Abstract
Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.
Highlights
Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria
To determine the effect of pH on the expression of the plc[1,2,3] genes, B. pseudomallei K96243 was incubated in LB broth which had been adjusted to pH 4.5, 5.0, 7.0, 8.0 or 9.0 before bacterial mRNA was extracted and tested
Using reverse transcription (RT)-PCR we first showed that expression of B. pseudomallei 23S rRNA was similar at all pH conditions tested
Summary
Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Plc[1] or plc[3] mutants showed no significant differences in plaque formation compared to wild-type bacteria These findings suggest that Plc[2], but not Plc[1] or Plc[3] are required for infection of host cells. B. pseudomallei has evolved mechanisms to evade phagocyte activities, including escape from phagosomes and entry into host cell cytosol where it multiplies and forms actin tails allowing cell-tocell spreading[4] This complex intracellular lifestyle is contributed by several bacterial virulence factors including type three secretion systems (T3SS), type six secretion systems (T6SS), polysaccharide capsule, lipopolysaccharide (LPS), and various secreted effector p roteins[5]. Dowling et al.[17] reported that Plc[3] might be a potential candidate vaccine requiring further study
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