase TLR4, TLR5 and TLR9 expression in both cell types. However, TLR2 expression was increased in RAW 264.7 macrophages, irrespective of isolate virulence. The levels of TNF-α and nitrate were significantly attenuated in RAW 264.7 macrophages, and no correlation was found between the level of virulence of the infecting strain and TLR expression, bacterial uptake, or killing. The ability of B. pseudomallei to evade detection by macrophages may in part be due to possible signal dampening of TLRs at very early stages of infection