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.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

     

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.     

Serotype variation in Bordetella pertussis is governed by cis-acting elements

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.     

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     

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.     

Iron stress increases Bordetella pertussis mucin-binding capacity and attachment to respiratory epithelial cells

Whooping cough is a reemerging infectious disease of the respiratory tract caused by Bordetella pertussis. The incomplete understanding of the molecular mechanisms of host colonization hampers the efforts to control this disease. Among the environmental factors that commonly determine the bacterial phenotype, the concentration of essential nutrients is of particular importance. Iron, a crucial and scarce nutrient in the natural environment of B. pertussis, has been found to induce substantial phenotypic changes in this pathogen. However, the relevance of this phenotype for the interaction with host cells was never investigated. Using an in vitro model for bacterial attachment, it was shown that the attachment capacity of B. pertussis to epithelial respiratory cells is enhanced under iron stress conditions. Attachment is mediated by iron-induced surface-exposed proteins with sialic acid-binding capacity. The results further suggest that some of these iron-induced surface-associated proteins are immunogenic and may represent attractive vaccine candidates.     

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.     

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.     

Use of Bordetella bronchiseptica and Bordetella pertussis as live vaccines and vectors for heterologous antigens

Bordetella pertussis and Bordetella bronchiseptica are respiratory pathogens of humans and animals respectively. Unlike many bacteria, they are able to efficiently colonise healthy ciliated respiratory mucosa. This characteristic of Bordetella spp. can potentially be exploited to develop efficient live vaccines and vectors for delivery of heterologous antigens to the respiratory tract. Here we review the progress in this area.     

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.     

百日咳鲍特菌感染婴幼儿口咽部的菌群变化

摘要：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。     

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

Abstract This paper attempts to provide an explanation for the effect of cyclodextrin on the yield of Bordetella pertussis soluble antigens. It was demonstrated that the addition of cyclodextrin to the synthetic Stainer-Scholte liquid medium enhances the level of the intracellular form of adenylate cyclase (200 kDa) in the supernate. In addition to this effect, it has been reported that cyclodextrin also enhances the levels of two other extracellular proteins, pertussis toxin and filamentous hemagglutinin. As these antigens are structurally different, it seems that the effect of cyclodextrin is not specific. With the use of different buffer systems of well-known action on outer membrane stability it was possible to determine a relationship between the presence of cyclodextrin, destabilisation of the outer membrane and the release of proteins. It was determined that the cyclodextrin did not modify the fluidity of B. pertussis cells but produced a change of outer membrane permeability.     

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.     

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.     

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.     

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.     

Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin

Heparin, a glycosaminoglycan synthesized in connective tissue-mast cells, appeared to inhibit the hemagglutination of rabbit erythrocytes induced by the filamentous hemagglutinin (FHA), a major adhesin of Bordetella pertussis. This inhibition suggested an interaction of heparin with the FHA region responsible for the hemagglutination activity. FHA-heparin interactions may play a role in bacterial attachment and persistence in the lungs during human pertussis. To confirm a direct FHA-heparin interaction, heparin was used as ligand in an affinity chromatography procedure. This technique allowed to purify FHA directly from the bacterial culture medium in a single-step using heparin-Sepharose CL-6B or Zetaffinity heparin 60 disks. The purified FHA was highly immunoreactive with anti-FHA monoclonal antibodies and showed no signs of degradation after 15 successive cycles of freezing-thawing. The described purification method is simple, and suitable for the rapid preparation of FHA.     

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.     

Serum resistance in bvg-regulated mutants of Bordetella pertussis

Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance. Here we examined whether other bvg-regulated proteins contribute to serum resistance. We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type. Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants. Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2–5-fold more resistant to serum killing.