Melanin Produced by Bordetella parapertussis Confers a Survival Advantage to the Bacterium during Host Infection.

Bordetella parapertussis causes respiratory infection in humans, with a mild pertussis (whooping cough)-like disease. The organism produces a brown pigment, the nature and biological significance of which have not been elucidated. Here, by screening a transposon library, we demonstrate that the gene encoding 4-hydroxyphenylpyruvate dioxygenase (HppD) is responsible for production of this pigment. Our results also indicate that the brown pigment produced by the bacterium is melanin, because HppD is involved in the biosynthesis of a type of melanin called pyomelanin, and homogentisic acid, the monomeric precursor of pyomelanin, was detected by high-performance liquid chromatography-mass spectrometry analyses. In an infection assay using macrophages, the hppD-deficient mutant was internalized by THP-1 macrophage-like cells, similar to the wild-type strain, but was less able to survive within the cells, indicating that melanin protects B. parapertussis from intracellular killing in macrophages. Mouse infection experiments also showed that the hppD-deficient mutant was eliminated from the respiratory tract more rapidly than the wild-type strain, although the initial colonization levels were comparable between the two strains. In addition, melanin production by B. parapertussis was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. Taken together, our findings indicate that melanin produced by B. parapertussis in a BvgAS-independent manner confers a survival advantage to the bacterium during host infection. IMPORTANCE In addition to the Gram-negative bacterium Bordetella pertussis, the etiological agent of pertussis, Bordetella parapertussis also causes respiratory infection in humans, with a mild pertussis-like disease. These bacteria are genetically closely related and share many virulence factors, including adhesins and toxins. However, B. parapertussis is clearly distinguished from B. pertussis by its brown pigment production, the bacteriological significance of which remains unclear. Here, we demonstrate that this pigment is melanin, which is known to be produced by a wide range of organisms from prokaryotes to humans and helps the organisms to survive under various environmental stress conditions. Our results show that melanin confers a survival advantage to B. parapertussis within human macrophages through its protective effect against reactive oxygen species and eventually contributes to respiratory infection of the bacterium in mice. This study proposes melanin as a virulence factor involved in the increased survival of B. parapertussis during host infection.

Shotgun proteomic analysis of Bordetella parapertussis provides insights into the physiological response to iron starvation and potential new virulence determinants absent in Bordetella pertussis.

Bordetella parapertussis is one of the pathogens that cause whooping cough. Even though its incidence has been rising in the last decades, this species remained poorly investigated. This study reports the first extensive proteome analysis of this bacterium. In an attempt to gain some insight into the infective phenotype, we evaluated the response of B. parapertussis to iron starvation, a critical stress the bacteria face during infection. Among other relevant findings, we observed that the adaptation to this condition involves significant changes in the abundance of two important virulence factors of this pathogen, namely, adenylate cyclase and the O-antigen. We further used the proteomic data to search for B. parapertussis proteins that are absent or classified as pseudogenes in the genome of Bordetella pertussis to unravel differences between both whooping cough causative agents. Among them, we identified proteins involved in stress resistance and virulence determinants that might help to explain the differences in the pathogenesis of these species and the lack of cross-protection of current acellular vaccines. Altogether, these results contribute to a better understanding of B. parapertussis biology and pathogenesis. SIGNIFICANCE: Whooping cough is a reemerging disease caused by both Bordetella pertussis and Bordetella parapertussis. Current vaccines fail to induce protection against B parapertussis and the incidence of this species has been rising over the years. The proteomic analysis of this study provided relevant insights into potential virulence determinants of this poorly-studied pathogen. It further identified proteins produced by B. parapertussis not present in B. pertussis, which might help to explain both the differences on their respective infectious process and the current vaccine failure. Altogether, the results of this study contribute to the better understanding of B. parapertussis pathogenesis and the eventual design of improved preventive strategies against whooping cough.

The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis.

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

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 AfuABpp , 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 AfuABpp 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.

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms.

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.

Concordancia entre las técnicas de IFD, PCR y ELISA para determinar la frecuencia de Bordetella parapertussis y Bordetella pertussis en un brote de tos ferina en el departamento de Antioquia (Colombia) en 2013 / Correlation among the DIF, PCR and ELISA techniques for determining the frequency of Bordetella parapertussis and Bordetella pertussis in an outbreak of whoopingcough in the department of Antioquia, Colombia, 2013

Objetivo: Obtener la concordancia de técnicas diagnósticas y confirmar el diagnóstico de los casos probables de tos ferina captados por inmunofluorescencia directa (IFD) durante un brote en 2013 en el departamento de Antioquia. Materiales y métodos: Se analizaron los datos demográficos, clínicos, epidemiológicos y de resultados de laboratorio de casos probables de tos ferina confirmados por IFD en un pico de tos ferina en 2013 en el departamento de Antioquia. Las muestras de aspirado nasofaríngeo y suero fueron recolectadas y recibidas entre los periodos epidemiológicos IV - VII de 2013. Todos los pacientes confirmados por IFD fueron confirmados por PCR o ELISA. El análisis de concordancia se realizó por índice kappa. Resultados: De las 180 muestras procesadas en el LSP de Antioquia, 134 (74%) fueron positivas por la técnica de IFD, de las cuales se confirmaron por PCR 109 muestras con 24 (22%) positivas para B. parapertussis , 3 (2,8%) para B. pertussis , 17 (15,6%) para Bordetella spp. y 18 (16,5%) con infección mixta por B. pertussis y B. parapertussis . De 81 casos que se confirmaron por ELISA, 31 (38,3%) fueron positivos. En el municipio de La Estrella la edad media de los casos confirmados fue de 6,6 años y la mediana de 3 años (rango: 2-4 años). Con respecto a los casos del municipio de Medellín, la edad media fue de 28,7 años y la mediana de 25 años (rango: 12-42 años). En su mayoría, en los síntomas no hubo diferencias significativas, excepto para la tos paroxística entre los casos confirmados de B. parapertussis y B. pertussis (p = <0,04) del municipio de La Estrella. De acuerdo con el índice kappa, los resultados mostraron una fuerza de concordancia pobre y sin grado de acuerdo con los resultados de las pruebas de PCR y ELISA comparados con IFD, índice kappa: (IFD/PCR: K = 0,0944) y (IFD/ELISA: K = - 0,4533). Conclusiones: Durante este análisis, en el 2013 la población de Antioquia fue afectada por la circulación de B. parapertussis y B. pertussis en población adolescente y adulta en Medellín y en la población de 2-4 años en La Estrella. Actualmente, la PCR y la ELISA son las técnicas adecuadas para el diagnóstico de tos ferina. La IFD por su subjetividad y baja concordancia se encuentra en desuso.

Objective: To determine the correlation between diagnostic techniques and to confirm the diagnosis of probable cases of whooping cough captured by direct immunofluorescence (DIF) during an outbreak in 2013 in the department of Antioquia. Materials and methods: We analysed the demographic, clinical, and epidemiological data and the laboratory results of probable cases of whooping cough confirmed by DIF at a peak of whooping cough in 2013 in the department of Antioquia. The nasopharyngeal aspirate and serum samples were collected and received between the epidemiological periods IV - VII of 2013. All patients confirmed by DIF were confirmed by polymerase chain reaction (PCR) and/or enzyme-linked immunosorbent assay (ELISA). The analysis of agreement was performed using the kappa index. Results: Of the 180 samples processed in the public health laboratory of Antioquia, 134 (74%) were positive using the DIF technique of which 109 samples were confirmed by PCR, with 24 (22%)samples positive for B. parapertussis , 3 (2.8%) for B. pertussis , 17 (15.6%) for Bordetella spp. and18 (16.5%) for mixed infection by B. pertussis and B. parapertussis . Of the 81 cases confirmed by ELISA, 31 (38.3%) were positive. In the municipality of La Estrella, the mean age of the confirmed cases was 6.6 years, and the median was 3 years (range, 2-4 years). For the municipality of Medellin, the mean age was 28.7 years, and the median was 25 years (range, 12-42 years). For most of the symptoms, there were no significant differences, except for paroxysmal cough among the confirmed cases of B. parapertussis and B. pertussis ( p = <.04) in the municipality of La Estrella. According to the kappa index, the results showed poor correlation strength and no agreement with the results of the PCR and ELISA tests compared with DIF, kappa index: (DIF/PCR: K = 0.0944) and (DIF/ELISA: K = - 0.4533). Conclusions: During this analysis in 2013, Antioquia was affected by the circulation of B. parapertussis and B. pertussis in the adolescent and adult population in Medellin and the 2-4 year-old population in La Estrella. Currently, PCR and ELISA are the recommended techniques for diagnosing whooping cough. Due to its subjectivity and low correlation, DIF is in disuse.

Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are three closely related pathogens. They all possess the gene coding for the Bordetella type three secretion system effector A (bteA) toxin that became a focus of interest since it was demonstrated that B. pertussis Japanese non-vaccine-type isolates produce BteA unlike vaccine-type isolates. We thus explored the in-vitro production of BteA in B. pertussis isolates collected in France during periods of different vaccine policy as well as in B. parapertussis and B. bronchiseptica isolates. We also analyzed the in-vivo induction of anti-BteA antibodies after infection with different isolates of the three species. We produced a recombinant His6-tagged BteA (rBteA) protein. Specific rBteA polyclonal serum was prepared which enabled us to screen Bordetella isolates for in-vitro BteA production: 99.0% (293/296) of tested B. pertussis isolates, including French vaccine strains, and 97.5% (79/81) of B. bronchiseptica isolates produced BteA in-vitro but only the latter was capable of inducing an in-vivo immune response. No in-vitro or in-vivo production of BteA was detected by any of the B. parapertussis isolates tested.

Re-emergence of pertussis: what are the solutions?

Whooping cough, due to Bordetella pertussis and Bordetella parapertussis, is an important cause of childhood morbidity and mortality. Despite widespread pertussis immunization in childhood, there are an estimated 50 million cases and 300,000 deaths due to pertussis globally each year. Infants who are too young to be vaccinated, children who are partially vaccinated and fully-vaccinated persons with waning immunity are especially vulnerable to disease. Since pertussis is one of the vaccine-preventable diseases on the rise, additional vaccine approaches are needed. These approaches include vaccination of newborns, additional booster doses for older adolescents and adults, and immunization of pregnant women with existing vaccines. Innovative new vaccines are also being studied. Each of these options will be discussed and their potential impact on pertussis control assessed.

Identification of a CO2 responsive regulon in Bordetella.

Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO(2). Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO(2) conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO(2) was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO(2). The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO(2), indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO(2) concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.

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.

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.

Patterns of Bordetella parapertussis respiratory illnesses: 2008-2010.

Clinical specimens from 9 states during 2008-2010 were tested by PCR for Bordetella pertussis and Bordetella parapertussis. Of the positive samples, 13.99% were identified as B. parapertussis. It was concluded that B. parapertussis infections are more common than previously realized and contribute to cases thought to be vaccine failures.

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.

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.

Acellular pertussis vaccination facilitates Bordetella parapertussis infection in a rodent model of bordetellosis.

Despite over 50 years of population-wide vaccination, whooping cough incidence is on the rise. Although Bordetella pertussis is considered the main causative agent of whooping cough in humans, Bordetella parapertussis infections are not uncommon. The widely used acellular whooping cough vaccines (aP) are comprised solely of B. pertussis antigens that hold little or no efficacy against B. parapertussis. Here, we ask how aP vaccination affects competitive interactions between Bordetella species within co-infected rodent hosts and thus the aP-driven strength and direction of in-host selection. We show that aP vaccination helped clear B. pertussis but resulted in an approximately 40-fold increase in B. parapertussis lung colony-forming units (CFUs). Such vaccine-mediated facilitation of B. parapertussis did not arise as a result of competitive release; B. parapertussis CFUs were higher in aP-relative to sham-vaccinated hosts regardless of whether infections were single or mixed. Further, we show that aP vaccination impedes host immunity against B. parapertussis-measured as reduced lung inflammatory and neutrophil responses. Thus, we conclude that aP vaccination interferes with the optimal clearance of B. parapertussis and enhances the performance of this pathogen. Our data raise the possibility that widespread aP vaccination can create hosts more susceptible to B. parapertussis infection.

Chemical synthesis of UDP-Glc-2,3-diNAcA, a key intermediate in cell surface polysaccharide biosynthesis in the human respiratory pathogens B. pertussis and P. aeruginosa.

In connection with studies on lipopolysaccharide biosynthesis in respiratory pathogens we had a need to access potential biosynthetic intermediate sugar nucleotides. Herein we report the chemical synthesis of uridine 5'-diphospho 2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid (UDP-Glc-2,3-diNAcA) (1) from N-acetyl-D-glucosamine in 17 steps and approximately 9% overall yield. This compound has proved invaluable in the elucidation of biosynthetic pathways leading to the formation of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid-containing polysaccharides.

Infection with Bordetella parapertussis but not Bordetella pertussis causes pertussis-like disease in older pigs.

The 3 major Bordetella species--namely, B. pertussis, B. parapertussis, and B. bronchiseptica--can be distinguished by their different host ranges. B. bronchiseptica infects a wide range of mammals (including humans), whereas B. pertussis infects only humans and, under experimental conditions, mice and pigs. In contrast, B. parapertussis, also a causative agent of pertussis, displays a unique host specificity with 2 subgroups, one infecting only humans and the other infecting only sheep. Here, we show that both strains of B. parapertussis also infect older piglets when delivered intrapulmonarily. Infected piglets displayed mild fever and respiratory symptoms, such as coughing and breathing difficulties. Importantly, transmission was observed between infected and noninfected piglets. In tracheal organ cultures, adherence to ciliated epithelial cells was observed. Furthermore, both strains of B. parapertussis displayed higher resistance than B. pertussis to neutralization by porcine beta-defensin 1 in the respiratory tract, which has been demonstrated to be associated with protection against B. pertussis disease in older pigs. The development of this new model will assist us in better understanding the pathogenesis of this disease and in the development of more-effective vaccines against pertussis.

Whooping cough due to Bordetella parapertussis: an unresolved problem.

Bordetella parapertussis is one of the bacteria that causes whooping cough. However, little attention has been paid to this bacterium because it causes a milder illness than Bordetella pertussis and the rate of detection is low, even though research suggests that pertussis vaccines have limited efficacy against B. parapertussis infection. However, recent studies have revealed high rates of detection in patients with whooping cough in some field studies. In this review, the relevant studies of B. parapertussis are summarized and it is demonstrated that it is now necessary to pay greater attention to infections by this bacterium.