Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction

Frontiers in Microbiology
Siroj JitprasutwitSunee Korbsrisate

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of ...Continue Reading

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Citations

Oct 30, 2020·Microorganisms·Jacob L Stockton, Alfredo G Torres
Oct 19, 2021·Molecular Biology and Evolution·Claire ChewapreechaJukka Corander

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Datasets Mentioned

BETA
PRJNA599542

Methods Mentioned

BETA
fluorescence-activated cell sorting
FACS
PCR
Illumina sequencing
flow cytometry

Software Mentioned

Artemis
ea
Artemis genome viewer
BWA
utils
GraphPad Prism

Related Concepts