A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production

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Abstract

Burkholderia dolosa caused an outbreak in the cystic fibrosis clinic at Boston Children’s Hospital and was associated with high mortality in these patients. This species is part of a larger complex of opportunistic pathogens known as the Burkholderia cepacia complex (Bcc). Compared to other species in the Bcc, B. dolosa is highly transmissible; thus understanding its virulence mechanisms is important for preventing future outbreaks. The genome of one of the outbreak strains, AU0158, revealed a homolog of the lafA gene encoding a putative lateral flagellin, which, in other non-Bcc species, is used for movement on solid surfaces, attachment to host cells, or movement inside host cells. Here, we analyzed the conservation of the lafA gene and protein sequences, which are distinct from those of the polar flagella, and found lafA homologs to be present in numerous ß-proteobacteria but notably absent from most other Bcc species. A lafA deletion mutant in B. dolosa showed a greater swimming motility than wild-type due to an increase in the number of polar flagella, but did not appear to contribute to biofilm formation, host cell invasion, or murine lung colonization or persistence over time. However, the lafA gene was important for cytokine production in human peripheral blood mononuclear cells, suggesting it may have a role in recognition by the human immune response.

Figures

  • Table 1. Primers used in this study.
  • Fig 1. Genomic loci of flagellin in B. dolosaAU0158, A. hydrophila, and V. parahemolyticus. A representation of the placement and conservation of B. dolosa genes involved in polar flagellin synthesis (above) and the proposed lateral flagellin biosynthesis genes (middle) are shown. For comparison, the well-studied genomic loci encoding lateral flagella proteins from A. hydrophila [45, 69] and V. parahaemolyticus [46] are shown. Gene names are given above the groupings and polar flagella genes begin with “fl” while lateral flagella genes begin with “lf” or “laf”. The first B. dolosa gene in each region has its AK34_RS designation shown. For example, the first gene in the lateral flagella gene set is shown as 27345 for AK34_RS27345. Conservation was determined dynamically using polar flagella homologs as thresholds for e-values. Groups of genes are color coded based on the A. hydrophila clusters. Asterisks indicate currently unannotated genes in the published B. dolosa genome sequence.
  • Fig 2. Presence of the lafA gene in outbreak and non-outbreak B. dolosa strains. PCR was performed with primers specific to the lafA gene (AK34_RS07895) and the resultant products run on a single 1% agarose gel. Multiple isolates from outbreak patients (patient number on top of lane) or single isolates (for patient 4) from 11 separate B. dolosa outbreak patients were tested. Also included: other clinical, non-outbreak strains; the sole environmental strain LMG21443 (2 different genome preparations); B. cenocepacia MC0-3 which contains a lafA gene; and a no template negative control. The marker (M) is a 2-log ladder with sizes noted. Gel shown represents three independent replicate PCR reactions.
  • Fig 3. Similarity and conservation of B. dolosa flagellin proteins. (A) Amino acid alignment between the proteins encoded by the B. dolosa fliC (AK34_RS27500) and lafA (AK34_RS07895) genes shows only weak similarity. Residues in black are conserved and those in lightly outlined boxes are similar. Dark lined boxes indicate the residues predicted to bind to the host immune receptor, TLR5 [71–75]. The alignment of additional homologs can be found in S1 Fig. (B) Phylogenetic network relationship between LafA protein homologs. Three gene clusters are represented by gray shaded areas: the β-Proteobacterial LafA protein sequences including those found in Burkholderia species (dark gray on left); a distinct cluster of γ-Proteobacteria LafA proteins (lighter gray on top); and the outgroup of polar flagellins from both β-Proteobacteria and γProteobacteria (light gray on right).
  • Table 2. Swimming and swarming motility of flagellin mutant strains.
  • Fig 4. Production of B. dolosa flagella in nutrient-rich media. B. dolosa strains were grown in either LB liquid medium (panels A, C, and E) or grown on TSA plates (panels B, D, F) then subjected to transmission electron microscopy. Representative images are shown for B. dolosa wild-type (panels A and B), B. dolosa ΔfliC (panels C and D), or B. dolosa ΔlafA (panels E and F). Black bars indicate 500 nm scale. Representative images of 10 replicate images are shown.
  • Table 3. Quantitation of the flagella from multiple TEM images.
  • Fig 5. Bacterial survival in murine pneumonia model. C57BL/6 mice were inoculated intranasally with 5 x 106 CFU/mouse and observed over time. At indicated times, the BAL fluid (panel A) was collected and the lungs homogenized (panel B), serially diluted, and plated for bacterial counts. Error bars represent one standard deviation of the data from 4–5 mice/group. Significant differences were assessed using one-way ANOVA. Asterisks indicate strain-based differences assessed using the Tukey’s multiple comparison test. p< 0.05, p< 0.001. There were no significant differences between strains in panel A. The levels of bacterial survival in the wild-type controls was published previously [50] as the bacterial survival analysis of the B. dolosa lafA deletion mutant was completed as part of a larger study.

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Roux, D., Schaefers, M., Clark, B. S., Weatherholt, M., Renaud, D., Scott, D., … Yoder-Himes, D. R. (2018). A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production. PLoS ONE, 13(1). https://doi.org/10.1371/journal.pone.0189810

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