Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis.

Adenylate cyclase (AC) toxin from Bordetella pertussis enters cells to cause supraphysiologic increases in cAMP. AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.

Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice.

Proliferation of Bordetella pertussis in the lungs of infant mice challenged by the intranasal route was examined. The bacteria rapidly proliferated in the lungs of mice challenged with a sublethal dose of a wild-type strain (BP338) or a filamentous hemagglutinin mutant (BPM409) from 500 at day 0 to 10(7) at day 15. The infection cleared in about 40 days. Pertussis toxin-deficient mutant BP357 gave a similar profile; however, the number of bacteria recovered was slightly reduced, suggesting that pertussis toxin is not essential for bacterial growth in the lungs. In contrast, adenylate cyclase toxin mutant BP348 was rapidly cleared from the lungs, with no viable bacteria remaining 10 days postchallenge, suggesting that the adenylate cyclase toxin is a colonization factor required for the bacteria to initiate infection.

Lethal infection by Bordetella pertussis mutants in the infant mouse model.

Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed.

Reasons given by female virgins for not having premarital intercourse.

By analyzing reasons given by virgin women for not engaging in premarital coitus, we can attain greater insight into the decision-making process involved in maintaining virginity as well as in the transition to nonvirginity. Among virgin women, there is a subgroup who are likely to have premarital coitus if they should meet the right person. On the other hand, women who give moral reasons for not engaging in coitus are the least likely to engage in premarital coitus even if they should become involved in a love relationship. Our results suggest that counsellors and educators should be aware of the diversity of reasons for not engaging in premarital intercourse as well as factors influencing these reasons in order to gain a more complete understanding of premarital sexual decision-making.

Perceived side effects of oral contraceptives among adolescent girls.

PIP: Knowledge and attitudes of adolescent females regarding the side effects of oral contraceptives were investigated. The data source was a large study of sexual and contraceptive attitudes and experience. The questionnaire responses of 486 single females attending 10 birth control and pregnancy counseling centers in Ontario, Canada were examined. The age range of the subjects was from 13-20; 71% were attending school and 69% were living at home. They were attending the centers in order to obtain contraceptives (55%), to renew OC prescriptions (20%), or to receive pregnancy counseling (25%). 29% of the subjects had used OCs before coming to the clinic, but 91% planned to use OC after their clinic visit. 8% were planning to use an IUD; 1% were planning to use a diaphragm; and less than 1% were planning to have their boyfriend use condoms. 85% of the subjects indicated that they had heard abut side effcts of OCs with weight gain as the best known side effect. Other side effects familiar to many included nausea, circulatory disorders, headaches, emotional changes, menstrual problems and cancer. About 1/2 of the subjects had learned about these side effects from the mass media or female friends, 25% from a school sex education class, 15% from their mothers, and 3% from a physician. Despite knowledge regarding side effects, most of the subjects had positive attitudes toward OCs with 59% believing that the advnatages outweighed any disadvantages.^ieng

Comparison of younger and older adolescent females attending birth control clinics.

Sexual and contraceptive attitudes and practices were assessed for 486 single females aged 13-20, attending ten birth control centres in Ontario. At last intercourse, 28% were not using any contraception and only 33% reported they had used contraceptives all of the time. Younger adolescents were less likely than older ones to have used effective methods of contraception or to have used contraceptive methods effectively. Younger adolescents expressed greater embarrassment about obtaining oral contraceptives, particularly from the family physician, and were more likely to perceive oral contraceptives as being difficult to obtain.

Self-esteem and sexual permissiveness.

Assessed the relationship between self-esteem and sexual permissiveness for 486 single females aged 13--20 who were attending 10 birth control centers in Ontario. Consistent with Stratton and Spitzer's social deviance model, which predicts that the nature of the permissiveness/self-esteem relationship is dependent on cultural norms, in this liberal sample high esteem Ss were accepting of premarital intercourse with affection. Those who deviated from the permissiveness with affection norm by endorsing sexual abstinence had lower esteem, but there was no relationship between esteem and endorsement of premarital intercourse without affection or esteem and number of coital partners. High self-esteem Ss were more willing to take sexual initiative and had lower sex guilt.

Self-esteem, locus of control, and adolescent contraception.

The relationships between locus of control, self-esteem, and attitudes to contraception and contraceptive behaviors were assessed for 486 single women of ages 13 to 20, attending 10 birth control centers in Southern Ontario, Canada. The Ss voluntarily filled out a questionnaire, and the data were measured with the Fatalism scale of Reid and Ware's I-E scale, a Likert-type self-esteem scale, and a semantic differential birth control pill scale. There were no significant relationships between locus of control and any of the contraception variables. Ss with high self-esteem were found to have positive attitudes toward using birth control pills, to be less embarrassed about obtaining contraception, and to be more effective and consistent contraceptive users.