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

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.

Galleria mellonella–intracellular bacteria pathogen infection models: the ins and outs

“…Most of these genes were located on chromosome 2 and associated with adaptation to and survival in intracellular environments (Ong et al 2004 ). Two genes, bpss1622 (a type III secretion system cluster 2 component) and bpss2104 (a T4SS cluster 3 component) were selected for deletion as they had not been studied previously (Jitprasutwit et al 2020 ). Infection of G. mellonella larvae with Δ bpss1622 or Δ bpss2104 resulted in attenuation, although the potential underlying mechanism(s) were not discussed (Jitprasutwit et al 2020 ).…”

Multinucleated Giant Cell Formation as a Portal to Chronic Bacterial Infections

“…The second stage of the intracellular life cycle involves Bpm replicating within the cytoplasm and the mobilization of host actin, while evading and/or subverting the bactericidal pathways being activated within the host cell. A recent study [ 17 ] demonstrated that Bpm upregulates genes associated with combating oxidative stress once free in the cytoplasm. This agrees with another study [ 18 ], that showed that Bpm induces host factor heme oxygenase 1 (HO-1), which corrals Reactive Oxygen Species (ROS) and promotes host cell survival.…”

Co-evolutionary Signals IdentifyBurkholderia pseudomalleiSurvival Strategies in a Hostile Environment

“…2 ). Expression profile of BPSL1661 was obtained from Ooi et al and cross-checked for consistency with ( Rodrigues et al 2006 ; Chin et al 2015 ; Jitprasutwit et al 2020 ) where conditions were overlapped.…”