ABSTRACT
Avirulent B. pertussis bacteria containing IS elements in the bvgAS operon were detected during the study of whooping cough patients and bacilli carriers. The present work is devoted to the study of the accumulation dynamics and the mechanisms of generation of persistent forms of the B. pertussis bacteria in lower monkeys as the most adequate model for extrapolation ofthe experiment results to humans. By means of the real-time PCR method, it was established that the B. pertussis bacteria lived more than three months in the upper respiratory tract after a single intranasal monkey infection; the period was reduced to 14-28 days during repeated infection. An increase in the portion of B. pertussis Bvg mutants in the population to tens of percent from the total number of registered bacteria was registered. The experimental confirmation ofthe development and accumulation of avirulent B. pertussis Bvg mutants during the development of the infectious process was obtained. Further study of the composition of the B. pertussis persistent bacteria population at different stages of the disease will make it possible to formulate new approaches to the whooping cough diagnostics and prevention and creation of fundamentally new drugs.
Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , DNA Transposable Elements/genetics , Transcription Factors/genetics , Whooping Cough/genetics , Animals , Bacterial Proteins/biosynthesis , Bordetella pertussis/pathogenicity , Gene Expression Regulation, Bacterial , Humans , Macaca mulatta , Mutagenesis, Insertional/genetics , Operon/genetics , Transcription Factors/biosynthesis , Whooping Cough/microbiology , Whooping Cough/pathologyABSTRACT
A growth of pertussis morbidity is observed in many countries of the world against the background of mass vaccindtion. Forms of the disease course have changed. Atypical forms of pertussis occur predominately in adolescents and adults. Asymptomatic carriage of the causative agent has been established. Infection of infants with. BordetelIa pertussis bacteria in more than 90% of cases occurs from parents and relatives. A prolonged persistence of the causative agent has been identified. Morbidity increase in developed countries is associated with the use of acellular vaccines, that do not protect from the infection, but reduce severity of the disease. A change of genotypes of the circulating bacteria strains is observed ubiquitously. Formation of a persistent form of B. pertussis is possible due to a reversible integration of IS-elements into bvgAS operon and other virulence genes. The results of studies of invasion and survival of B. pertussis bacteria in eukaryotic cells, a change in B. pertussis bacteria population after experimental infection of laboratory mice and monkeys are presented, accumulation of avirulent insertion Bvg mutants of B. pertussis was detected. The data obtained are in accordance with the results of analysis of causative agent population in patients with typical and atypical forms of pertussis in humans. More than 50% of the population of B. pertussis bacteria in practically healthy carriers was shown to be presented by avirulent insertion Bvg mutants. B. pertussis virulence reducing as a result of inactivation of single or several virulence genes probably provide long-term persistence of bacteria in host organism and formation of apparently healthy vehicles. Follow-up studies on that front would help to formulate new attitudes to preventive measures of pertussis and lead to development of fundamentally new pharmaceuticals (vaccines) preventing formation of bacterial persistence.
Subject(s)
Bacterial Proteins/immunology , Bordetella pertussis/immunology , Whooping Cough/microbiology , Adolescent , Adult , Animals , Bacterial Proteins/genetics , Bordetella pertussis/genetics , Bordetella pertussis/pathogenicity , Carrier State , Genotype , Haplorhini , Humans , Immune Evasion , Interspersed Repetitive Sequences/immunology , Mice , Mutation , Operon , Pertussis Vaccine/administration & dosage , Pertussis Vaccine/immunology , Whooping Cough/immunology , Whooping Cough/pathology , Whooping Cough/prevention & controlABSTRACT
The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3.
Subject(s)
Bordetella pertussis/genetics , Pertussis Vaccine/genetics , Transglutaminases/genetics , Virulence Factors, Bordetella/genetics , Whooping Cough/prevention & control , Administration, Intranasal , Animals , Bordetella pertussis/immunology , Freeze Drying , Gene Silencing , Guinea Pigs , Lethal Dose 50 , Mice , Mutation , Pertussis Vaccine/administration & dosage , Pertussis Vaccine/immunology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
The roles of yadA, invA, and psaA genes introduced into the genetic background of the Y. pseudotuberculosis strain possessing the large p VM82 plasmid in virulence and invasion capacity were studied. Isogenic single mutants as well as double and multiple mutants of these genes were constructed and used. LD50 was used as a measure of virulence and the estimation of the ability to invade mammalian cells and the effect of infection on the weight changes of infected mice were used as additional indicators of pathogenicity. It was shown that the YadA had a major effect on the bacterial virulence when compared with the effects of PsaA and InvA. InvA appears to mediate the main pathway of the cellular invasion. YadA is responsible for the weight loss after infection of mice with sublethal doses of Y. pseudotuberculosis. The effects of YadA on virulence and of InvA on bacterial invasion were independent of the expression of the other genes studied. To our knowledge, this study showed for the first time the direct involvement of YadA in the virulence of Y. pseudotuberculosis in mice. Further pathomorphological studies are required to reveal the differences in the pathogenesis of pseudotuberculosis caused by yadA mutants or yadA+ bacteria of Y. pseudotuberculosis.
Subject(s)
Adhesins, Bacterial/metabolism , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Yersinia pseudotuberculosis Infections/metabolism , Yersinia pseudotuberculosis/pathogenicity , Adhesins, Bacterial/genetics , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Body Weight , Cell Line , Humans , Mice , Mutation , Virulence , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/metabolism , Yersinia pseudotuberculosis Infections/microbiologyABSTRACT
A method of monitoring the sequential events of IS481 transposition into the ctag site of bvg operon of Bordetella pertussis has been developed. Reproduction of virulent B. pertussis cells in vitro is accompanied by intrachromosomal site-specific IS481 transposition, which, in turn, results in inactivation of bvg operon of the causative agent and cell avirulent state. Avirulent bvg mutants of B. pertussis are incapable of intramolecular IS481 transposition. The frequency of the transposition increases when MgSO4 and nicotinic acid are present the culture medium. In the absence of these modulating factors. IS481 transposition along B. pertussis chromosome is inhibited but not arrested completely. Negative regulation of the bvg-repressed genes of B. pertussis seems to be a mechanism that controls bvg-dependent IS481 transposition.
