Translocation of Hcp1 protein in type Ⅵ secretion system of Burkholderia pseudomallei mediates formation of multinucleated giant cells

Abstract

Objective To analyze the mechanism that Hcp1 protein in type Ⅵ secretion system of Burkholderia pseudomallei (B. pseudomallei) mediates the formation of multinucleated giant cells (MNGCs) when host cells are infected by the bacterium. Methods The mutant strain (BPC006 Δhcp1) and complementation strain (BPC006 Δhcp1:: hcp1) were constructed by homologous recombination and plasmid complement technology, respectively. After RAW264.7 cells were infected with B. pseudomallei, the localization of Hcp1 in host cells was analyzed by immunofluorescence staining. The localization was further verified by cytoplasmic-membrane isolation in 293T cells after transfecting pCDNA4.1-Hcp1. The biological significance and effect of Hcp1 were explored by the anti-Hcp1 polyclonal antibody blocking and the formation of MNGC was detected by Giemsa staining. Results Western blotting showed that BPC006 Δhcp1 could not express Hcp1, while BPC006 Δhcp1:: hcp1 restored Hcp1 expression. The above results proved that the mutant and complement strains were successfully constructed. Both cellular immunofluorescence co-localization and cytoplasmic-membrane isolation experiments showed that Hcp1 localized to host cell membranes. Last but not least, compared with the control group, anti-Hcp1 polyclonal antibodies inhibited the formation of MNGC (P < 0.01). Conclusion Hcp1 protein in type Ⅵ secretion system of B. pseudomallei is able to translocate to the RAW264.7 cell membranes and plays an important role in the formation of MNGCs.

Keywords

• burkholderia pseudomallei
• hcp1
• multinuclear giant cells
• gene knockout and complementation