The Bps polysaccharide of Bordetella pertussis promotes colonization and biofilm formation in the nose by functioning as an adhesin

Many 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.     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

The Effect of 2,4-Dinitrophenol on the Growth of Bordetella pertussis in Chick Tracheal Organ Culture

Among various metabolic inhibitors tested, only 2, 4-dinitrophenol inhibited the growth of Bordetella pertussis in chick tracheal organ culture at concentrations nontoxic both for bacterial organisms and for ciliary motility of the tracheal fragments. Although this effect of 2, 4-dinitrophenol was reversible in its early stage, longer treatment with this inhibitor resulted in an irreversible inhibition of bacterial growth due to secondary damage of the tracheal fragments. From these observations, it was postulated that the energy required for bacterial growth might be derived from cellular metabolism sensitive to inhibition with 2, 4-dinitrophenol.     

Natural‐host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C‐terminal domain,but not the RGD motif,during infection

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human‐adapted (e.g. Bordetella pertussis) strains produce a surface‐exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg‐Gly‐Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural‐host animal models should apply to B. pertussis FHA as well. We also show that the C‐terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage‐like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.     

Adherence of ovine and human Bordetella parapertussis to continuous cell lines and ovine tracheal organ culture

The adherence of ovine and human isolates of Bordetella parapertussis to ovine and human continuous culture cell lines and to ovine tracheal organ culture was compared. Adherence to non-ciliated respiratory continuous culture cells did not reveal any host-specificity of the isolates. In contrast, adherence of ovine B. parapertussis strains to ciliated ovine tracheal organ culture was significantly greater than that of human strains. These results indicate that tracheal organ culture is a useful tool for studying host-specific adherence of B. parapertussis and suggest that adherence of B. parapertussis to ciliated epithelia is species-specific making it unlikely that the transfer of B. parapertussis between humans and sheep will result in an infection.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Effect of hydromechanical stress on cellular antigens ofBordetella pertussis

Cells ofBordetella pertussis grown in a bioreactor under stirring conditions were studied to investigate the effect of shear stress on cellular-bound filamentous haemagglutinin (FHA). FHA attached to the bacterial surface, unlike extracellular FHA, was not affected at the shear levels tested. Moreover, no other cellular immunogen involved in the whole-cell protective activity seemed to be affected by hydromechanical forces.     

Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin

Summary Large quantities of purified Bordetella pertussis toxin were required to develop an investigational toxoid vaccine. To overcome the difficulties associated with the large scale growth of Bordetella pertussis in submerged culture, we employed an aeration strategy using compressed oxygen and an antifoam agent. The microorganism grown in this way in 1001 batches for 20–24 h yielded 4–6 mg·1^-1 pertussis toxin.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

Analysis of Swedish Bordetella pertussis isolates with three typing methods: characterization of an epidemic lineage

Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and the Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.     

Increased incidence of pertussis and parapertussis in HIV-1-positive adolescents vaccinated previously with whole-cell pertussis vaccine

We investigated 296 adolescents (11–18 years), who had been immunized previously with the three doses of DPT vaccines. 48 were diagnosed positive for HIV-1. Nasopharyngeal swabs were obtained from 296 adolescents who presented with persistent cough and nasopharyngeal secretions. Nasopharyngeal swabs (calcium alginate) specimens were collected by passing the swabs through the nares into the posterior nasopharynx and rotating the swabs for a few seconds. The swabs were plated for culture of Bordetella organisms in charcoal cephalexin blood agar (CCBA). The CCBA plates were incubated for 2–6 days at 35 °C in a humid aerobic atmosphere. The suspected, shiny (mercury-like) colonies were tested by slide agglutination with antisera to B. pertussis and B. parapertussis, and urease, oxidase activities were performed. Results indicate that out of 48 HIV-1-positive adolescents, 18 had positive cultures for Bordetella organisms (14, Bordetella pertussis, and 4, Bordetella parapertussis). Of 248 HIV-1-negative subjects, 3 had Bordetella organisms (2, Bordetella pertussis, 1, Bordetella bronchiseptica). One of the subjects, a boy, aged 14 years, with Bordetella bronchiseptica had a dog as pet, which was found to be infected. The results indicate that adolescents with HIV-1 infection, despite being vaccinated against pertussis have a higher rate of infection when exposed to pertussis bacteria than HIV-1-negative adolescents. This revised version was published online in August 2006 with corrections to the Cover Date.     

Temporal signaling and differential expression of Bordetella iron transport systems: the role of ferrimones and positive regulators

The bacterial respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ multiple alternative iron acquisition pathways to adapt to changes in the mammalian host environment during infection. The alcaligin, enterobactin, and heme utilization pathways are differentially expressed in response to the cognate iron source availability by a mechanism involving substrate-inducible positive regulators. As inducers, the iron sources function as chemical signals termed ferrimones. Ferrimone-sensing allows the pathogen to adapt and exploit early and late events in the infection process.     

Isolierung von Bordetella-Phagen aus Bakterienstämen der Impfstoffproduktion

It has been demonstrated that strains of Bordetella pertussis used for vaccine production contain temperate phages. It can be conducted from many experiments performed in our laboratory. that 10–100 phages per 10¹⁰ bacteria are released. However, the production of bacterial mass is not markedly influenced by lysogeny. Strains of Bordetella bronchiseptica used for production of vaccine against Rhinitis atrophicans of pigs have temperate phages too. These phages may cause a complete lysis during a submerse cultivation. The phages of Bordetella pertussis and Bordetella bronchiseptica can be propagted on Bordetella parapertussis.     

Iron and pH‐responsive FtrABCD ferrous iron utilization system of Bordetella species

A putative operon encoding an uncharacterized ferrous iron transport (FtrABCD) system was previously identified in cDNA microarray studies. In growth studies using buffered medium at pH values ranging from pH 6.0 to 7.6, Bordetella pertussis and Bordetella bronchiseptica FtrABCD system mutants showed dramatic reductions in growth yields under iron‐restricted conditions at pH 6.0, but had no growth defects at pH 7.6. Supplementation of culture medium with 2 mM ascorbate reductant was inhibitory to alcaligin siderophore‐dependent growth at pH 7.6, but had a neglible effect on FtrABCD system‐dependent iron assimilation at pH 6.0 consistent with its predicted specificity for ferrous iron. Unlike Bordetella siderophore‐dependent and haem iron transport systems, and in agreement with its hypothesized role in transport of inorganic iron from periplasm to cytoplasm, FtrABCD system function did not require the TonB energy transduction complex. Gene fusion analysis revealed that ftrABCD promoter activity was maximal under iron‐restricted growth conditions at acidic pH. The pH of human airway surface fluids ranges from pH 5.5 to 7.9, and the FtrABCD system may supply ferrous iron necessary for Bordetella growth in acidic host microenvironments in which siderophores are ineffective for iron retrieval.     

Quantitation of Adenylate Cyclase ofBordetella pertussisby Enzyme Linked Immunosorbent Assay

Bordetella pertussisproduces extracytoplasmic adenylate cyclase toxin (AC) which has received considerable attention as a potential vaccine candidate. Great interest from laboratories involved in production, purification and quality control of acellular pertussis vaccine is focused on finding an appropriate technique for rapid and accurate quantitation of AC antigen. In this paper a competitive ELISA is proposed. A polystyrene microplate coated with purified AC was incubated with the sample to be tested plus anti-AC serum. The bound anti-AC antibodies were measured by sequential reaction with alkaline phosphatase-labelled anti-mouse IgG and p-nitrophenylphosphate. This method showed high specificity, with the 50% inhibition corresponding to 4 μg/ml of AC. It also proved to be useful to assess the presence of AC in culture supernatants, with high reproducibility.     

High level of soluble expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and characterisation of the CyaA pore-forming fragment from a <Emphasis Type="Italic">Bordetella pertussis</Emphasis> Thai clinical isolate

Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys⁹⁸³. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality.     

Experimental allergic orchitis in mice

Susceptibility to the induction of murine autoimmune orchitis was found to be associated with the locus controlling Bordetella pertussis-induced sensitivity to the vasoactive amine, histamine. Only those inbred and H-2 congenic strains of mice possessing both the H-2 ^d haplotype and the locus for susceptibility to B. pertussis-induced sensitivity to histamine developed autoimmune orchitis. In addition, segregation analysis of backcross generation mice also demonstrated a high degree of correlation between susceptibility both to disease and to histamine sensitization, which was indicative of additional multigene control. Pertussigen-histamine sensitization factor (P-HSF) was only effective in eliciting disease when it was administered on the same day, or within a period up to 6 days following sensitization with mouse testicular homogenate-emulsified in complete Freund's adjuvant. P-HSF induced sensitivity to histamine was not found to be associated with an increase in the vascular permeability of target tissue. Thus, B. pertussis-induced sensitivity to histamine appears to play a more crucial role during the sensitization phase of autoimmune orchitis induction, rather than at the inflammatory or effector phase of the disease.