Architecture and matrix assembly determinants of Bordetella pertussis biofilms on primary human airway epithelium

Author summaryDespite widespread vaccination, pertussis or whooping cough caused by the gram-negative obligate human pathogen Bordetella pertussis (Bp) is resurging in many countries. The mechanisms by which Bp infects human hosts, causes disease, and persists in the nasopharynx are ill-defined. We show herein that Bp forms aggregates, clusters, and highly structured biofilms colocalized with cilia on well-differentiated and ciliated primary human bronchial epithelial cells (HBE). The biofilm extracellular matrix (ECM) on HBE cells was heterogenous and consisted of the polysaccharide Bps, extracellular DNA, and bacterial cells arranged in different complexes (mono-, bi-, and tri-partite). Utilizing mutant strains, we discovered positive roles of three Bp virulence factors, Bps, filamentous hemagglutinin, and adenylate cyclase toxin in matrix production, cell cluster formation and biofilm maturation. Our study provides a human-relevant model for studying bacterial biofilms, reveals the nature of ECM and bacterial determinants required for biofilm formation on primary human airway cells. Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.