Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.     

Genomic analysis of the adenylate cyclase-hemolysin C-terminal region of Bordetella pertussis, Bordetella parapertussisand Bordetella bronchiseptica

Adenylate cyclase-hemolysin plays an important role in the virulence of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species. Its C-terminal region carries protective epitopes and receptor binding site for human cells. Genomic analyses of this region indicate no polymorphism in B. pertussis and B. parapertussis regions, but substantial variability in B. bronchiseptica that might be linked to the various niches of this species.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin

Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.     

Vag8, a Bordetella pertussis bvg-Regulated Protein

Bordetella pertussis expresses a bvg-regulated 95-kDa protein, Vag8, encoded by vag-8. Southern blot analysis indicates that strains of Bordetella bronchiseptica and Bordetella parapertussis have DNA homologous to vag-8. Antiserum raised to a fusion of maltose binding protein to an N-terminal 60-kDa fragment of Vag8 recognizes the native 95-kDa protein in immunoblots of B. pertussis and B. bronchiseptica but not B. parapertussis. A 95-kDa protein-negative derivative of B. pertussis 18323 containing a deletion of vag-8 colonized mice as efficiently as the parent B. pertussis strain in a mouse aerosol model of pertussis.     

Evaluation of PCR methods for the diagnosis of pertussis by the European surveillance network for vaccine-preventable diseases (EUVAC.NET)

This study aimed to evaluate the performance of polymerase chain reaction (PCR) methods used for the diagnosis of pertussis in laboratories within Europe in 2011. National reference laboratories in 25 European countries were contacted and a total of 24 laboratories from 19 countries agreed to participate in the study. A panel of seven samples of DNA from Bordetella pertussis, Bordetella parapertussis and Bordetella holmesii plus a negative control were distributed and analysed according to the routine PCR methods in each laboratory. The study took place in 2011. Nineteen laboratories used a real-time PCR approach, four laboratories used block-based PCR and one laboratory used a combination of methods. Six different combinations of amplification targets were used, and ten laboratories tested only for the presence of B. pertussis DNA. All laboratories (24/24) correctly identified a sample with high concentration of B. pertussis DNA, while three misidentified the B. parapertussis DNA as B. pertussis and 15 misidentified the B. holmesii DNA as either B. pertussis or B. parapertussis. There was a wide variation in the methods used for PCR-based diagnosis of pertussis among the European laboratories. Several laboratories were not able to discriminate between DNA samples from different Bordetella species.     

Mixed outbreak ofBordetella pertussis andBordetella parapertussis infection in Finland

The epidemiology of whooping cough in a vaccinated population was studied during an outbreak of paroxysmal cough in an elementary school with 258 pupils in Turku, Finland. Nasopharyngeal specimens for isolation ofBordetella pertussis and/or sera for ELISA detection of antipertussis immunoglobulin M, A and G antibodies were taken from 94 % of children who were prospectively followed for two months.Bordetella pertussis was isolated in six patients, and 17 culture-positive cases withBordetella parapertussis were identified. Patients withBordetella pertussis orBordetella parapertussis were found simultaneously in the same classrooms. Comparison of immunoglobulin M responses toBordetella pertussis andBordetella parapertussis was used for differential diagnosis of these two infections. Twenty-six cases with pertussis and 27 cases with parapertussis were diagnosed. The results of this prospective study suggest thatBordetella parapertussis is a more common etiologic agent of mild respiratory tract infection among vaccinated school-aged children than is generally recognised. The possibility thatBordetella pertussis was converted toBordetella parapertussis during this outbreak is discussed.     

Bordetella parapertussis Survives the Innate Interaction with Human Neutrophils by Impairing Bactericidal Trafficking inside the Cell through a Lipid Raft-Dependent Mechanism Mediated by the Lipopolysaccharide O Antigen

Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen.     

Bordetella parapertussis Infection in Children: Epidemiology, Clinical Symptoms, and Molecular Characteristics of Isolates

The clinical trial conducted in Italy to evaluate the efficacy of acellular pertussis vaccines provided an opportunity to estimate the frequency of clinical infections with Bordetella parapertussis and to compare the clinical characteristics of children suffering from Bordetella pertussis illness with those of children with B. parapertussis illness. This study dealt with 76 B. parapertussis infections diagnosed from a population of 15,601 children participating in the follow-up of suspected cases of pertussis. An overall incidence of 2.1 cases of laboratory-confirmed parapertussis per 1,000 person-years was observed. Children affected by B. parapertussis infections showed a less severe clinical picture both in the duration of symptoms and in the percentage of patients affected, even when compared with vaccinated children with pertussis. To characterize the isolated strains, we performed assays for susceptibility to erythromycin and sulfamethoxazole-trimethoprim, and we examined the genomic DNAs by pulsed-field gel electrophoresis. The results showed a high degree of genetic stability among B. parapertussis strains regardless of time of collection and geographical distribution.     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

High-resolution melting analysis for the detection of two erythromycin-resistant Bordetella pertussis strains carried by healthy schoolchildren in China

Two erythromycin-resistant strains of Bordetella pertussis were isolated from nasopharyngeal specimens of two asymptomatic schoolchildren in China. High-resolution melting and sequencing analyses confirmed the homogeneous A2047G mutation in 23S rRNA genes of the two isolates. High-resolution melting (HRM) analysis is a useful assay for the rapid detection of erythromycin-resistant B. pertussis. The appearance of erythromycin-resistant B. pertussis strains in China is alarming.     

Comparison of media for agar dilution susceptibility testing ofBordetella pertussis andBordetella parapertussis

Antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis andBordetella parapertussis is not standardized. In an attempt to find the optimal medium for agar dilution testing, the activity of erythromycin againstBordetella pertussis andBordetella parapertussis (34 isolates each) was assessed using homologous broth/agar combinations of Bordet-Gengou, charcoal, Iso-Sensitest (Oxoid) and Mueller-Hinton media. Each medium was supplemented with 5 % and 20 % whole defibrinated horse blood. Mueller-Hinton medium supplemented with 5 % horse blood performed best overall.     

Role of pertussis toxin in causing symptoms ofBordetella parapertussis infection

Whooping cough can be caused by eitherBordetella pertussis orBordetella parapertussis. Although the two species share an almost complete DNA identity,Bordetella parapertussis does not produce pertussis toxin, which is thought to be the main virulence factor ofBordetella pertussis. In order to elucidate the role of pertussis toxin in causing the typical symptoms of whooping cough, clinical information from 33 patients with culture-positiveBordetella parapertussis infection was collected and compared to that from 331 patients with infection caused byBordetella pertussis. Isolated strains ofBordetella parapertussis lacked pertussis toxin expression, as was demonstrated by negative tests for histamine sensitization. This was further substantiated in vivo by a significantly lower leukocyte count in the parapertussis group as compared to the pertussis group. Frequencies of typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting, were almost identical in the two groups. Nocturnal coughing and contact anamnesis were noted more often in theBordetella pertussis group. Children in the parapertussis group were significantly more often vaccinated with whole-cell pertussis vaccine than children infected withBordetella pertussis. The results indicate that pertussis toxin may not play a decisive role in causing the typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting.     

Activity of new macrolides againstBordetella pertussis andBordetella parapertussis

MICs and MBCs of four new macrolides (azithromycin, clarithromycin, dirithromycin and roxithromycin) and two older macrolides (erythromycin and josamycin) forBordetella pertussis andBordetella parapertussis were determined. The activity of the new macrolides was as good as that of erythromycin, while josamycin was slightly less active.Bordetella parapertussis was more resistant thanBordetella pertussis.     

Bordetella parapertussis isolates not expressing pertactin circulating in France

Surprisingly, most Bordetella parapertussis isolates collected in France since 2007 do not express pertactin, owing to mutations in the structural gene encoding this protein. We used a 454 pyrosequencing strategy to study and compare the genetics of two B. parapertussis isolates (one expressing pertactin and one not expressing pertactin) and the reference strain. No region of difference was detected between the genomes of the two isolates and the genome of the reference strain. No increase in repeated sequences between both isolates was found, and there were very few sequence differences. Using cellular and animal models, we found no substantial difference between the pathogenicity of these B. parapertussis isolates, which is consistent with clinical data. The emergence of these isolates, indicating that pertactin expression is not essential for virulence for B. parapertussis, is discussed.     

Invasion of HeLa cells by Bordetella bronchiseptica

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis , is able to invade HeLa cells. Comparison of the invasiveness of Bvg⁺ and Bvg^-B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg^- (flagellated) phase was ten-fold lower than when Bvg⁺ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica , which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.     

The O Antigen Is a Critical Antigen for the Development of a Protective Immune Response to Bordetella parapertussis

Despite excellent vaccine coverage in developed countries, whooping cough is a reemerging disease that can be caused by two closely related pathogens, Bordetella pertussis and B. parapertussis. The two are antigenically distinct, and current vaccines, containing only B. pertussis-derived antigens, confer efficient protection against B. pertussis but not against B. parapertussis. B. pertussis does not express the O antigen, while B. parapertussis retains it as a dominant surface antigen. Since the O antigen is a protective antigen for many pathogenic bacteria, we examined whether this factor is a potential protective antigen for B. parapertussis. In a mouse model of infection, immunization with wild-type B. parapertussis elicited a strong antibody response to the O antigen and conferred efficient protection against a subsequent B. parapertussis challenge. However, immunization with an isogenic mutant lacking the O antigen, B. parapertussis Δwbm, induced antibodies that recognized other antigens but did not efficiently mediate opsonophagocytosis of B. parapertussis. The passive transfer of sera raised against B. parapertussis, but not B. parapertussis Δwbm, reduced B. parapertussis loads in the lower respiratory tracts of mice. The addition of 10 μg of purified B. parapertussis lipopolysaccharide (LPS), which contains the O antigen, but not B. parapertussis Δwbm LPS drastically improved the efficacy of the acellular vaccine Adacel against B. parapertussis. These data suggest that the O antigen is a critical protective antigen of B. parapertussis and its inclusion can substantially improve whooping cough vaccine efficacy against this pathogen.Bordetella pertussis and B. parapertussis are the causative agents of whooping cough, resulting in approximately 50 million cases and 300,000 deaths annually worldwide (28). While whooping cough is considered by the CDC to be a reemerging disease (5), the relative incidences of B. pertussis and B. parapertussis are not clear (50). It is known, however, that the resurgence of whooping cough roughly correlates with the introduction of acellular pertussis vaccines (5). These vaccines contain only B. pertussis-derived antigens and confer little to no protection against B. parapertussis (9, 14, 15, 23, 27, 28). Current acellular pertussis vaccines contain some combination of filamentous hemagglutinin, pertactin, and fimbriae 2 and 3, all of which are expressed by both B. pertussis and B. parapertussis, and pertussis toxin, which is B. pertussis specific (33, 34). Based on genome sequences, the levels of amino acid sequence identity between B. pertussis and B. parapertussis filamentous hemagglutinin, pertactin, and fimbria 2 and 3 proteins are about 98, 91, 71, and 92% (35), and antibodies raised against these antigens from B. pertussis cross-react with B. parapertussis (17, 31). However, immunization with purified B. pertussis filamentous hemagglutinin or pertactin does not confer protection against B. parapertussis (17). B. pertussis fimbriae confer some protection against B. parapertussis, but at much lower levels than they protect against B. pertussis (52). Based on these observations and the fact that B. parapertussis infection induces protective immunity to itself (56, 58), we hypothesized that the lack of protective antigens from B. parapertussis may be part of the reason why current whooping cough vaccines are ineffective against this bacterium.Although B. pertussis and B. parapertussis are very closely related (8, 35, 48), they differ in the structure of their lipopolysaccharides (LPS) (1, 2, 39, 40, 47). B. pertussis produces a lipooligosaccharide containing lipid A and a branched-chain core oligosaccharide with a complex trisaccharide modification but lacks the O antigen due to a natural deletion of the wbm locus responsible for its synthesis (39, 47). B. parapertussis LPS is similar to B. pertussis LPS but lacks the trisaccharide modification and includes an O antigen (39, 40). In addition to conferring serum resistance by inhibiting C3 deposition onto the surfaces of bacteria (11), the O antigen enables B. parapertussis to avoid B. pertussis-induced immunity by preventing antibody binding to cross-reactive antigens on the surfaces of B. parapertussis cells (56, 59). Since the O antigen is one dominant surface antigen recognized by B. parapertussis immune sera (56) and has been shown previously to be a protective antigen of various pathogenic bacteria (22, 36), we hypothesized that the O antigen is a protective antigen of B. parapertussis.To assess the role of the O antigen in the generation of an adaptive immune response to B. parapertussis, the immunity and protection generated by B. parapertussis infection or vaccination were compared to those generated by an isogenic mutant of B. parapertussis lacking the O antigen (Δwbm) (39). Animals immunized with B. parapertussis, but not B. parapertussis Δwbm, were protected against subsequent challenge with B. parapertussis. Mice immunized with B. parapertussis Δwbm were also deficient in the production of B. parapertussis-specific antibodies, and sera collected from these mice were less effective at reducing B. parapertussis colonization upon passive transfer than sera raised against B. parapertussis. The inclusion of LPS from B. parapertussis, but not from B. parapertussis Δwbm, rendered the acellular B. pertussis vaccine Adacel efficacious against B. parapertussis challenge. Together, these data indicate that the O antigen is an important protective antigen of B. parapertussis.     

Expression of P.69/ppertactin from Bordetella pertussis in a baculovirus/insect cell expression system: protective properties of the recombinant protein

The surface antigen P.69/pertactin of Bordetella pertussis has been expressed using the polyhedron promoter of baculovirus in cultured insect cells. Either full-length or truncated prn DNA was used to express P.69 pertactin. The full-length gene gave rise to low levels of P.93 precursor protein, some of which was processed to P.69. The shortened prn expressed P.69 pertactin directly at levels up to 3.5 mg per litre. P.69 vaccinated animals were protected against aerosol challenge with virulent B. pertussis bacteria.     

The Bpel locus encodes type III secretion machinery in Bordetella pertussis

Type III secretory genes(Bscl, J, K, L, N and O) have recently been identified in Bordetella bronchiseptica and shown to be under the control of the BvgAS locus. We examined a 35 616 byte DNA sequence amplified from Bordetella pertussis Tohama I for homology with known type III secretory genes in Yersinia spp. and Pseudomonas sppand a total of 20 homologous open reading frames were detected. Putative type III secretion proteins in B. pertussis were designated according to their homology with type III secretion proteins in B. bronchiseptica, Yersinia and Pseudomonas. These ORFs were arranged in two putative operons, which together we have designated as the BpeI locus. The first spans nucleotides 23385–7888 and encodes the putative proteins LcrH1, BopD, BopB, LcfH2, BscI, BscJ, BscK, BscL, BscN, BscO, BscQ, BscR, BscS, BscT, BscU, and BscC, in this order. The second spans nucleotides 23580–29863 and encodes the putative proteins LcrE, LcrD, BscD and BscF, in this order. The homology of these proteins to type III secretory proteins was B. bronchiseptica (73–99%),Yersinia spp. (17–65%), Pseudomonas spp. (18–64%). The B. pertussis proteins were similar to their homologues in B. bronchiseptica, Yersinia and Pseudomonas in terms of length, molecular weight and isoelectric point. Coiled-coil domains were detected in putative translocation proteins, BopB and BopD. BopB and BopD were similar to each other, to the RTX toxin family and to cyaA, cyaB, cyaD and cyaE. The percentage G+C content of the sequence analysed was 66.16%, which is similar to the published percentage G+C (67–70%) for the B. pertussis chromosome.