SummaryMany respiratory pathogens establish persistent infection or a carrier state in the human nasopharynx without overt disease symptoms but the presence of these in the lungs usually results in disease. Although the anatomy and microenvironments between nasopharynx and lungs are different, a virulence factor with an organ-specific function in the colonization of the nasopharynx is unknown. In contrast to the severity of pertussis and mortality in non-vaccinated young children, Bordetella pertussis results in milder and prolonged cough in vaccinated adolescents and adults. Individuals harbouring bacteria in the nasopharynx serve as reservoirs for intrafamilial and nosocomial transmission. We show that the Bps polysaccharide of B. pertussis is critical for initial colonization of the mouse nose and the trachea but not of the lungs. Our data reveal a biofilm lifestyle for B. pertussis in the nose and the requirement of Bps in this developmental process. Bps functions as an adhesin by promoting adherence of B. pertussis and Escherichia coli to human nasal but not to human lung epithelia. Patient serum specifically recognized Bps suggesting its expression during natural human infections. We describe the first bacterial factor that exhibits a differential role in colonization and adherence between the nasopharynx and the lungs.

Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, densitydependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.Bacteria belonging to the genus Bordetella cause respiratory tract infections in both humans and animals (42). Bordetella pertussis is the etiological agent of pertussis, cases of which are steadily increasing in number, even in vaccinated populations (9). It has been proposed that the resurgence of pertussis is due in part to carriage within adolescent and adult populations because of waning immunity (3,4,9). Bordetella bronchiseptica has a broad host range and naturally infects a wide variety of nonhuman animals. It typically establishes asymptomatic infections but can cause atrophic rhinitis in pigs, kennel cough in dogs, snuffles in rabbits, and bronchopneumonia in guinea pigs (18,42).B. bronchiseptica is capable of establishing a chronic and asymptomatic infection and can be harvested from the nasal cavities of rats and mice for extended periods (1, 37). A convincing and frequently proposed hypothesis to explain long-term carriage is the ability of microorganisms to exist as biofilms. Bacterial biofilms are increasingly recognized as important contributors to chronic or persistent diseases. A biofilm is generally defined as a surface-attached population of one or more types of bacteria encased in a polymeric matrix, which can be composed of a number of different macromolecules, including nucleic acids, proteins, and polysaccharides (5). Numerous studies have documented the ability of biofilm bacteria to be recalcitrant to antibiotic treatments and to the host immune system (31,39,40,53).We and others have recently demonstra...

The Escherichia coli cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3 or 5 extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-1573Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the csdA null mutant at 15°C. Plasmid-encoded CsdA⌬444, which lacks most of the carboxy-terminal extension, enhanced the growth of a csdA null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.RNA helicases are involved in various cellular processes that require modulation of RNA structure, such as RNA splicing, ribosome biogenesis, translational initiation, mRNA degradation, and cell division (9,23,30). Driven by nucleoside triphosphate hydrolysis, these enzymes catalyze unwinding of RNA duplexes and disruption of RNA-protein interactions (9,23,30). Based upon the conservation of several motifs, RNA helicases are grouped into related families. Members of superfamily 2 (SF2) share eight conserved motifs and include the DExD/H helicase family, comprising the DEAD, DEAH, DExH, and DExD families (5, 34). The DEAD box family of ATP-dependent helicases, consisting of at least 500 eukaryotic and prokaryotic proteins, is the largest family (reviewed in reference 8). The prototype is eukaryotic initiation factor 4A (eIF4A), which exhibits helicase activity (21, 28).Proteins in the DEAD box family contain a core of nine conserved sequence motifs, including the Q motif, which is unique to this subset of SF2 helicases (33), and the Asp-GluAla-Asp (DEAD) motif that gives the family its name (21). The DEAD motif has been demonstrated to be essential for ATPase and/or RNA unwinding activity of several helicases, including the mammalian and yeast initiation factor 4A, the yeast protein Ded1p, and the Escherichia coli enzyme RhlB (3,14,26,36). The recent crystal structure of the RNA-bound Drosophila melanogaster Vasa DEAD box helicase demonstrated that the DEAD sequence participates with residues of other conserved motifs to bind ATP (32). An intricate network of interactions between canonical helicase motifs serves to couple ATP binding and hydrolysis with RNA binding and unwinding activities in a manner consistent with roles for these motifs previously established by biochemical and genetic studies (8).In addition to the core of conserved motifs, DEAD box proteins contain variable amino-and carboxy-terminal sequences. It has been suggested that these flanking sequences aid in the binding of substrates and cofactors or regulate t...

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