pubmed.ncbi.nlm.nih.gov

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand - PubMed

️Mon Jan 01 2024

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

Charlene Mae Salao Cagape et al. Sci Rep. 2024.

Abstract

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei. B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimA_Bp and BimA B. mallei-like (BimA_Bm). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei. This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimA_Bp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimA_Bp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimA_Bp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimA_Bp and BimC variants and their implications for B. pseudomallei virulence.

Keywords: Burkholderia pseudomallei; Actin-based motility; BimA; BimC; Melioidosis; Variation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

The authors have declared that no conflict of interests exists.

Figures

**Figure 1**
Alignment of the BimA_Bp and BimC types identified in clinical *B. pseudomallei* isolates. (a) Alignment of *B. pseudomallei* BimA_Bp types depicting the variations observed between types 1 and 10 compared to BimA_Bm MSHR668 located at the bottom of the alignment. (b) Alignment of *B. pseudomallei* BimC types depicting the variations observed between types 1 and 5. *B. pseudomallei* K96243, classified as type 1 was used as the reference strain. Multiple sequence alignment was performed using ClustalW.

**Figure 2**
Number of *B. pseudomallei* genomes and Pairwise Single Amino acid Polymorphism (SAP) of BimA_Bpand BimC types in clinical *B. pseudomallei* isolates. (a) and (b) Heat maps for pairwise SAP distances between BimA_Bp and BimC variants. The color and number correspond to the number of SAP distances between each variant type in reference to BimA_Bp and BimC types 1 (*B. pseudomallei* K96243, classified as type 1 was used as the reference strain). (c) and (d) Number of *B. pseudomallei* genomes harboring the BimA_Bp and BimC variant types.

**Figure 3**
Maximum-likelihood phylogenomic tree of 1,294 *B. pseudomallei* clinical isolates rooted on MSHR5619. The innermost ring (1) represents the PopPUNK lineages. The second (2) and middle (3) rings represent the BimA_Bp and BimC variant types, respectively. The fourth (4) and outermost (5) rings represent the country sources based on patients’ home addresses and the year of sample collection, respectively. The tree scale indicates 0.01 nucleotide substitutions per site.

**Figure 4**
Geographical distribution of *B. pseudomallei* clinical genomes. (a) Geographical map of northeast Thailand with study sites highlighted in gray. (b) Geographical distribution of the three dominant PopPUNK lineages. (c) Geographical distribution of the ten BimA_Bp types. (d) Geographical distribution of the five BimC types. The spatial distribution of genomes was represented by the patients’ home addresses in Thailand, Laos and Cambodia.

**Figure 5**
3D structural models of ten BimA_Bptypes and BimA_Bmof Australian strain MSHR668 of *Burkholderia pseudomallei*. (a) 3D structural model of BimA_Bp type 1 (*B. pseudomallei* K96243, classified as type 1 was used as the reference strain) as described by Stevens et al.. The yellow-colored ribbon represents the predicted signal peptide (residues 1–53); green, NIPVPPPMPGGGA direct repeat (residues 63–75); violet, proline-rich motif (residues 78–84); pink, WH and WH₂-like domains (residues 155–158); orange, PDASX repeats (residues 244–268); blue, transmembrane domain (residues 458–516). (**b – j**) 3D structural models of BimA_Bp types 2, 3, 4, 5, 6, 7, 8, 9 and 10. Mutation positions are represented in pink Corey-Pauling-Koltun (CPK) models and labeled accordingly. The red-colored ribbons represent the insertion. (k) 3D structural model of BimA_Bm MSHR668 . All BimA_Bp and BimA_Bm models were built using I-TASSER. The C-score of the models were: -0.96 for BimA_Bp type 1; -1.16 for type 2; -0.71 for type 3; -0.74 for type 4; -0.99 for type 5; -0.73 for type 6; -1.02 for type 7; -1.12 for type 8; -1.13 for type 9; -0.71 for type 10; and -0.93 for BimA_Bm MSHR668. Figures were generated by Discovery Studio Visualizer version 21.1.

**Figure 6**
3D structural models of five major BimC types of *B. pseudomallei*. (a) The 3D structural model of BimC type 1 (*B. pseudomallei* K96243, classified as type 1 was used as the reference strain) was built based on a template TibC of *E. coli* H10407 (4RB4) using SWISS-MODEL, with 43.85% sequence identity and 93% coverage and GMQE score of 0.77. The iron-finger motif (C354, C357, C373 and C385) is shown as an inset. (**b – e)** 3D structural models of BimC types 2, 3, 4 and 5. Mutation positions are represented in pink Corey-Pauling-Koltun (CPK) models and labeled accordingly. Figures were generated by Discovery Studio Visualizer version 21.1.

**Figure 7**
Plaque-forming efficiencies of representative strains harboring different BimA_Bptypes in A549 cells. (a) Photographic representation of plaques. (b) Plaque-forming efficiencies of *B. pseudomallei* isolates harboring the different BimA_Bp types in A549 cells. The cells were infected with *B. pseudomallei* strains representative of BimA_Bp type 1 (*B. pseudomallei* K96243, classified as type 1 was used as the reference strain), type 2 (DR10025A, DR20021A and DR40130A), type 4 (DR40111A, DR80025A and DR90085A), type 6 (DR10008A, DR40025A and DR50053A), type 9 (DR50173A, DR70003A and DR90006A), and type 10 (DR20062A, DR50003A and DR60054A) at MOI of 0.1:1. Plaques were stained with 2% (w/v) crystal violet at 24 h post-infection. Plaque-forming efficiency (PFU/ml) was counted as the number of plaques (plaque-forming units: PFU) divided by the CFU (colony-forming units) of bacteria added per well (CFU/ml). Error bars represent means ± standard deviation of data from three independent experiments (one-way ANOVA; P < 0.05).

**Figure 8**
Confocal microscopy images of A549 cells infected with *B. pseudomallei* isolates with different BimA_Bptypes. The representative strains of *B. pseudomallei* (a) BimA_Bp type 1 (*B. pseudomallei* K96243, classified as type 1 was used as the reference strain), (b) BimA_Bp type 2 (DR40130A), (c) BimA_Bp type 4 (DR40111A), (d) BimA_Bp type 6 (DR10008A), (e) BimA_Bp type 9 (DR50173A), and (f) BimA_Bp type 10 (DR50003A) were used to infect A549 cells (g) at MOI of 30:1. Immunofluorescence staining was performed at 8 h post-infection using 4B11 monoclonal antibody specific to *B. pseudomallei* capsular polysaccharide to visualize the bacteria in green; phalloidin for F-actin in red; and Hoechst 33258 for the host DNA in blue. (h) The length of actin tails (white arrow) was determined using Zen Zeiss 3.0 SR (black) software tools. The mean lengths were: 3.1605 μm, BimA_Bp type 1; 1.988 μm, BimA_Bp type 2; 2.043 μm, BimA_Bp type 4; 1.807 μm, BimA_Bp type 6; 1.465 μm, BimA_Bp type 9; and 1.603 μm, BimA_Bp type 10 (one-way ANOVA; P < 0.05). Scale bar, 10 μm.

References

1. Cheng, A. C. & Currie, B. J. Melioidosis: epidemiology, pathophysiology, and management. Clin. Microbiol. Rev.18, 383–416. 10.1128/CMR.18.2.383 (2005). - PMC - PubMed
1. Seng, R. et al. Prevalence and genetic diversity of Burkholderia pseudomallei isolates in the environment near a patient’s residence in Northeast Thailand. PLoS Negl. Trop. Dis.13, e0007348. 10.1371/journal.pntd.0007348 (2019). - PMC - PubMed
1. Shaw, T. et al. Environmental factors associated with soil prevalence of the melioidosis pathogen Burkholderia pseudomallei: a longitudinal seasonal study from South West India. Front. Microbiol.13. 10.3389/fmicb.2022.902996 (2022). - PMC - PubMed
1. Wiersinga, W. J., Currie, B. J., Peacock, S. J. & Melioidosis N Engl. J. Med.367, 1035–1044 10.1056/NEJMra1204699 (2012). - PubMed
1. Gassiep, I., Armstrong, M., Norton, R. & Human Melioidosis Clin. Microbiol. Rev.33. 10.1128/cmr.00006-00019 (2020). - PMC - PubMed

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand - PubMed