pH6 Antigen in Plague Vaccine
Investigation of pH6 Antigen Role in Live Plague Vaccine Immune-Genesis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Melnikov V G
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
Project summaryStudying the molecular mechanisms of pathogen-host interactions is one of the most urgent problems in biology and medicine. It is necessary to solve this problem in order to develop effective means for prevention and treatment of infectious diseases.
The main goal of this project is to elucidate the role of pH6 antigen at the initial stages of plague infectious process. The causative agent of plague, Yersinia pestis, is one of the most virulent microorganisms. It provides a convenient model to study the fundamental problems of pathogenicity.
The virulence of Y. pestis is determined by a wide spectrum of its features initially denominated as "determinants of virulence" [Burrows T.W., 1957, 1963; Burrows T.W., Bacon G.A., 1958]. The "classic" determinants include the ability of bacterial cells to absorb exogenous dyes and hemin (Pgm+), dependence of growth at 37 °C on Ca2+ ions present in the medium (Ca), synthesis of VW antigens, "murine" toxin and capsule antigen F1 (Tox+ and Fra+), concomitant synthesis of pesticin, fibrinolysin and plasmocoagulase (Pst+, Fb+, and Cg+), purine independence, or the ability to produce endogenous purines (Pur+). After the four decennaries of research and deliberation lasting from the moment of "virulence determinants" postulation some of them such as W antigen, pesticin, and ability to produce endogenous purines are no more accounted as pathogenicity factors [Perry R.D., Fetherston J.D., 1997; Brubaker R.R., 1991, 2000].
Recently, a number of additional virulence factors have been identified, they include outer membrane proteins (Yop's) [Brubaker, 1984; Rosqvist R. et al., 1988, 1990, 1991; Leung K.Y., Straley S.C., 1989; Reisner B.S., Straley S.C., 1992; Galyov E.E. et al., 1993; Straley S.C. et al., 1993; Cornelis G.R., 1998; Cornelis G.R. et al., 1998, etc.], adhesion pili (pH6 antigen) [Vodop'yanov S.O., Mishan'kin B.N., 1985; Vodop'yanov S.O. et al., 1990; Lindler L.E., Tall B.D., 1993; Fosberg A. et al., 1997, 1998, 1999], adenylate cyclase, neuraminidase [Mishan'kin B.N., 1987], and some proteins of siderophore-dependent iron assimilation system [Carniel E. et al., 1992; Perry R.D., Fetherston J.D., 1997].
Lately cardinal changes have taken place in understanding the molecular mechanisms of virulence of Gram-negative bacterial pathogens. These changes are caused, first of all, by the discovery of the type III secretion system, providing direct communication between the bacterium cytoplasm and the host target-cell cytoplasm when they are in tight contact. With the assistance of this system bacteria secrete effector proteins, disturbing normal functioning of the target-cell, into the host cell.
To realize the function of the type III secretion system, the pathogen and the host cell must have a close contact, which is obtained through specialized surface structures, bacterial adhesins (pili, fimbria, protein capsules and others). An obvious issue is that the adhesin YadA (as well as Inv and Ail) have been strongly implicated in favoring type III translocation of Yops in the enteropathogenic yersiniae (probably by assuring tight host cell-bacterium contact). Since Yersinia pestis lacks these three "adhesins" [Perry R.D., Fetherston J.D., 1997], some alternative structure may substitute for this purpose. The only known adhesins in Y. pestis are Pla and pH6 antigen (although other as yet undefined homologues exist as judged by inspection of the genome). In different strains and different studies mutation loss of Pla may both results in dramatic reduction and/or in absence of any alteration of virulence. pH6-mutants also may have both only modest reduction of virulence and/or can be completely avirulent.
pH6 antigen was described by Ben-Efraim S. et al. in 1961 as an antigen synthesized by Y. pestis at the temperature close to body temperature of mammals and pH values close to pH of phagolysosomes in macrophages or abscesses. It was found that pH6 antigen expression is a necessary prerequisite of plague microbe virulence, although its role in the pathogenesis remains unclear [Lindler L.E. et al., 1990]. pH6 antigen was shown to be cytotoxic for peritoneal [Bichowsky-Slomnicki L., Ben-Efraim S., 1963] and alveolar [Stepanshina V.N. et al., 1993] macrophages, suggesting its involvement into the macroorganism-microbe interactions. L.E. Lindler et al.  studied in detail the molecular organization of pH6 antigen. It has been demonstrated that the protein is synthesized in the form of subunits with a molecular weight of 15 kDa, which readily aggregate into macromolecular complexes. The synthesis of pH6 antigen is coded by psa operon having a structure similar to that of fra operon coding for F1 antigen. Elimination of the structural gene coding for pH6 antigen from the chromosome of virulent Y. pestis plague cells leads to the loss of virulence [Lindler L.E. et al., 1990; Cherepanov P.A. et al., 1991; Panfertsev E.A. et al., 1991]. Recently it has been demonstrated that after growth at 37 °C and pHЈ 6.0 the cells of plague pathogen are "coated" into capsules consisting of pH6 antigen [Cherepanov P.A. et al., 1991; Cherepanov P.A. et al., 1998].
Thus, at different periods of its existence within host organism, plague pathogen has two different types of capsules determined by operons similar to those encoding biogenesis of pilus and nonpilus bacterial adhesins [Hultgren S.J., Jones C.H., 1995]. In the present project, we set the task of elucidation of the role of pH6 antigen in the infection of macroorganism by derivatives of Y. pestis vaccine strain EV line NIIEG (Culture Collection of SRCAM) and wild-type strain 231 (Culture Collection of SRCAM).
The tasks of project are next:
1. To isolate pH6 antigen and characterize its biochemical properties.
2. To produce monoclonal antibodies against pH6 antigen.
3. To estimate the synthesis of pH6 antigen at various periods of infectious process in vitro, using macrophages of lines RAW or JA774.
4. To estimate the synthesis of pH6 antigen at various periods of infectious process in vivo, using murine (or guinea-pig) model.
5. To assess the possibility of application of pH6 antigen as a second (after F1 antigen) marker for laboratory diagnostics of plague and detection of the pathogen.
6. To assess the contribution of F1 and pH6 antigens into the induction of T lymphocytes after immunization of laboratory animals with live plague vaccines.
7. To construct PsaA-negative mutants and to perform comparative study of PsaA-negative and their wild-type isogenic Y. pestis variants ability to survive in macrophages of lines RAW or JA774, organisms of laboratory animals, and to develop generalized infectious process.
8. To construct Y. pestis mutants able to produce pH6 antigen at 37 °C but in the pH-independent manner and to perform comparative study of such mutants and their parent isogenic Y. pestis variants ability to develop protective immunity or generalized infectious process.