Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.     

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.     

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.     

The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica

Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase. Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B. bronchiseptica.     

Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis

Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-β-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of traqueal secretions, which might additionally contribute to the effective colonization of the respiratory tract.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Role of a putative polysaccharide locus in Bordetella biofilm development

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc), we provide evidence for the production of poly-beta-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.     

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.     

Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.     

Pseudomonas aeruginosa attachment and biofilm development in dynamic environments

Biofilm formation by Pseudomonas aeruginosa is hypothesized to follow a developmental pattern initiated by attachment to a surface followed by microcolony formation and mature biofilm development. Swimming and twitching motility are important for attachment and biofilm development in P. aeruginosa. However, it is clear that many P. aeruginosa strains lacking swimming motility exist as biofilms in the lungs of cystic fibrosis patients. Consequently, we have developed a dynamic attachment assay to identify motility-independent attachment-defective mutants. Using transposon mutagenesis, we identified 14 novel dynamic attachment-deficient (dad) mutants including four mutants specific to dynamic assay conditions (dad specific). Two of the dad-specific mutants contain insertions in genes involved in sensing and responding to external stimuli, implying a significant impact of external factors on the biofilm developmental pathway. Observations of initial attachment and long-term biofilm formation characterized our dad mutants into two distinct classes: biofilm delayed and biofilm impaired. Biofilm-delayed mutants form wild-type biofilms but are delayed at least 24 h compared with the wild type, whereas biofilm-impaired mutants never form wild-type biofilms in our assays. We propose a dynamic model for attachment and biofilm formation in P. aeruginosa including these two classes.     

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

Approaches to prevention, removal and killing of biofilms

Biofilms contribute to hygiene problems in the food industry and in the medical field. Biofilms are diverse and due to the development of special phenotypes, biofilm organisms are not as susceptible to biocides as planktonic microorganisms. Biofilms may be prevented by regular disinfection. Since the attachment of microbes to surfaces and the development of biofilm phenotypes is a very fast process, it is, however, almost impossible, to prevent biofilm formation completely. The removal and killing of established biofilms requires harsh treatments, mostly using oxidising biocides. Depending on the nature of the biofilms, different biocides may be useful and the best biocide for a specific biofilm still has to be determined under practical conditions. Another approach is the prevention of biofilm formation by selection of materials that do not support the attachment of microorganisms. Some materials like glass and stainless steel show less biofilm formation than others. The ranking of materials, however, depends on the conditions, under which they are tested. A novel approach is biofilm inhibition by supplementation of systems with nutrients, to inhibit attachment. First results on inhibition of biofouling in reversed osmosis systems are presented.     

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.     

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Enhancement of Bordetella parapertussis infection by Bordetella pertussis in mixed infection of the respiratory tract

The epidemiological and pathogenic relationship between Bordetella pertussis and Bordetella parapertussis, the two causes of whooping cough (pertussis), is unclear. We hypothesized that B. pertussis, due to its immunosuppressive activities, might enhance B. parapertussis infection when the two species were present in a coinfection of the respiratory tract. The dynamics of this relationship were examined using the mouse intranasal inoculation model. Infection of the mouse respiratory tract by B. parapertussis was not only enhanced by the presence of B. pertussis, but B. parapertussis significantly outcompeted B. pertussis in this model. Staggered inoculation of the two organisms revealed that the advantage for B. parapertussis is established at an early stage of infection. Coadministration of PT enhanced B. parapertussis single infection, but had no effect on mixed infections. Mixed infection with a PT-deficient B. pertussis strain did not enhance B. parapertussis infection. Interestingly, the depletion of airway macrophages reversed the competitive relationship between these two organisms, but the depletion of neutrophils had no effect on mixed infection or B. parapertussis infection. We conclude that B. pertussis, through the action of PT, can enhance a B. parapertussis infection, possibly by an inhibitory effect on innate immunity.     

Characteristics of azithromycin action on bacterial biofilms of pneumonia pathogens]

Azithromycin (Zitrolid) action on biofilms formed by gramnegative and grampositive microorganisms, isolated from patients with respiratory tract infections was investigated. The drug was effective at the stage of the biofilm formation and on exposition to the already formed biofilms.     

Virulence factors of Bordetella pertussis

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.