Pertussis Toxin S1 Research Articles

Expression of Bordetella pertussis Antigens Fused to Different Vectors and Their Effectiveness as Vaccines.

Pertussis is an acute respiratory tract infection caused by Bordetella pertussis. Even though its current vaccine coverage is relatively broad, they still have some shortcomings such as short protection time and might be incapable of blocking the spread of the disease. In this study, we developed new pertussis vaccine candidates by separately fusing three pertussis antigens (B. pertussis fimbriae 2 “Fim2”, pertussis toxin S1 subunit “PtxS1”, and filamentous hemagglutinin “FHA1877–2250”) to each of two immune-boosting carrier proteins (B subunits of AB5 toxin family: cholera toxin B subunit “CTB” and shiga toxin B subunit “StxB”). We then immunized mice with these fusion antigens and found that they significantly increased the serum antibody titers and elicited high bactericidal activity against B. pertussis. After CTB-or StxB-fused antigen-immunized mice were challenged with a non-lethal dose of B. pertussis, the bacterial loads in different tissues of these mice were significantly reduced, and their lung damage was nearly invisible. Furthermore, we also demonstrated that these candidate vaccines could provide strong prophylactic effects against a lethal challenge with B. pertussis. Overall, our candidate vaccines conferred better immune protection to mice compared with pertussis antigen alone. This B5 subunit-based vaccine strategy provides a promising option for vaccine design.

Subcutaneous administration of a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis induces mucosal and systemic immune responses.

Objective(s):After decades of containment, pertussis disease, caused by Bordetella pertussis seems to be re-emerging and still remains a major cause of reported vaccine-preventable deaths worldwide. The current licensed whole-cell vaccines display reactogenicity while acellular vaccines are expensive and do not induce Th1-type immune responses that are required for optimum protection against the disease. Thus, there is an urgent need to develop new vaccines and the recombinant technology seems to be the method of choice for this purpose. The present study was an attempt to develop a new, simplified, cost-effective and well-defined vaccine against Bordetella pertussis, with capacity to induce a Th1 response. Materials and Methods:A fusion DNA fragment encoding the N-terminal region of pertussis toxin S1 subunit and filamentous hemagglutinin type 1 immunodominant domain was constructed and the corresponding fusion protein (F1S1) was produced in Escherichia coli. F1S1 in conjunction with imiquimod was administered by subcutaneous (SC) and intranasal (IN) routes to BALB/c mice. Results:This vaccine formulation could elicit high levels of IFN-γ, serum IgG (with higher IgG2a/IgG1 ratio) and lung IgA after the SC and, to a lesser extent, following the IN administration. Conclusion:Our results indicate that the above-mentioned important proteins of B. pertussis could be successfully produced in E. coli as a single fusion protein. Furthermore, this protein could induce proper systemic and mucosal immune responses after administration via SC or IN routes.

Polymorphism in Bordetella Pertussis Virulence Factors in Serbia before Switch from Whole Cell to a Cellular Pertussis Vaccine

Background: Mass vaccination has significantly reduced the incidence of pertussis; however, the disease is re-emerging even in some countries with high vaccination coverage. In Serbia, whole cell pertussis vaccine was introduced in 1957 and it has been used since 2014. Methods: To monitor changes in bacterial population, 77 isolates collected from 1953 to 2013 were studied. The methods included serotyping of fimbriae (Fim), genotyping of pertactin (prn), pertussis toxin S1 subunit (ptxA) and pulsed-field gel electrophoresis analysis (PFGE). Results: Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. In the period 1961-1979 genotype ptxA1 became as common as genotype ptxA2. After that, during the period of 1980-1989, the predominant ptx genotype was ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981. The prn2 allele was only found in one strain isolated in 1984, two of the four strains isolated in 2000 and three strains from 2011. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The four vaccine strains represented four PFGE profiles. Twenty-two (40%) isolates tested by PFGE produced 22 distinct profiles. Twenty-four (43%) isolates had unique Serbian profiles (BpSBR). Conclusion: The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used for 57 years.

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation.

Pertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established. We addressed these issues with the use of a surface plasmon resonance system to quantify the cytosolic pool of PTS1 from intoxicated cells. Only 3% of surface-associated PTS1 reached the host cytosol after 3 h of toxin exposure. This represented, on average, 38,000 molecules of cytosolic PTS1 per cell. Cells treated with a proteasome inhibitor contained larger quantities of cytosolic PTS1. Stabilization of the dissociated PTS1 subunit with chemical chaperones inhibited toxin export to the cytosol and blocked PT intoxication. ERAD-defective cell lines likewise exhibited reduced quantities of cytosolic PTS1 and PT resistance. These observations identify the unfolding of dissociated PTS1 as a trigger for its ERAD-mediated translocation to the cytosol.

Sequence Variation of the Pertussis Toxin S1 Subunit Encoding Gene in the Clinical Isolates of Bordetella pertussis in Iran

Background: Whooping cough (pertussis) is an acute respiratory disease caused by Bordetella pertussis (B. pertussis). Pertussis toxin is an important virulence factor of B. pertussis and plays a major role in the immune and inflammatory responses. Likewise, allelic variations in the genes of virulence factors have led to the non-responsiveness of the new strains to both whole-cell and acellular vaccines. Given the importance of pertussis vaccine, we sought to address the lack of fundamental studies on the polymorphisms of the virulence genes of B. pertussis in Iran. Objectives: The aim of this study was to identify the polymorphisms of the pertussis toxin S1 subunit (ptxS1) gene in the circulating strains and compare them to the vaccine strain. Patients and Methods: In this study, 50 strains of B. pertussis isolated from patients with pertussis were investigated in the pertussis reference laboratory of Pasteur institute of Iran. Cultivation, biochemical tests, and the specific antisera were used to confirm B. pertussis. The sequencing of the polymerase chain reaction products was performed to determine the ptxS1 alleles, and B. pertussis 134 was studied as the vaccine strain. Results: The results showed that all the strains had the dominant allele ptxS1A. There were differences between the alleles of the clinical strains and the vaccine strain. Conclusions: In recent years, a significant increase in the incidence of pertussis has been reported worldwide. Our findings regarding the allelic shift of the ptxS1 gene are similar to those reported in many European and American countries showing the difference of the dominant allele of ptxS1 between the circulating isolates and the vaccine strains.

Changes in genetic diversity of the Bordetella pertussis population in Serbia between 1953 and 2011

Mass vaccination has significantly reduced the incidence of pertussis, however, the disease is re-emerging, even in some countries with high vaccination coverage. In Serbia, whole cell pertussis vaccine was introduced in 1957. To monitor changes in bacterial population, 77 isolates collected from 1953 to 2011 were studied. The methods included serotyping of fimbriae (Fim), genotyping of pertactin (prn) and pertussis toxin S1 subunit (ptxA). A shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. In the period 1961-1979, the genotype ptxA1 became as common as genotype ptxA2. After that, during the period 1980-1989, the predominant ptx genotype was ptxA1. The reappearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981. The prn2 allele was only found in one strain isolated in 1984, two of the four strains isolated in 2000 and in three isolated strains from 2011. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant thereafter. The results of this study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used.

Direct molecular typing of Bordetella pertussis from clinical specimens submitted for diagnostic quantitative (real-time) PCR

Molecular typing of Bordetella pertussis is routinely performed on bacterial isolates, but not on DNA extracted from nasopharyngeal aspirates or pernasal swabs submitted for diagnostic real-time PCR (qPCR). We investigated whether these DNA extracts were suitable for multilocus variable-number tandem repeat analysis (MLVA) and DNA sequence-based typing. We analysed all the available qPCR-positive samples received by our laboratory from patients <1 year of age between January 2008 and August 2010. Eighty-one per cent (106/131) of these generated a complete MLVA profile. This rose to 92 % (105/114) if only samples positive for both of the two targets used for the B. pertussis PCR (insertion element IS481 and pertussis toxin promoter ptxP) were analysed. Sequence-based typing of the pertactin, pertussis toxin S1 subunit and pertussis promoter regions (prn, ptxA and ptxP) was attempted on 89 of the DNA extracts that had generated a full MLVA profile. Eighty-three (93 %) of these produced complete sequences for all three targets. Comparison of molecular typing data from the 89 extracts with those from 111 contemporary bacterial isolates showed that the two sources yielded the same picture of the B. pertussis population [dominated by the MLVA-27 prn(2) ptxA(1) ptxP(3) clonal type]. There was no significant difference in MLVA type distribution or diversity between the two sample sets. This suggests that clinical extracts can be used in place of, or to complement, bacterial cultures for typing purposes (at least, in this age group). With small modifications to methodology, generating MLVA and sequence-based typing data from qPCR-positive clinical DNA extracts is likely to generate a complete dataset in the majority of samples from the <1 year age group. Its success with samples from older subjects remains to be seen. However, our data suggest that it is suitable for inclusion in molecular epidemiological studies of the B. pertussis population or as a tool in outbreak investigations.

Role of the Cell Wall Microenvironment in Expression of a Heterologous SpaP-S1 Fusion Protein by Streptococcus gordonii

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.

Fusion expression and immunogenicity of Bordetella pertussis PTS1-FHA protein: implications for the vaccine development

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.

Characteristics of the antigenic genes of four clinical Bordetella pertussis strains

Objectives To analyze the characteristics of antigenic genes of clinical Bordetella pertussis strains recently isolated by analyzing the sequence of pertussis toxin S1 subunit(ptxS1) , pertactin (Prn) , fimbriae 2 (Fim2) and fimbriae 3 (Fim3 ) genes of four clinical isolates. Methods The 4 clinical isolates were collected in 2002 in Shijiazhuang of Hebei province. Four strains were isolated from pertussis patient's nasopharyngeal aspirate. ptxS1, Prn, Fim2 and Fim3 genes of these strains were amplified and sequenced. The sequences of those genes were compared with those of the isolates in GenBank and the isoaltes used in the production of pertussis vaccine in China. Results The results of the gene sequencing showed the four clinical isolates belonged to ptxS1 A type, which were different from those in vaccine strains. In addition, three Prn and three Fim'3 variants were observed in the four clinical isolates. Sequence analysis showed that the nucleotide sequence and deduced amino acid sequence of those strains had more than 99% identity with those in vaccine strains. The phylogenetic trees of those genes also showed these strains had a higher level of similarity with other Bordetella pertussis strains. Conclusion The four clinical isolates are different from vaccine strains in four antigenic genes, which laid a foundation for further studies on pertussis epidemiology,quality control and development of pertussis vaccine in China. Key words: Bordetella pertussis; Pertussis toxin; Bacterial outer membrane proteins; Virulence factors,bordetella; Antigens,bacterial; Fimbriae proteins; Sequence analysis

Bordetella pertussis vaccine strains and circulating isolates in Serbia

In Serbia, whole cell pertussis vaccine was introduced in 1957. Current composition of the vaccine has been used since 1985 and contains four autochthonous strains of Bordetella pertussis isolated from 1957 to 1984. To monitor changes in bacterial population, 70 isolates collected from 1953 to 2000 were studied together with the vaccine strains. The methods included serotyping of fimbriae (Fim), genotyping of pertactin ( prn) and pertussis toxin S1 subunit ( ptxA), and pulsed-field gel electrophoresis analysis. Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. All isolates from 1980 to 1984 harbored ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981 and 1984. The allele prn2 was found only in two strains isolated in 2000. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The Serbian vaccine strains showed differences in ptxA and prn. The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used.

Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

Role of PCR in the diagnosis of pertussis infection in infants: 5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007

As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged <or=6 months admitted to a paediatric intensive care unit or paediatric ward with a respiratory illness compatible with pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9 % ptxA-pr and IS481; 3.3 % IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of <or=6 months, most patients (130/138) with evidence of Bordetella spp. by PCR were <or=3 months old. The overall percentage increase in laboratory-confirmed cases due to PCR compared with culture for the 5 year period described ranged from 9 to 26 % per year (mean 19 %). Real-time PCR is an invaluable tool both for enhanced epidemiological surveillance and for the provision of a rapid diagnosis of pertussis where results can affect patient and contact management.

The Role of Gi Signaling in Hematopoiesis.

Hematopoiesis implies a number of different cellular events from self-renewal of stem cells and proliferation to differentiation and migration. All these steps require tight regulation by signaling, and many cytokine signaling pathways have been described. However the signaling mechanisms that govern trafficking, proliferation, survival and differentiation of hematopoietic stem cells and other hematopoietic cell types are poorly understood. G-protein coupled receptors (GPCRs) can regulate many of these processes, and the Gi-coupled receptor CXCR4 is known to play a key role. Roles for other Gi-coupled receptors are just beginning to be uncovered, and given the functions of Gi-signaling in regulation of cell homing, proliferation and survival, it seems likely that Gi-coupled GPCR signaling plays an important role in hematopoiesis. By using a genetic approach – knockout of Gi function in hematopoietic lineages – we are probing the role of Gi-coupled GPCR's in hematopoiesis to determine which receptors are involved. There are 6 sub-types in the Gi family, and it has been difficult to eliminate all Gi function by gene knockout. However, all Gi family members, except for Gz, are sensitive to inhibition by pertussis toxin (PTX). To ablate Gi signaling in specific cell lineages, we utilized a mouse in which the ROSA26 gene was modified to drive expression of pertussis toxin S1 subunit (PTX) after Cre-mediated excision of a floxed stop cassette. To study the effect of inhibition of Gi signaling in hematopoiesis, we crossed mice carrying one allele of the hematopoietic lineage-specific Cre transgene Vav-iCre with the ROSA26PTX/PTX mice. Half the offspring from this cross carry Vav-Cre Tg+/o: ROSA26PTX/+ (Vav-PTX mice), the other half transgene-negative ROSA26PTX/+ (controls). Vav-PTX mice were born at the expected Mendelian rate. Pups were grossly normal but consistenly displayed a smaller thymus. However, all Vav-PTX mice died between day 2 and 14, presumably from pneumococcal pneumonia. Neonatal Vav-PTX mice showed fewer hematopoietic cells in bone marrow, which was also observed in Vav-PTX embryos collected at E18.5. Thus, defective hematopoiesis was not secondary to postnatal infection or general failure to thrive. Competitive transplantation studies using fetal liver cells from Vav-PTX embryos or control embryos collected at E14.5 showed decreased short-term reconstitution (5weeks) and severely impaired long-term reconstitution (10 and 16 weeks) compared to control fetal liver cells. In contrast, fetal liver cells from CXCR4−/− embryos still showed partial reconstitution after 16 weeks. These data are consistent with defective homing of hematopoietic stem cells (HSCs) from fetal liver to bone marrow, but also with decreased proliferation, survival, and/or differentiation of HSCs once they reach their marrow niche. When fetal liver cells were grown in vitro, cells from Vav-PTX embryos showed decreased survival and proliferation when stimulated with cytokine promoting either B-cell, erythroid or granulocyte/macrophage lineages. This suggested a role for Gi signaling in proliferation in all hematopoietic lineages. In addition, early data suggest that the population of Lin−, c-kit+, Sca1+ cells in fetal liver from Vav-PTX embryos is decreased compared to their littermate controls. Our results suggest a key role for Gi and hence Gi-coupled GPCRs in several mechanisms required for normal hematopoiesis.

Abstract 939: Galpha-i Signaling in Neural Crest Cells is Required for Normal Migration of Cardiac Neural Crest Cells, Cardiac Development, and Survival

G protein coupled receptors (GPCRs) have long been known to play crucial roles in transducing environmental signals to the adult cardiovascular system. In recent years, the roles of G protein-mediated signaling pathways in orchestrating the interactions of different tissues during cardiovascular development have become increasingly evident. To analyze the role of G protein signaling pathways in vivo we have generated mice where the function of the heterotrimeric G alpha subunit Gai can be ablated in a cell type specific manner utilizing the Cre-loxP system. We have mated these mice to two different neural crest-specific Cre lines in order to probe the effects of loss of Gai mediated signaling on the ability of neural crest cells (NCC) to contribute to the developing outflow tract and aortic arch arteries. METHODS: We have generated mice that express the Gai-inhibiting pertussis toxin S1 subunit (PTX) from the ROSA26 locus in a Cre recombination dependent manner (ROSA-PTX mice). These were mated to mice expressing either the Wnt1 Cre or P0 Cre transgene. Wnt1Cre is active in both premigratory and migratory NCC, whereas P0Cre is active only in migratory NCC and their derivatives. RESULTS: P0Cre-ROSA-PTX mice were normal at birth and demonstrated no structural heart defects. In contrast, Wnt1Cre-ROSA-PTX mice were present in normal numbers at late gestation but died perinatally due in part to cardiac outflow tract defects. Excision reporter and in situ hybridization studies suggest this is secondary to a delay/blockage of cardiac NCC migration into the developing outflow tract. NCC migration into the pharyngeal arches was unaffected in these mice and no craniofacial, thymic, or aortic arch abnormalities were observed. CONCLUSIONS: These results indicate that Gai-mediated signaling is required in premigratory or early migratory cardiac NCC for normal development of the outflow tract. In contrast, endothelin A receptor knockout mice (currently the only GPCR knock out with a neural crest phenotype) are thought to exhibit defects of postmigratory NCC function. RNA profiling of NCC for GPCRs involved in this Gai-dependent pathway has revealed several potential candidate receptors, including orphan receptors. Further analysis of these receptors is underway.

The Pertussis Toxin S1 Subunit Is a Thermally Unstable Protein Susceptible to Degradation by the 20S Proteasome

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.

Development of safer pertussis DNA vaccine expressing non-toxic C180 polypeptide of pertussis toxin S1 subunit

A toxic N-terminal 180-amino-acid fragment (C180) of pertussis toxin S1 subunit has the most potent ability to induce protective immunity against pertussis toxin (PT) following DNA-based immunization [Kamachi K, Arakawa Y. Infect Immun 2004;72:4293–6]. For the development of a safer pertussis DNA vaccine, three plasmids encoding mutant C180 (C180-R9K, C180-E129G and C180-R9K/E129G) were constructed and tested for their protective immunogenicity and cytotoxicity. All of the gene gun delivery of the plasmid, performed by inserting the mutant C180 gene into a mammalian expression vector pcDNA3.1, successfully induced anti-PT IgG antibody production without the loss of immunogenicity in mice. The immunizations of mice with the plasmids significantly inhibited leukocytosis-promoting activity by PT. Among stably transfected Chinese hamster ovary (CHO) cells expressing mutant C180, the expression of C180-R9K and C180-R9K/E129G was non-toxic to the transfectants, confirming that these mutant C180s have no cytotoxicity to mammalian cells. These results indicate that C180-R9K and C180-R9K/E129G genes, especially C180-R9K/E129G, are candidates for safe and effective antigen DNAs in the development of pertussis DNA vaccine.

Population dynamics of Bordetella pertussis in Finland and Sweden, neighbouring countries with different vaccination histories

Pertussis is an infectious disease of the respiratory tract in humans caused by Bordetella pertussis. Despite extensive vaccinations, pertussis has remained endemic and re-emerged. In Finland, a whole-cell pertussis vaccine has been used since 1952 with high coverage. In Sweden, whole-cell vaccinations were introduced in 1953 but ceased in 1979, and pertussis vaccinations with acellular vaccines were introduced in 1996. Two epidemic peaks occurred in Sweden in 1999 and 2002 and in Finland in 1999 and 2003. We compared Finnish ( N = 193) and Swedish ( N = 455) B. pertussis isolates circulating in 1998–2003 together with vaccine strains used in these neighbouring countries with different vaccination histories. The isolates were analysed by serotyping, genotyping of pertussis toxin S1 subunit and pertactin, and pulsed-field gel electrophoresis. The results suggest that the sequential epidemics were caused by clonal expansion of a certain B. pertussis strain possibly transmitted from Sweden to Finland. The roles of antigenic variation in immunity-driven evolution of B. pertussis in both countries are discussed.

Expression of a pertussis toxin S1 fragment by inducible promoters in oralStreptococcusand the induction of immune responses during oral colonization in mice

Previous work aimed at developing a live oral vaccine expressing pertussis toxin S1 fragment on the surface of the bacterium Streptococcus gordonii elicited a lower than expected antibody response, perhaps because of low antigen expression. In this study, in-frame promoter fusions were constructed to investigate whether an increase in antigen production by the streptococcal vaccine strain results in a better antibody response. The promoters tested were (i) the Streptococcus mutans sucrose-inducible fructosyltransferase (ftf) promoter and (ii) the Bacillus subtilis/Escherichia coli chimeric tetracycline-inducible xyl/tetO promoter. Each of these two promoters was placed upstream of the spaP/s1 fusion gene to drive its expression. The constructs were introduced into S. gordonii DL1 and S. mutans 834. The inducibility of the promoters was confirmed through the determination of SpaP/S1 production via Western blottings. Induced production of SpaP/S1 was observed in S. gordonii and S. mutans with each of the promoters, but the level of expression was the highest in S. mutans, using the xyl/tetO promoter. Thus, S. mutans carrying the xyl/tetO/spaP/s1 construct (S. mutans PM14) was used in oral colonization studies in BALB/c mice. Streptococccus mutans PM14 was able to colonize the animals for the 14-week duration of experimentation. A mucosal IgA response was observed in all the treatment groups but was highest in mice receiving tetracycline induction. In the mouse model of Bordetella pertussis respiratory infection, animals colonized with S. mutans PM14 showed a decreased in B. pertussis lung colony count (P = 0.03) on day 3 compared with control mice colonized by the parent S. mutans 834.

ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens

Unlike many bacterial pathogens, Mycoplasma pneumoniae is not known to produce classical toxins, and precisely how M. pneumoniae injures the respiratory epithelium has remained a mystery for >50 years. Here, we report the identification of a virulence factor (MPN372) possibly responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in humans. We show that M. pneumoniae MPN372 encodes a 68-kDa protein that possesses ADP-ribosyltransferase (ART) activity. Within its N terminus, MPN372 contains key amino acids associated with NAD binding and ADP-ribosylating activity, similar to pertussis toxin (PTX) S1 subunit (PTX-S1). Interestingly, MPN372 ADP ribosylates both identical and distinct mammalian proteins when compared with PTX-S1. Remarkably, MPN372 elicits extensive vacuolization and ultimate cell death of mammalian cells, including distinct and progressive patterns of cytopathology in tracheal rings in organ culture that had been previously ascribed to infection with WT virulent M. pneumoniae. We observed dramatic seroconversion to MPN372 in patients diagnosed with M. pneumoniae-associated pneumonia, indicating that this toxin is synthesized in vivo and possesses highly immunogenic epitopes.