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.     

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.     

Bordetella pertussis attachment to respiratory epithelial cells can be impaired by fimbriae-specific antibodies

Bordetella pertussis attachment to host cells is a crucial step in colonization. In this study, we investigated the specificity of antibodies, induced either by vaccination or infection, capable of reducing bacterial adherence to respiratory epithelial cells. Both sera and purified anti-B. pertussis IgG or IgA fractions efficiently reduced attachment. This effect was found to be mediated mainly by fimbriae-specific antibodies. Antibodies with other specificities did not significantly interfere in the interaction of B. pertussis with respiratory epithelial cells, with the exception of antifilamentous hemaglutinin antibodies, which reduced bacterial attachment. However, this effect was smaller in magnitude than that observed in the presence of fimbriae-specific antibodies. The strong agglutinating activity of antifimbriae antibodies seems to be involved in this phenomenon.     

Failure to convert Bordetella parapertussis to Bordetella pertussis with a pertussis phage

To analyze the described lysogenic conversion of Bordetella parapertussis to a Bordetella pertussis-like form we used the phage 134 to lysogenize a B. parapertussis strain. Southern blot analysis of the isolated ‘lysogens’ showed that they were not true lysogens, but rather chronically infected strains. These pseudo-lysogens did not show any changes in virulence properties compared with the parental strain. The only difference we could show was a change in the LPS-structure: the pseudolysogens had a rough LPS, like B. pertussis, whereas the parental B. parapertussis strain was smooth.     

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.     

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.     

Antimicrobial effect of human milk on Bordetella pertussis

It has been demonstrated that human milk, unlike bovine milk, can reduce the viability of Bordetella pertussis. This antibacterial activity was not due to the presence of antibiotics or antibodies in the human milk. Reducing the level of available iron or increasing the concentration of lysozyme in bovine milk did not induce anti-B. pertussis activity. Analysis of total fatty acids revealed that human milk contained significantly more linoleic acid than bovine milk. However, the addition of linoleic acid to bovine milk did not inhibit the growth of B. pertussis.     

Growth and siderophore production by Bordetella pertussis under iron-restricted conditions

It has been demonstrated that under iron-restricted conditions Bordetella pertussis can obtain iron from iron-saturated human transferrin. Direct contact between B. pertussis and transferrin was not required as B. pertussis was able to acquire iron from transferrin when they were separated by a dialysis membrane. Siderophore activity was detected in supernatants from iron-restricted cultures of B. pertussis, B. bronchiseptica and B. parapertussis. Siderophores were identified as hydroxamates and were produced by both virulent and avirulent strains of B. pertussis.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

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.     

Proteomic Adaptation of Australian Epidemic Bordetella pertussis

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non‐ptxP3/non‐prn2). Cluster I was mostly responsible for the 2008–2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high‐resolution multiple reaction monitoring (MRM‐hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.     

Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.     

Activation of the complement cascade by Bordetella pertussis

Bordetella pertussis must survive the defenses of the human respiratory tract including the complement system. The BrkA (Bordetella resistance to killing) protein prevents killing by the antibody-dependent classical pathway. In this study, the ability of B. pertussis to activate the human complement cascade by other pathways was examined. B. pertussis was not killed in serum depleted of C2, however serum depleted for factor B killed B. pertussis as efficiently as intact serum, suggesting complement activation occurred exclusively by the classical pathway. B. pertussis was not killed by serum depleted of antibody, suggesting the bacteria fail to activate the antibody-independent branches of the classical pathway, including the mannose binding lectin pathway. Mutants lacking the terminal trisaccharide of lipopolysaccharide retained the complement-resistant phenotype, suggesting this structure does not influence activation of complement.     

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants

Abstract Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A. 1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.     

百日咳鲍特菌感染婴幼儿口咽部的菌群变化

目的 通过比较健康婴幼儿与百日咳鲍特菌感染婴幼儿口咽部的菌群相对丰度,探讨百日咳鲍特菌感染对婴幼儿口咽部的菌群影响。方法 采用高通量测序技术,对53例百日咳鲍特菌感染婴幼儿和21例健康婴幼儿口咽标本进行16S rDNA测序,对测序序列进行分析,比较两组间的菌群多样性及在门、属水平上菌群结构差异。结果 健康婴幼儿与百日咳鲍特菌感染患儿在性别和年龄方面差异没有统计学意义。百日咳鲍特菌感染婴幼儿比健康婴幼儿口咽部菌群多样性显著增加。变形菌门（Proteobacteria）作为主要的门在百日咳鲍特菌感染患儿组中相对丰度显著高于健康婴幼儿组;两组排在相对丰度前15位的属,共有3个主要的属在百日咳鲍特菌感染患儿中显著增加,分别为盐单胞菌属（Halomonas）、嗜血杆菌属（Haemophilus）和鲍特菌属（Bordetella）;而罗氏菌属（Rothia）显著减少。结论 百日咳鲍特菌感染婴幼儿口咽部的菌群发生了显著的变化,百日咳鲍特菌感染患儿口咽部的菌群多样性比健康婴幼儿显著增加,在门和属水平百日咳鲍特菌感染患儿与健康婴幼儿主要组成方面均有显著性不同。     

Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera

Abstract To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli α-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with α-hemolysin.     

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli

Abstract Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (lon protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.     

Investigation of the effects of oxidative stress-inducing factors on culturing and productivity of Bordetella pertussis

The stress response of Bordetella pertussis during fermentation was assessed by means of fluorescence-based techniques. During the manufacturing of vaccines, B. pertussis is subjected to stress during adaptation to a new environment and operating conditions in the bioreactor, which can have harmful consequences on growth and protein yield. In this study, stress was imposed by varying the percentage of dissolved oxygen (DO) and inoculum size, and by adding rotenone and hydrogen peroxide. In this study, fluorescence spectroscopy is used as a tool for measuring oxidative stress. High levels of DO during fed-batch operation had no detrimental effect on growth, but the specific productivity of pertactin (PRN) decreased. Cultures that were started with an inoculum size that was 10 times smaller than the control resulted in significantly less PRN as compared to controls where reduction was more significant in flasks as compared to bioreactors. A comparison of filtered to heat-sterilized media revealed that filtered media offered a protective effect against H₂O₂. Heat sterilization of the media might result in the destruction of components that offer protection against oxidative stress. Nonetheless, filter sterilization on its own would be insufficient for large-scale manufacturing. It should be emphasized that the effects of these stressors while investigating for other microorganisms have not been studied for B. pertussis.