Bordetella pertussis Can Be Motile and Express Flagellum-Like Structures - PubMed (original) (raw)

Bordetella pertussis Can Be Motile and Express Flagellum-Like Structures

Casandra L Hoffman et al. mBio. 2019.

Abstract

Bordetella bronchiseptica encodes and expresses a flagellar apparatus. In contrast, Bordetella pertussis, the causative agent of whooping cough, has historically been described as a nonmotile and nonflagellated organism. The previous statements that B. pertussis was a nonmotile organism were consistent with a stop codon located in the flagellar biosynthesis gene, flhA, discovered when the B. pertussis Tohama I genome was sequenced and analyzed by Parkhill et al. in 2003 (J. Parkhill, M. Sebaihia, A. Preston, L. D. Murphy, et al., Nat Genet, 35:32-40, 2003, https://doi.org/10.1038/ng1227). The stop codon has subsequently been found in all annotated genomes. Parkhill et al. also showed, however, that B. pertussis contains all genetic material required for flagellar synthesis and function. We and others have determined by various transcriptomic analyses that these flagellar genes are differentially regulated under a variety of B. pertussis growth conditions. In light of these data, we tested for B. pertussis motility and found that both laboratory-adapted strains and clinical isolates can be motile. Upon isolation of motile B. pertussis, we discovered flagellum-like structures on the surface of the bacteria. B. pertussis motility appears to occur primarily in the Bvg(-) phase, consistent with regulation present in B. bronchiseptica Motility can also be induced by the presence of fetal bovine serum. These observations demonstrate that B. pertussis can express flagellum-like structures, and although it remains to be determined if B. pertussis expresses flagella during infection or if motility and/or flagella play roles during the cycle of infection and transmission, it is clear that these data warrant further investigation.IMPORTANCE This report provides evidence for motility and expression of flagella by B. pertussis, a bacterium that has been reported as nonmotile since it was first isolated and studied. As with B. bronchiseptica, B. pertussis cells can express and assemble a flagellum-like structure on their surface, which in other organisms has been implicated in several important processes that occur in vivo The discovery that B. pertussis is motile raises many questions, including those regarding the mechanisms of regulation for flagellar gene and protein expression and, importantly, the role of flagella during infection. This novel observation provides a foundation for further study of Bordetella flagella and motility in the contexts of infection and transmission.

Keywords: Bordetella; Bordetella bronchiseptica; Bordetella pertussis; flagella; flagellar motility; flagellar structure; motility.

Copyright © 2019 Hoffman et al.

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Figures

FIG 1

FIG 1

B. bronchiseptica and B. pertussis are motile in the Bvg(−) phase. Bacteria were grown overnight as shaking cultures in Stainer-Scholte Medium (SSM) and diluted to an optical density at 600 nm (OD600) of 0.800. Two microliters of diluted cultures was stabbed into 0.4% SSM agar plates. B. bronchiseptica strains were grown for 24 h at 37°C and ambient CO2 levels, B. pertussis strains were grown for 72 h under the same conditions. (A) B. bronchiseptica WT RB50, Bvg(−) RB54, and Bvg(+) RB53 were tested for motility. (B) B. pertussis WT BP338 and Bvg(−) BP347 were tested for motility. The B. pertussis motility zone increases when the bacteria are modulated to the Bvg(−) phase with 40 mM MgSO4. B. pertussis WT BP338 cells were grown overnight as shaking cultures in SSM and diluted to an OD600 of 0.800. Two microliters of diluted cultures was stabbed into 0.4% SSM agar plates. B. pertussis strains were grown for 72 h at 37°C at ambient CO2 levels. (C and D) Representative images of BP338 grown without (C) and with (D) 40 mM MgSO4. The experiment was repeated 6 times, and the radius was quantitated each time. WT BP338 has dashed outlines in panel F to better show the radius of the spreading zone. (E) The mean radius with standard deviation was graphed for each condition (±40 mM MgSO4). P < 0.0001. Serum increases B. pertussis motility. (F and G) Representative images of BP338 grown without (F) and with (G) 10% fetal bovine serum (FBS) in motility agar. The experiment was repeated 6 times, and the radius was quantitated each time. (H) The mean radius with standard deviation was graphed for each condition (±10% FBS). P < 0.0001. Lab-adapted and clinical isolates demonstrate a motile phenotype under Bvg(−)-modulated conditions. Bacteria were grown overnight as shaking cultures in SSM and diluted to an OD600 of 0.800. Two microliters of diluted cultures was stabbed into motility agar plates containing 0.4% SSM plus 40 mM MgSO4. B. pertussis strains were grown for 72 h at 37°C at ambient CO2 levels. (I) WT UT25, WT BPSM, and GMT1 (J) Clinical isolates V015 and V145.

FIG 2

FIG 2

Negative-stain TEM of B. bronchiseptica and B. pertussis shows flagellar structures on bacterial surface. Presumably motile B. bronchiseptica strains were isolated from the outer edges of the spreading zones in 0.4% SSM agar plates plus 40 mM MgSO4. (A) WT RB50, (B) Bvg(−) RB54, (C and D) lab-adapted WT BP338, (E and F) the BP338-derived Bvg(−) BP347, (G) recent clinical isolate V015, and (H) lab-adapted WT UT25 were isolated for negative-stain TEM as described in the methods and imaged with a JEOL 1230 transmission electron microscope. Representative images of flagellated bacteria were selected; not all observed bacteria were flagellated. The experiment was repeated 3 times for WT RB50, Bvg(−) RB54, WT BP338, and BP338-derived Bvg(−) BP347. The experiment was repeated twice for clinical isolate V015 and WT UT25. (I) Western blot analysis of flagellin protein expression of motile B. bronchiseptica and B. pertussis strains. Presumably motile B. pertussis strains were isolated from the outer edges of the spreading zones in plates containing 0.4% SSM agar plus 40 mM MgSO4. Samples were prepared as described in the methods. Nitrocellulose membranes were probed with a variety of flagellin antibodies. (I, row 1) BioLegend monoclonal anti-FliC antibody. (I, row 2) Anti-Salmonella Typhi flagellin antibody. (I, row 3) Anti-Shigella sonnei flagellin antibody. (I, row 4) Anti_-_Vibrio cholerae flagellin antibody. Noncommercial antibodies were obtained from Jorge Giron and used previously to characterize Shigella flagella.

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