Adenylate cyclase activity during phenotypic variation of Bordetella pertussis

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.     

Effect of histamine-sensitizing factor of Bordetella pertussis on pharmacologic response of rat atria

The effects of sensitization with the histamine-sensitizing factor (HSF) of Bordetella pertussis as well as Bordetella vaccines on a pharmacologic response in rat heart preparations were determined. In normal rats the spontaneous beating of atria in vitro through the positive inotropic action produced by the addition of epinephrine was inhibited immediately by addition of acetylcholine, whereas in the B. pertussis vaccine-treated rats the exciting atria were scarcely inhibited by acetylcholine. Neither B. parapertussis nor B. bronchiseptica vaccines induced such an altered atrial response in rats. Of the B. pertussis cell components purified HSF induced the altered response at the minimal dose of 0.1 microgram per rat, and a dose of 1 microgram or more produced the maximal change. This altered atrial-inducing activity of HSF was inactivated by heating at 63 C for 30 min, and was neutralized by anti-HSF rabbit serum. The altered response rose quickly in 1 day after i.v. injection of 1 microgram of HSF, reached a plateau in 3 to 5 days, which lasted at least 14 days, and disappeared completely 56 days later. HSF failed to produce directly any functional damage to the beating atria in vitro, and to induce the altered response of the normal rat atria by incubation with as much as 10 microgram of HSF per bath (50 ml) for 4 hr. A trace stimulation was found in the normal rat atria as well as in perfused frog hearts, if HSF was given directly at a dose of 20 microgram per bath.     

Characteristics of the biological properties of a cell-free pertussis preparation

The biological properties of Bordetella pertussis antigenic complex, obtained by a technologically simple method from the medium used for the cultivation of B. pertussis, were studied. The preparation was characterized by pronounced hemagglutinating activity, toxicity, histamine-sensitizing and leukocytosis-stimulating activity and produced a cytopathogenic effect on the culture of Chinese hamster ovary cells. The detoxified preparations showed pronounced protective activity in experiments on the active and passive protection of mice. The ED50 of the preparation was 0.146 microgram of protein. In the proposed human immunization dose containing 10 micrograms of protein the detoxified preparation showed no hemagglutinating, leukocytosis-stimulating or histamine-sensitizing activity and proved to be nontoxic in the weight loss test on mice.     

Antigens of Bordetella pertussis. IV. Effect of heat, merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussi.s

Munoz, J. (Rocky Mountain Laboratory, Hamilton, Mont.), and B. M. Hestekin. Antigens of Bordetella pertussis. IV. Effect of heat, Merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussis. J. Bacteriol. 91:2175-2179. 1966.-Both histamine-sensitizing and protective activities of soluble preparations from Bordetella pertussis cells are destroyed by heating at 80 C for 0.5 hr. The histamine-sensitizing activity appeared to be more susceptible to inactivation by heat than the protective activity. Formaldehyde in a final concentration of 0.5% rapidly diminished the histamine-sensitizing ability of saline extract (SE) held at 37 C. The protective activity was clearly more resistant to inactivation by formaldehyde at similar temperature. The inactivating action of formaldehyde was slower when the concentration of SE was increased or when the mixture was kept at 2 to 5 C. Merthiolate in a final concentration of 1:10,000 had no demonstrable deleterious effects on either protective or histamine-sensitizing activity of SE.     

Effect of monoclonal antibodies on the adherence of Bordetella pertussis to Vero cells

Abstract An in vitro assay involving the adherence of Bordetella pertussis to Vero cells was used to quantify the inhibitory effects of monoclonal antibodies (McAb) on adherence. McAbs to agglutinogens (fimbriae), filamentous haemagglutinin (FHA), lymphocytosis promoting factor (LPF) and to an X-mode specific outer membrane protein were tested. X-mode cells adhered to the Vero cells to a greater extent than C-mode cells. McAb to agglutinogen 2 inhibited the adherence of B. pertussis bearing homologous agglutinogen. McAbs to LPF, FHA, agglutinogen 3 and X-mode specific outer membrane protein also partially inhibited the adherence of B. pertussis to Vero cells.     

The effect of growth conditions on adenylate cyclase activity and virulence-related properties of Bordetella pertussis

Growth of Bordetella pertussis in Stainer & Scholte medium in which the NaCl had been replaced by one of several inorganic or organic salts resulted in a large decrease in adenylate cyclase activity, histamine-sensitizing activity and in the amounts of two cell-envelope polypeptides of Mr 28000 and 30000. Although some variation between strains was observed, there was never a case where one of these properties was lost independently of the others. Cultures in which these properties were lost had decreased amounts of extracellular cAMP when compared to NaCl-grown cultures. Adenylate cyclase activity was detected in three locations of B. pertussis cultures (extracellular, extracytoplasmic but cell-associated, and cytoplasmic). After growth in medium containing high concentrations of MgSO4, enzyme activity was decreased to a similar extent in all three locations.     

Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. I. Extraction and partial purification of phagocytosis- and chemotaxis-inhibitory activities.

A new factor that inhibited phagocytosis to opsonized targets and chemotaxis of PMN was extracted from B. pertussis cells, and named PMN-inhibitory factor (PIF). Cells in phase I produced 10 times more PIF than those in phase III, and like other phase I-associated components--the hemagglutinin, the histamine-sensitizing factor and agglutinogens--PIF showed degenerative, phenotypic variation during in vitro culture of phase I bacteria. PIF was partially purified by four steps, including adsorption chromatography on Dansyl-aminononamethylene Sepharose. The resulting fraction was heterogeneous but showed little histamine-sensitizing and cytotoxic activities and was free from LPS, the hemagglutinin and a leukocyte agglutinin. The inherent resistance of B. pertussis cells, in either phase I or III, as demonstrated also in the present study, and PIF-mediated defiance against immunological defense mechanism may constitute a complex host-parasite relation in experimental infections with B. pertussis.     

Histamine-sensitizing Factor, Mouse-protective Antigens, and Other Antigens of Some Members of the Genus Bordetella

The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.     

The pyrogenicity of pertussis vaccine in mice and the factors in the vaccine responsible for this effect

The injection of whole cell pertussis vaccine into mice produced a biphasic fever reaction with two peaks appearing after about one and four hours, respectively. A method for the quantitative determination of each peak fever activity was developed and the factor responsible for each activity was investigated. The first and the second peak fever activities did not parallel each other in individual vaccines. The earlier fever activity appeared to correlate with endotoxin activity in individual vaccines while the later appeared to correlate with histamine-sensitizing factor (HSF) activity. The later peak fever activity was greatly reduced by heating the vaccine at 100 degrees C for 30 min while the first was little affected by such treatment. It was concluded that the fever activity of pertussis vaccine in mice may be ascribed to the combined actions of endotoxin and a heat-labile substance, possibly HSF.     

Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine sensitizing factor.

The effect of an extract containing the histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of mice to ovalbumin was investigated with respect to dose of antigen and adjuvant. Of particular interest was the enhancement of reaginic antibody production. In comparison to the Al(OH)3 induced production of reaginic antibody where low doses of antigen and adjuvant yield high titers of reagin, the HSF extract demonstrated optimal adjuvant activity at high doses of both adjuvant and antigen. The reaginic antibody response was maximal usually by 2 to 3 weeks post-immunization and persisted for long periods of time. The hemagglutinating antibody response was maximal at 8 to 10 weeks post-immunization. The initial treatment of mice with HSF extract plus antigen resulted in the production of memory cells since a subsequent immunization with ovalbumin alone evoked a secondary reaginic response. These observations may have implications in clinical allergy since substances similar to the pertussis factor might be produced by other microbial organisms and these substances could modulate the immunologic response of individuals to common allergens.     

In Vitro Induction of Altered Response of Rat Atria to Acetylcholine with Histamine-Sensitizing Factor of Bordetella pertussis

Purified histamine-sensitizing factor (HSF) of Bordetella pertussis induced in vitro an alteration in the pharmacologic response of rat atria to acetylcholine. Spontaneously beating atrial preparations isolated from rats were exposed to HSF at a concentration of 50 ng/ml at 37 C for 30 min to 4 hr and washed with Krebs-Ringer solution, and then tested at 1-hr intervals up to 28 hr during incubation for their responses to epinephrine and acetylcholine. At 13 hr after exposure to HSF, irrespective of the exposure period, the HSF-treated atria, in which positive-inotropic action of epinephrine was manifested, depressed the negative-inotropic response to acetylcholine. The activity of HSF was neutralized by anti-HSF serum only in the first 3 hr of exposure. Only 1.8% of the added ¹²⁵I-labeled HSF bound “specifically” to one pair of atria for the manifestation of the altered response.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.     

Interaction of Bordetella pertussis virulence components with neutrophils: effect on chemiluminescence induced by a chemotactic peptide and by intact bacteria

The effect of secreted virulence components of Bordetella pertussis on chemiluminescence (CL) of rabbit peritoneal neutrophils was determined with the chemotactic peptide N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or intact B. pertussis as the stimulus. Pertussis toxin (PT) inhibited the response to fMLP in a dose-dependent manner, although only after the neutrophils had been exposed to the toxin for greater than 15 min. Both filamentous haemagglutinin (FHA) and lipopolysaccharide (LPS) markedly enhanced the CL response to fMLP after greater than or equal to 15 min incubation with the neutrophils. Similar effects to those of B. pertussis LPS were also seen with smooth and rough LPS from Salmonella minnesota. With the lowest dose of each component which elicited a maximal effect on CL, the inhibitory effect of PT overrode the enhancing effect of FHA and B. pertussis LPS. Pre-incubation of neutrophils with PT, FHA or B. pertussis LPS caused a slight reduction in the subsequent CL response to virulent B. pertussis Tohama. Virulent (phase I, or X-mode) organisms of B. pertussis 18334 and B. pertussis Tohama induced greater neutrophil CL than their avirulent (C-mode) derivatives. There appeared to be an inverse correlation between bacterial hydrophilicity and the ability to induce neutrophil CL: X-mode bacteria were significantly less hydrophilic than C-mode organisms. Three mutants, the adenylate cyclase (AC)- and haemolysin (HLY)-deficient B. pertussis BP348, the FHA-deficient B. pertussis BP353, and the PT-deficient B. pertussis BP357, generated similar levels of CL and had similar hydrophilicity values. The hydrophilicity value of the avirulent mutant B. pertussis BP347 (deficient in AC, HLY, FHA and PT) and the CL induced by this strain were similar to those of B. pertussis C-mode organisms. Thus, the interaction of B. pertussis with neutrophils appears to be complex, reflecting both the alteration of leucocyte function by secreted virulence components of the organism and, in the absence of opsonins, the surface properties of the bacterium.     

Comparison between two types of pertussis vaccines]

We have produced pertussis vaccines with laboratory and industrial methods. The characteristic of laboratory cultivation of microorganisms is, in this context, growth on Hornibrook medium in low form flask and in stationary culture. Industrial cultivation is done in homogenous culture on a B-2 medium in fermentor. The strains utilized were isolated from whooping-cough cases in the Montreal region. The yield (org. x 10(9)/ml) obtained with an industrial cultivation of B. pertussis was 4 to 7 times higher than that reached with a laboratory cultivation of this microorganism. The non-toxicity as expressed in weight gain of mice was shown for both types of vaccine. The vaccines produced in fermentor were less histamino sensibilizing for mice than the one produced in stationary flash culture. The quality of the vaccines achieved by industrial method is easily reproducible due to the fact that enough variables can be measured.     

Comparative characteristics of the toxic properties of the whooping cough microbes]

The author presents materials concerning the study of the toxic properties of 12 pertussis cultures used in the USSR for the production of vaccines. As a result of studying the toxic properties of the microbes in the test of the change of the weight of mice, and in experiments of testing the anaphylactogenic and histamine-sensitizing activities it was revealed that the strains of pertussis microbes differed by the quantitative content in them of the thermolabile toxin, endotoxin, lymphocytosis-stimulating and histamine-sensitizing factors; therefore, the period of keeping the pertussis suspensions for 1.5 months from the moment of detoxication proved to be inadequate for some of the strains. Since the toxic properties of the strains failed to correlate with their protective activity, in recommendation of pertussis strains for the production of pertussis vaccine preference should be given to the strains with lesser toxic properties.     

The development of a new generation of pertussis vaccine

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.     

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.     

Effects of Additives on the Survival of Lactic Streptococci in Frozen Storage

Three single-strain cultures, Streptococcus lactis C₂, S. cremoris R₁, and S. diacetilactis DRC₂, were frozen and stored in skim milk, in skim milk containing apple juice, and in skim milk containing one of the following additives: glycerol (10%, v/v), dimethyl sulfoxide (10%, v/v), l-malic acid (0.5 and 2.0%, w/v), acetamide (0.5 and 2.0%, w/v), or succinimide (0.5 and 2.0%, w/v). Cultures were frozen and stored at -23.3 C, frozen and stored at -196 C in liquid nitrogen, or frozen at -196 C and stored at -23.3 C. Cultures frozen and stored at -196 C in liquid nitrogen gave the greatest recovery of viable cells. The number of cells surviving after storage at -23.3 C was greater when the cells had been frozen in liquid N₂ than when they had been frozen at -23.3 C. All strains stored at -23.3 C showed a decrease in numbers of surviving cells; additives, particularly l-malic acid and apple juice, were advantageous in preserving the viability of the S. lactis C₂ and S. cremoris R₁ strains, but had little or no effect on the survival of S. diacetilactis DRC₂. l-Malic acid and apple juice stimulated acid production for all cultures in activity tests following incubation after thawing, whereas glycerol and dimethyl sulfoxide retarded its development.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.