Modeling the effect of oxidative stress on Bordetella pertussis fermentations.

A mathematical model is proposed for Bordetella pertussis with the main goal to better understand and describe the relation between cell growth, oxidative stress and NADPH levels under different oxidative conditions. The model is validated with flask experiments conducted under different conditions of oxidative stress induced by high initial glutamate concentrations, low initial inoculum and secondary culturing following exposure to starvation. The model exhibited good accuracy when calibrated and validated for the different experimental conditions. From comparisons of model predictions to data with different model mechanisms, it was concluded that intracellular reactive oxidative species only have an indirect effect on growth rate by reacting with NADPH and thereby reducing the amount of NADPH that is available for growth.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches.

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

A New Vaccine Delivery Vehicle and Adjuvant Candidate: Bordetella pertussis Inactivated Whole Cells Entrapped in Alginate Microspheres.

There is no doubt about the whole cell pertussis vaccine efficacy, but it is necessary to improve the vaccine quality specially to decrease its toxicity by obtaining good immunogenicity with low bacterial content. In this work, under optimum condition inactivated B. pertussis bacteria cells entrapped with alginate microparticles were fabricated and in vivo immunogenicity and ptency of new microparticle based vaccine were evaluated in mice. Microspheres loaded with inactive B. pertussis bacterium cells were prepared via an emulsification method and analyzed for morphology, size, polydispersity index, loading efficiency, loading capacity, release profile and in vivo potency. The inactivated bacterial suspension mixture prepared in this work was nontoxic and showed potent ED50 (1:333 of human dose) and preserved agglutinins 1, 2, 3. The optimum conditions for the preparation of microparticles were achieved at alginate concentration 3.8% (w/v), CaCl2 8% (w/v), PLL 0.1% (w/v), lipophilic surfactant 0.22 (%w/v), hydrophilic surfactant 3.6 (%w/v), cross linking time 3min, homogenization rate 600 rpm, and alginate to CaCl2 solution ratio 4. Both empty and B. pertussis loaded microparticles exhibited smooth surface texture and relatively spherical shape. The B. pertussis encapsulated microspheres fabricated under optimized conditions showed mean particle size 151.1 µm, polydispersity index 0.43, loading efficiency 89.6%, loading capacity 36.3%, and relatively constant release rate lasted to 15 days. In vivo immunogenicity and protection study against wild type challenge showed strongly higher potency (approximately 2.5 fold) of encapsulated B. pertussis organisms than non-encapsulated conventional aluminum hydroxide adsorbed vaccine. It can be concluded that microencapsulation of inactive B. pertussis cells appears to be a suitable approach for improving the wP vaccine quality, specially by obtaining good immunogenicity with low bacterial content.

An In Vitro Assay for Substrate Translocation by FhaC in Liposomes.

The two-partner secretion (TPS) pathway is used by gram-negative bacteria to secrete a large family of virulence exoproteins. Its name is derived from the fact that it involves two proteins, a secreted TpsA protein and a cognate TpsB transporter in the outer membrane. A typical TPS system is represented by the filamentous hemagglutinin FhaB (TpsA protein) and its transporter FhaC (TpsB protein) of Bordetella pertussis. Results from mutational analysis and heterologous expression experiments suggested that FhaC is essential for FhaB translocation across the outer membrane of bacteria. We have devised a cell-free biochemical assay to reconstitute in vitro the translocation of FhaB into reconstituted membrane vesicles. Thereby the clearest evidence has been provided that the single ß-barrel FhaC protein serves as the sole translocator to transport FhaB across the outer membrane. This is the first in vitro assay for protein secretion across the Escherichia coli outer membrane and the detailed protocol described here should be amenable to modifications and application to the analysis of related protein transport events occurring at the outer membranes of gram-negative bacteria.

Dose-response models for selected respiratory infectious agents: Bordetella pertussis, group a Streptococcus, rhinovirus and respiratory syncytial virus.

BACKGROUND: Dose-response assessment is one step in quantitative microbial risk assessment (QMRA). Four infectious microbes capable of causing respiratory diseases important to public health, and for which dose-response functions have not been available are: Bordetella pertussis (whooping cough), group A Streptococcus (pharyngitis), rhinovirus (common cold) and respiratory syncytial virus (common cold). The objective of this study was to fit dose-response functions for these microbes to published experimental data. METHODS: Experimental infectivity data in human subjects and/or animal models were identified from the peer-reviewed literature. The exponential and beta-Poisson dose-response functions were fitted using the method of maximum likelihood, and models compared by Akaike's Information Criterion. RESULTS: Dose-response functions were identified for each appropriate data set for the four infectious microbes. Statistical and graphical measures of fit are presented. CONCLUSIONS: With the fitted dose-response functions it will be possible to perform QMRA for these microbes. The dose-response functions, however, have a number of limitations associated with the route of exposure, use of animal hosts, and quality of fit. As a result, thoughtfulness must be used in selecting one dose-response function for a QMRA, and the function should be recognized as a significant source of uncertainty. Nonetheless, QMRA offers a transparent, systematic framework within which to understand the mechanisms of disease transmission, and evaluate interventions.

The ins and outs of pertussis toxin.

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the most complex soluble bacterial proteins. It is actively secreted through the B. pertussis cell envelope by the Ptl secretion system, a member of the widespread type IV secretion systems. The toxin is composed of five subunits (named S1 to S5 according to their decreasing molecular weights) arranged in an A-B structure. The A protomer is composed of the enzymatically active S1 subunit, which catalyzes ADP-ribosylation of the α subunit of trimeric G proteins, thereby disturbing the metabolic functions of the target cells, leading to a variety of biological activities. The B oligomer is composed of 1S2:1S3:2S4:1S5 and is responsible for binding of the toxin to the target cell receptors and for intracellular trafficking via receptor-mediated endocytosis and retrograde transport. The toxin is one of the most important virulence factors of B. pertussis and is a component of all current vaccines against whooping cough.

Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice.

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

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.

Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller.

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis.

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.

Online automatic tuning and control for fed-batch cultivation.

Performance of controllers applied in biotechnological production is often below expectation. Online automatic tuning has the capability to improve control performance by adjusting control parameters. This work presents automatic tuning approaches for model reference specific growth rate control during fed-batch cultivation. The approaches are direct methods that use the error between observed specific growth rate and its set point; systematic perturbations of the cultivation are not necessary. Two automatic tuning methods proved to be efficient, in which the adaptation rate is based on a combination of the error, squared error and integral error. These methods are relatively simple and robust against disturbances, parameter uncertainties, and initialization errors. Application of the specific growth rate controller yields a stable system. The controller and automatic tuning methods are qualified by simulations and laboratory experiments with Bordetella pertussis.

Crystal structures of two Bordetella pertussis periplasmic receptors contribute to defining a novel pyroglutamic acid binding DctP subfamily.

Gram-negative bacteria have developed several different transport systems for solute uptake. One of these, the tripartite ATP independent periplasmic transport system (TRAP-T), makes use of an extracytoplasmic solute receptor (ESR) which captures specific solutes with high affinity and transfers them to their partner permease complex located in the bacterial inner membrane. We hereby report the structures of DctP6 and DctP7, two such ESRs from Bordetella pertussis. These two proteins display a high degree of sequence and structural similarity and possess the "Venus flytrap" fold characteristic of ESRs, comprising two globular alpha/beta domains hinged together to form a ligand binding cleft. DctP6 and DctP7 both show a closed conformation due to the presence of one pyroglutamic acid molecule bound by highly conserved residues in their respective ligand binding sites. BLAST analyses have revealed that the DctP6 and DctP7 residues involved in ligand binding are strictly present in a number of predicted TRAP-T ESRs from other bacteria. In most cases, the genes encoding these TRAP-T systems are located in the vicinity of a gene coding for a pyroglutamic acid metabolising enzyme. Both the high degree of conservation of these ligand binding residues and the genomic context of these TRAP-T-coding operons in a number of bacterial species, suggest that DctP6 and DctP7 constitute the prototypes of a novel TRAP-T DctP subfamily involved in pyroglutamic acid transport.

Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques.

This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.

Temporal expression of pertussis toxin and Ptl secretion proteins by Bordetella pertussis.

Pertussis toxin is an AB(5) toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.

Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years.

We compared Bordetella pertussis isolates collected in France over the last 10 years, the vaccine strains used for more than 30 years, and isolates collected before the introduction of generalized vaccination. The analysis included serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the pt S1 gene, encoding the S1 subunit of pertussis toxin, and the prn gene, encoding the adhesin pertactin. We found that the incidence of infection increases every 3 years. Ninety-five per cent of the isolates analyzed express type 3 fimbriae. Most of the isolates circulating since 1991, unlike the vaccinal strains, express a type A pertussis toxin and a type 2 pertactin. The isolates could be classified into five major groups by pulsed-field gel electrophoresis. Most of these groups correlated with the pertactin type expressed by the isolates. Pulsed-field gel electrophoresis is more discriminative than sequencing particular genes since it could differentiate isolates expressing type 2 pertactin into two subgroups: those circulating in 1993 to 1997 and those circulating in 1997 to 2001. This observation suggests that there has been continuous evolution of the B. pertussis population.

Effect of heptakis (2,6-0-dimethyl)beta-cyclodextrin on cell growth and the production of pertussis toxin and filamentous hemagglutinin in Bordetella pertussis.

Heptakis (2,6-0 dimethyl)beta-cyclodextrin (MeCD) which permits the growth of single colonies of Bordetella pertussis Tohama phase I on Stainer-Scholte medium solidified with agar also enhanced the production of pertussis toxin (PT). More than one hundred times the amount of PT was produced in Stainer-Scholte medium with MeCD in shake culture than was produced in MeCD-free medium. A maximum of 50 mg PT protein was produced per liter of culture broth as estimated by in vitro and in vivo assays. The production of filamentous hemagglutinin (FHA) was several hundred times greater when B. pertussis was grown in shake cultures with MeCD than when growth was in MeCD-free shake cultures. The FHA content of the production medium was confirmed by enzyme-linked immunosorbent assays and electron microscopy. Evaluation of an acellular vaccine containing PT and FHA detoxified with formaldehyde showed that it was protective in the intracerebral challenge mouse potency assay.

Isolation and characterization of isogenic pairs of domed hemolytic and flat nonhemolytic colony types of Bordetella pertussis.

Four different serotype strains of Bordetella pertussis, 3779BL(2)S(4), Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D(+)H(+)) wild-type colonies. Cloned D(+)H(+) colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and these subsequently grew as flat, nonhemolytic (D(-)H(-)) colonies when transferred back onto Bordet-Gengou agar. The frequency of D(-)H(-) organisms within a population of cloned D(+)H(+) was determined to be between 5 x 10(-5) and 5 x 10(-6). The D(-)H(-) colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D(+)H(+) and D(-)H(-) colony types from the four strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity, and presence of agglutinogens by agglutination. In all cases the D(-)H(-) colony types showed reduced activities or amounts of antigen compared with their D(+)H(+) parents. Freely diffusible antigens were markedly different between the two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor, and anti-353/Z adsorbed with autoclaved 353/Z. In addition, autoradiographs of (125)I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D(-)H(-) organisms appear to fulfill the criteria for phase III or phase IV in the system of Leslie and Gardner (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1954).

[Vaccine from the cell fragments of Bordetella pertussis. I. Protective, sensitizing properties and morphological characteristics of the vaccine]. / Vaktsina iz kletochnykh fragmentov Bordetella pertussis. Soobshchenie I. Zashchitnye, sensibiliziruiushche svoistva i morfologicheskaia kharacteristika vaktsiny

The authors present the results of studying the protective and sensitizing properties of a new preparation made of a ultrasonic disintegrate of pertussis microbes treated by ethyl ether. As shown by electron microscopy, the preparation consisted of the cell wall elements (the membrane), remnants of the cytoplasm and protectosome, i.e. it represented a vaccine consisting of cell fragments. In crude and sorbed condition it possessed marked protective properties (a test on mice). The content of protective units in the adsorbed preparation increased 1.5-3 times. The vaccine produced no sensitizing action, and its histamine-sensitizing activity was 3-5 times lower by protein and 5-10 times--by IOU than that of the whole-cell vaccine prepared form the same microbial suspension.