Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells

Abstract

Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.

Fig 1. Burkholderia pseudomallei colony morphotypes.

(A) Wild Type, WT and (B) small colony variant, SCV morphotypes grown for 4 days at 37°C on Ashdown agar.

Fig 2. Intracellular survival of Burkholderia pseudomallei WT and SCV in A549 cells.

Monolayers were infected with B. pseudomallei WT and SCV at a MOI of 1:10 and 1:100 and the intracellular loads of bacteria were quantified over 12 hours post-infection. The values are the means of each of three independent experiments, each performed in triplicate and error bar represents the standard deviation.

Fig 3. Percentage of cytotoxicity to A549 epithelial cells.

Cells were infected with the bacteria at a MOI 1:10 and 1:100 for 0, 2, 4, 6, 8, 10, and 12 hours. The values are the means ± the standard deviation of each of three independent experiments, each performed in triplicate.

Fig 4. Proteomic profiles of Burkholderia pseudomallei.

Regulation of protein spots of SCV post-exposure (Panel B) in comparison with WT post-exposure (Panel A). WT post-exposure gel (Panel C) showing regulation of protein spots in comparison to WT pre-exposure (18) and SCV post-exposure gel (Panel D) showing regulation of protein spots in comparison to SCV pre-exposure (18). In the post-exposure, the bacteria was exposed to A549 cells for 2 hours, then the recovered bacteria were subjected to sonication to lyse the bacteria followed by proteins extraction to be used in 2-D-GE. A total of 550 mg of proteins was separated on an IPG strip pH 3–10 in the first dimension, followed by the separation on SDS-12% PAGE for the second-dimension separation. Coomassie blue R-350 stain was used to visualize the separated proteins. The up-regulated protein spots are indicated by red circles and down-regulated spots indicated by green circles. Protein spot numbers relate to information provided in the text and Tables 1–3.

Fig 5. Protein categories and the subcellular localisation of Burkholderia pseudomallei WT vs SCV (post-exposure to A549 cells).

Functional protein categories were predicted using COG, while subcellular localisation was predicted using PSORT. Panels A and B refer to functions and cellular location of the up-regulated proteins, respectively. Panels C and D refer to functions and cellular location of the down-regulated proteins, respectively.

Fig 6. Protein categories and the subcellular localisation of Burkholderia pseudomallei WT (pre-exposure vs post-exposure to A549 cells).

Functional protein categories were predicted using COG, while subcellular localisation was predicted using PSORT. Panels A and B refer to functions and cellular location of the up-regulated proteins, respectively. Panels C and D refer to functions and cellular location of the down-regulated proteins, respectively.

Fig 7. Protein categories and the subcellular localisation of Burkholderia pseudomallei WT (pre-exposure vs post-exposure to A549 cells).

Functional protein categories were predicted using COG, while subcellular localisation was predicted using PSORT. Panels A and B refer to functions and cellular location of the up-regulated proteins, respectively. Panels C and D refer to functions and cellular location of the down-regulated proteins, respectively.