Role of pH6 Antigen in Plague Infectious Process
Study of Roles of pH6 Antigen in Promoting Virulence and Immunity in Yersiniae
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 and Biotechnology, Russia, Moscow reg., Obolensk
- University of Kentucky / Department of Microbiology and Immunology, USA, KY, Lexington\nLudwig-Maximilians-Universität / Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Germany, Munich\nDept. of Medical Microbiology and Immunology, University of Alberta, Canada, AB, Edmonton\nCanadian Food Inspection Agency
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 in plague infectious process. The causative agent of plague, Yersinia pestis, is one of the most virulent microorganisms and 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 FI (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; Anisimov A.P, 1999, 2002].
Recently, a number of additional virulence factors have been identified, they include secreted Yersinia outer proteins (Yops) [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 into the host cell, disturbing their normal functioning.
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 or in absence of any alteration of virulence [Anisimov A.P, 2002; Anisimov et al., 2004]. pH6- mutants also may have both only modest reduction of virulence or can be completely avirulent [Anisimov A.P, 2002; Anisimov et al., 2004].
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 abscesses or phagolysosomes in macrophages. 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 host-pathogen 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. Recently it has been demonstrated that after growth at 37°C and pH≤ 6.0 the cells of the plague pathogen are "coated" by the capsules consisted of pH6 antigen [Cherepanov P.A. et al., 1991; Cherepanov P.A. et al., 1998]. The synthesis of pH6 antigen is encoded by psa operon having a structure similar to that of fra operon coding for FI antigen. Elimination of the structural gene coding for pH6 antigen from the chromosome of virulent Y. pestis cells leads to the slight reduction [Lindler L.E. et al., 1990] or total loss of virulence [Panfertsev E.A. et al., 1991]. R.D. Perry supposes (personal communication) that this difference might be due to additional unidentified mutations carrying by the Russian Y. pestis strain 231 and absent in the strain KIM5+. Microbial pathogenesis is usually complex and multifactorial. Several virulence factors may act inpidually or in concert to produce infection. Removal of any one of these components may or may not render the organism avirulent. The loss of pH6-antigen production can be compensated in KIM5+ by the products of other unidentified genes, whereas unidentified mutations decreasing the potential store of Y. pestis virulence factors in the strain 231 might be the main reason of dramatic decrease of virulence in the case of elimination of one more pathogenicity factor from such potentially-weakened strain.
It was shown that under certain circumstances pH6-antigen may form a capsule, in Y. pestis similar to that formed by fraction I antigen. The role of pH6-antigen as an adhesin in Y. pestis has been assumed. Y. pseudotuberculosis, a microorganism closely related to Y. pestis, possess functional YadA fibrillae, mediating adhesion of bacteria to the enterocites of gut mucosa. In contrast Y. pestis possesses no YadA-fibrillae, but harbor nonfunctional yadA gene knocked down by frameshift and missense mutations. Thus one can assume that in Y. pestis pH6-antigen in the absence of YadA fibrillae may play a role of active adhesin. In Y .pseudotuberculosis YadA fiblillae most probably are carrying out the main function of adhesion, while pH6 activity may be hidden, or repressed, or complementary to the YadA.
The authors of the project had a good experience in studying microbial pathogenesis on the model of Y. pestis and Y .pseudotuberculosis (relevant publications enclosed in the section, 12. Supporting Information, please find).
In the present project, we set the task of elucidation of the roles 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 initial strains, EV line NIIEG and wild-type strain 231, were obtained from the Culture Collection of the Russian Institute for Plague Control "Microbe" (Saratov).
The tasks of project are:
1. To perform molecular cloning and sequencing of psa operon from Y. pestis 231.
2. To perform sequencing and analyzing of several yad-like, fimbrial-chaperone, and inv genes from Y. pestis 231.
3. To estimate biological properties of Y. pestis and Y. pseudotuberculosis psa mutants.
4. To estimate biological properties of Y. pestis pH-independent mutants.
5. To estimate pH6-antigen interaction with YadA in Y. pseudotuberculosis.
Molecular cloning and DNA sequencing will be performed with the use of PCR technology and available genome sequence data (http://www.sanger.ac.uk/Projects/Y_pestis/ and http://www.genome.wisc.edu/sequencing/pestis.htm).
Construction of Y. pestis and Y. pseudotuberculosis mutants will be performed with the use of site directed mutagenesis with the help of suicide vector pCVD442 (http://medschool.umaryland.edu/infeMSD/som.html) or as described by Datsenko, K.A. & Wanner, B.L. (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-5).
Virulence testing of yersiniae strains will be performed with the use of mouse model.
pH6-antigen expression in yadA+ and yadA- Y. pseudotuberculosis and Y. pestis variants will be estimated with the use of ELISA and RT-PCR.
Estimation of the Y. pestis and Y. pseudotuberculosis variants ability to survive in macrophage-like cells will be performed with the use of macrophage-like cells J774.A as target-cells and derivatives of Y. pestis vaccine strain or Y. pseudotuberculosis variants. Determination of cell infection will be done by means of plates and microscopy.
Expected Results and Their Application.
– Data on possible unidentified mutations causing different rates of attenuation in the Y. pestis strains 231 and KIM5+.
– Data on roles of pH6 antigen in promoting virulence and immunity in Y. pestis.
– Elucidation of the role of pH6 antigen in the adhesion step of pathogenesis of pseudotuberculosis.
– Revealing of the relationship between YadA adhesin and pH6-antigen.
– Input in understanding of evolution of adhesive function in Yersinia pathogenic for humans.
The project realization will be an essential contribution into development of applied biology and medicine as well as into solution of significant social problem, control of bioterrorism, being of serious danger for world community. Moreover, performance of the project will allow the scientists and engineers of the Stare Research Center for Applied Microbiology, engaged previously in studies in the area of defense, to reorient the circle of their scientific interests and to use the gained experience to carry out fundamental and applied studies, connected with solution of international scientific and technical problems in the fields of biology and medicine to serve peace purposes, to create long-term prospects for fruitful activity within the framework of International Scientific Association.