doi: 10.1371/journal.pone.0028811. Epub 2011 Dec 22.
FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea
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- PMID:22216115
- PMCID: PMC3245231
- DOI: 10.1371/journal.pone.0028811
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FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea
Diego O Serra et al. PLoS One.2011.
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Abstract
Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.
© 2011 Serra et al.
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Figures
Biofilm cultures were performed in glass column systems containing polypropylene beads. (A) Biofilm biomass accumulated by each strain over the polypropylene beads after 72 and 96 h of cultivation was stained with CV 0.1% (v v−1). The CV stain associated with cells was solubilized in ethanol/acetone (80∶20) and the resulting solution was subjected to measurement of the absorbance at 590 nm. The data are the means ± standard deviations of three independent experiments. An asterisk indicates significant differences between the WT and the ΔfhaB mutant (P value <0.05; Student'st-test). (B) Viable cell counts of the biofilm cells of each strain. The adhered cells were gently washed and detached from the polypropylene beads by slight agitation in PBS buffer. Serial dilutions of cell suspensions were then plated on BGA plates. Results are expressed as the number of colony-forming units per unit area (CFUs cm−2). The data are the means ± standard deviations of three independent experiments. An asterisk indicates significant differences between the WT and the ΔfhaB mutant (P value <0.05; Student'st-test). (C) Viable cell counts of the planktonic cells in the bulk-liquid phase of biofilm cultures of each strain. The cells were drained out of the biofilm column system at the end of the experiment (96 h). Serial dilutions of cell suspensions were then plated on BGA plates. Results are expressed as the number of colony-forming units per unit volume (CFUs ml−1). The data are the means ± standard deviations of three independent experiments.
(A) Attachment of WT and ΔfhaB strains to glass. Inoculated bacteria (1×109 CFUs ml−1) were allowed to attach to the surface under static conditions for 4 h at 37°C. Fluorescence images of WT and ΔfhaB GFP-tagged cells that remained adhered to the glass coverslips. (B) Attachment and subsequent biofilm growth by the WT and ΔfhaB B. pertussis strains. WT and ΔfhaB biofilm cultures were initiated with high inoculum levels (1×109 CFUs ml−1). Bacteria were allowed to attach for 4 h followed by incubation under normal biofilm culture conditions. The effect of low-level attachment on subsequent biofilm formation by the WTB. pertussis strain was also examined. The number of WT bacteria in the inoculum was reduced from 1×109 to 4×108 CFUs ml−1 so as to decrease the population of cells initially adhered to the level displayed by the ΔfhaB strain with a high inoculum (1×109 CFUs ml−1), and then incubated under normal biofilm culture conditions. Adhered biomass was stained with CV 0.1% (v v−1). The CV stain associated with cells was solubilized in ethanol/acetone (80∶20) and the resulting solution was subjected to measurement of the absorbance at 590 nm. The data are the means ± standard deviations of three independent experiments. An asterisk indicates significant differences between attachment and biofilm growth (P value <0.05; Student'st-test). (C) Viable cell counts of the adhered cells of each strain to polypropylene beads under the conditions described in B. The adhered cells were gently washed and detached from the polypropylene beads by slight agitation in PBS buffer. Serial dilutions of cell suspensions were then plated on BGA plates. Results are expressed as the number of colony-forming units per unit area (CFUs cm−2). The data are the means ± standard deviations of three independent experiments. An asterisk indicates significant differences between attachment and biofilm growth (P value <0.05; Student'st-test).
GFP-tagged strains were inoculated directly in the continuous-flow chambers. Bacteria were allowed to attach for 4 h at 37°C, and then the sterile SS medium was pumped through the flow chambers at a flow rate of 0.1 ml min−1. The experiment was repeated at least three times. Biofilms were visualizedin situ by fluorescence and CLSM microscopy. (A) Representative fluorescence micrographs of biofilms taken every 24 h for 3 days at a magnification of ×400. (B) Volumetric 3D reconstructions of representative Z-section image stacks of biofilms taken at 72 h of growth at a magnification of 630×. For each strain, images are presented in two different perspectives.
GFP-tagged planktonic bacteria of the WT (BPSM) strain, pre-incubated with SS medium (A) or with (B) dilutions of anti-FHA serum, followed by incubation for 4 h with 24-h-old WT biofilms. (C) Twenty four-h-old WT biofilms pre-incubated with dilutions of anti-FHA serum, and then subjected to a 4-h incubation period with planktonic GFP-tagged WT bacteria. GFP-expressing bacteria attached to the biofilms were visualized by acquiring multi-channel (transmitted light and fluorescence) CLSM images. At least six microscopic fields per coverslips and four coverslips per condition were analysed. A representative merged image for each condition is shown. The data indicated in the top right of each image are the means ± standard deviations of the numbers of GFP-tagged bacteria per 100 µm2 of pre-formed biofilm microcolony derived from the analysis of twenty four microscopic fields on four coverslips per condition. The numbers of attached bacteria was determined by examining multi-channel images using ImageJ in conjunction with the ITCN plug-in.
GFP-tagged planktonic bacteria of the WT (BPSM) and ΔfhaB (BPGR4) strain pre-incubated with SS medium, various concentrations of purified FHA or BSA (20 µg ml−1) followed by incubation for 4 h with 24-h-old WT biofilms. GFP-expressing bacteria attached to biofilms were visualized by acquiring multi-channel (transmitted light and fluorescence) CLSM images. The numbers attached bacteria was determined by examining multi-channel images using ImageJ in conjunction with the ITCN plug-in. The data are the means ± standard deviations of the numbers of GFP-tagged bacteria per 100 µm2 of preformed biofilm microcolony derived from the analysis of twenty four microscopic fields on four coverslips per condition. An asterisk indicates significant differences between the conditions compared (P value <0.05; Student'st-test).
Groups of five, 6 to 8 week old C57BL/6 mice were intranasally inoculated with containing 5×105 CFUs of either the WT or the ΔfhaB strain in 50 µl. After 1 (A) or 7 (B) days, mice were sacrificed and colonization was assessed for the nasal septum, trachea, and lungs. Horizontal bars represent the mean for each group. The dashed lines indicate the limit of detection. The experiment was performed in duplicate with all mice from both experiments being represented. An asterisk indicates significant differences between the WT and the ΔfhaB mutant (P value <0.05; unpaired two-tailed Student'st-test).
Groups of 6-week-old C57BL/6 mice were intranasally inoculated with 50 µl containing 5×105 CFUs of either the WT or ΔfhaB strain grown under Bvg+ phase conditions. Sections of trachea and nasal septum were harvested at 1 or 7 days post-infection from infected animals, immediately fixed, and probed with rat anti-Bordetella serum followed by a donkey anti-rat secondary antibody conjugated to Alexa Flour 488 (which stains bacteria green). To determine the localization of the host epithelium, specimens were stained for F-actin using phalloidin conjugated to Alexa Fluor 633 (which stains the epithelium red). For visualization with CLSM, those tissues corresponding to infected animals that showed similar levels for colonization by the WT and the ΔfhaB mutant were chosen. For each specimen, image stacks were obtained from at least ten areas of the trachea and nasal septum. (A–H) Each micrograph is a Z reconstruction produced from a representative stack of Z-section images.
References
- He Q, Mertsola J. Factors contributing to pertussis resurgence. Future Microbiol. 2008;3:329–339. - PubMed
- Birkebaek NH, Kristiansen M, Seefeldt T, Degn J, Moller A, et al. Bordetella pertussis and chronic cough in adults. Clin Infect Dis. 1999;29:1239–1242. - PubMed
- Halperin SA. The control of pertussis–2007 and beyond. N Engl J Med. 2007;356:110–113. - PubMed
- Nelson JD. The changing epidemiology of pertussis in young infants. The role of adults as reservoirs of infection. Am J Dis Child. 1978;132:371–373. - PubMed
- Cherry JD. Epidemiological, clinical, and laboratory aspects of pertussis in adults. Clin Infect Dis. 1999;28(Suppl 2):S112–117. - PubMed
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