Genetic Variation of Pertussis Pathogene
Bordetella Pertussis Phase Variations Induced by Translocation of IS-Elements into Virulence Genes is a Proposed Mechanism for Microbial Adaptation
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Gamalei Institute of Epidemiology and Microbiology, Russia, Moscow
- National Institute of Public Health and the Environment, The Netherlands, Bilthoven\nPublic Health Agency of Canada / National Microbiology Laboratory, Canada, ON, Ottawa\nPublic Health Laboratories, Canada, ON, Etobicoke
Project summaryBordetella pertussis, the causative agent of whooping cough is transmitted via respiratory droplets. Pertussis prevention is included in Russian national calendar of planned vaccination and is provided by mass infant and childhood immunization schedules. However, the circulation of the pathogen is not decreasing. The yearly incidence of reported pertussis is 48.5 million cases all over the world, and the annual pertussis death rate is 300 000 in infants and children (Croweroft N.S.,et al. 2003). In infants pertussis occurs more often and severe, but there were marked laboratory confirmed cases of B.pertussis infection in adolescents and adults recently (Cherry J.D. 2004, Heininger U., 2004). The study of pertussis epidemiology showed that the main reservoir of infection in susceptible to disease infants were often adolescents and adults. Also the cases of B.pertussis asymptomatic carriage were recorded (Heininger U.W. et al., 2004). The mechanisms of B.pertussis infection in human organism were studied. It is considered that survival of B.pertussis in eukaryotic cells depends on expression of B.pertussis virulence factors (Bassinet L., 2000).
One of the particular properties of B.pertussis is high rate of phenotypic variability. Bacteria with different virulence, forms of colonies and ability for erythrocyte hemolysis were isolated from infected laboratory animals on different stages of disease and also from bacteria incubated in vitro (Preston N.W., 1988). Such bacteria were named phase-variants (Lacey B. 1960), and transition from one phase to another as phase transition. S. Stibitz et al (1989) demonstrated that phase variant can be a result of the frame shift mutation bvgAS operon B.pertussis, but, real mechanism of phase variability is unknown.
The structure of genes responcible for virulence of B.pertussis and their expression have been studied during last 20 years in vitro. It was found that two-component regulatory system BvgAS, consists of a sensor protein BvgS and transcriptional activator BvgA (Cotter PA, MillerJF, 2001; Cummings CA et al.,2006) regulated the most of B.pertussis virulence genes.
It was shown, that B.pertussis chromosome contains the repeated IS-like sequences. We have found that these sequences have main properties of mobile genetic elements (MGE) (Karataev G.I. 2008). It is considered the events of spontaneous transposition IS elements into operon bvgAS may regulate B.pertussis virulence, and B.pertussis Bvg- insertion mutants were isolated (Sinyashina LN. et al, 2005; Sinyashina LN et al., 2008).
The test-systems for detection of B.pertussis bacteria in clinical samples and detection and analysis B.pertussis bacteria containing IS insertion into bvgAS operon were developed (Karataev G.I., 2008).
Authors have experience in the study of Bordetella pathogeneses, mobile genetic elements, and pertussis vaccine development, identification and epidemiology of Bordetella. The authors have c.a. 20 published papers, in the field mentioned above. They took part in the work of the project of ISTC 2923, 2223.
The aim of studding: The study of interrelation between B.pertussis phase variability and IS-elements integration into virulence genes of bacterial cultured in vitro or isolated from experimental animals or from clinical samples from patients with typical and atypical form of whooping cough.
Schemes of studding:
- The selection of primers and PCR conditions for the registration of IS481 and IS1002 transposition into B.pertussis virulence genes;
- Registration of the IS481 and IS1002 transpositions events into B.pertussis virulence genes. Determination of the factors influenced on the IS transposition frequency;
- Studying of the dynamic of IS transposition into virulence genes of B.pertussis DNA isolated from lungs of laboratory animals infected by virulent B.pertussis bacteria;
- Aanalysis of B.pertussis bacteria phase state in the patients at different stage of disease by PCR and sequencing of PCR products;
- The development of the test-systems for identification of IS481 and IS1002 transposition in B.pertussis virulence genes.
The results of the work will make possible to elaborate new methods for pertussis diagnosis and to estimate the persistence of bacteria in healthy humans. Test-systems will allow to determine the dynamic of IS induced avirulent B.pertussis mutants formation and to estimate of the relationships between the infecting agent virulence, and the balance between the initiation of disease and establishment of persistence. Additionally it can help to identify persons with latent forms of infection and to lower the incidences of pertussis infection by excepting contacts between latent infected inpiduals and uninfected ones, especially in the children institutions.
The prophylactic measures based o B.pertussis identification will be provide commercial and social effects.
Expected Results and Their Application:
- The methods of detection of IS481 and IS1002 transposition events into B.pertussis virulence genes in vitro and in vivo will be developed;
- Iidentification and characterization of B.perussis in clinical probes, obtained from patients presenting acute and convalescent stage of decease and also from patients presenting atypical respiratory infections as well as from healthy persons, contacted with sick people will be carried out;
- Test-systems for identification of B.pertussis bacteria containing IS in virulence genes will be constructed;
- Results obtained will provide new data about pertussis epidemiology.
The project will help Russian researchers engaged previously in studies in the area of defense, to reorient the circle of their scientific interests and to be involved in the current project with the possibility to gain more experience in performing fundamental, as well as applied studies, within the framework of international science and technology programs for biology and medicine.
The work is planned for 3 years. To determine the events of IS 481 and IS 1002 transpositions into B.pertussis virulence genes selection of primers and PCR protocol working out will be performed during the first two quarters. At the same time the work on 2 and 3 tasks will be performed based on the tests developed for determination IS integration into bvgAS operon. After demonstration of IS integrations into of PT and FHA genes (proposed 3d and 4th quarters) we will start the task 4. The task 5 will be fulfilled during 8-12quarters.
The collaborators will provide technical advice and cowork with the Russian scientists on generation, collection and analysis of scientific data. Collaborator will monitor the project and review progress reports in process of conference and meetings.
IS481 and IS1002 transposition events into B.pertussis virulence genes will be detected by the analysis of PCR products with pair of primers one of which is complementary to the IS sequences and the second one - to the locus near expected integration site (fig 1). The PCR products specificity will be confirmed by sequencing. This experimental approach is based on facts of high specificity of IS transposition into NctagN sites of B.pertussis chromosome. Standard softwares will be used for the primers’ design and analysis of DNA sequence data. For intracerebral and intranasal infection linear laboratory animals will be used. Taking clinical samples from patients is intended to perform with nasopharyngeal tampons and by mucus aspiration from rhinopharynx. The DNA from clinical material will be isolated by guanidinchloride and magnetic sorbent.
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