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

Siroj Jitprasutwit,Veerachat Muangsombut,Nattawat Onlamoon,Niramol Jitprasutwit,Joanne M Stevens,Claudia M Hemsley,Mark P Stevens,Catherine Ong,Richard W Titball,Patoo Withatanung,Paiboon Vattanaviboon,Sunee Korbsrisate

doi:10.3389/fmicb.2020.00072

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 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Highlights

Burkholderia pseudomallei is a saprophytic bacterial pathogen that causes melioidosis, primarily in Southeast Asia and Northern Australia (Wuthiekanun et al, 2005; Wiersinga et al, 2006)
Following depletion of clones expressing enhanced green fluorescent protein (eGFP) during culture on LB agar, J774A.1 murine macrophages were infected with the remaining B. thailandensis E264 pTrapL-K9 library using an optimal MOI
For subsequent library screening, J774A.1 cells were infected with the B. thailandensis E264 pTrapL-K9 library at an MOI of 20

Summary

Introduction

Burkholderia pseudomallei is a saprophytic bacterial pathogen that causes melioidosis, primarily in Southeast Asia and Northern Australia (Wuthiekanun et al, 2005; Wiersinga et al, 2006). Humans often acquire melioidosis through wounds or by the inhalation of contaminated dust or water droplets (Wiersinga et al, 2006). B. pseudomallei is resistant to many antibiotics, making it difficult to treat, and currently, there is no vaccine to protect against melioidosis (Dance, 2000). In the United States, B. pseudomallei is a Tier 1 select agent, owing to its potential to cause a mass casualty event after a deliberate release (Wagar, 2016). B. pseudomallei is listed in Schedule five pathogens and toxins controlled under the Anti-Terrorism, Crime and Security Act (ATCSA) in the United Kingdom

Methods

Results

Conclusion