Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells

Abstract

Objective To construct the fluorescence-labeled Burkholderia pseudomallei (B. pseudomallei) to study the pathogenesis of the interaction between the bacteria and host cells. Methods Gene recombination technique was carried out to recombine the green fluorescent protein (GFP), red fluorescent protein (RFP) and blue fluorescent protein (BFP) genes into the prokaryotic expression vector pUCP28T, respectively, in order to obtain the fluorescence-labeled B. pseudomallei. Furthermore, the biological characteristics of fluorescence-labeled bacteria were detected in bacterial culture in vitro, and inflammatory factor IL-8 was detected to evaluate the changes in the ability of fluorescence-labeled bacteria infecting host cells. Then, the dynamic process of fluorescent-labeled B. pseudomallei being enveloped by host cell autophagosome was observed with aid of living cell workstation. Results Three fluorescence-labeled B. pseudomallei were constructed respectively, and were stably expressed in 20 passages in vitro. The expression of exogenous fluorescent proteins did not affect the growth of B. pseudomallei and infection of host cells. Meanwhile, the dynamic changes of co-localization between RFP-labeled B. pseudomallei and GFP-LC3 were observed by live cell imaging system. Conclusion Three different fluorescence-labeled B. pseudomallei are successfully constructed. And they are important practical tools for the pathogenesis of the bacteria and the screening of antimicrobial agents of melioidosis.

Keywords

• burkholderia pseudomallei
• gene recombination
• fluorescence labeling
• autophagy