Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-10-13
2004-01-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S350000, C435S069100, C435S006120, C435S091100, C435S091500, C435S170000, C435S007950, C435S007100, C536S023100, C536S024300, C424S262100, C514S263370
Reexamination Certificate
active
06677439
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for isolation of rare proteins from bacterial samples. More particularly, this invention relates to a method for isolating rare outer membrane proteins from the family Spirochaetaceae, such as genus Treponema and to the use of such proteins in diagnosis and prophylaxis of related diseases.
2. Description of Related Art
The genus Treponema (order Spirochaetales, family Spirochaetaceae), a type of gram-negative bacteria, contains four human pathogens as well as at least six nonpathogens. The pathogens are characterized by an extreme sensitivity to environmental conditions that renders them impossible to culture in vitro. Due to DNA homology the agents that cause syphilis, yaws and endemic syphilis have been combined into one species and three subspecies:
T. pallidum
subsp.
pallidum
(syphilis);
T. pallidum
subsp.
pertenue
(yaws); and
T. pallidum
subsp.
endemicum
(endemic syphilis).
T. carateum
, which is the causative agent of pinta, remains a separate species. Syphilis is found worldwide, yaws is endemic in the tropics, pinta is prevalent in tropical areas of Central and South America, and endemic syphilis is restricted to desert regions. These treponemal infections are very complex, each exhibiting distinct stages of symptomatic manifestations followed by asymptomatic periods. Without antibiotic therapy, these diseases are chronic and may last for 30 to 40 years.
To date, the four pathogens have been considered antigenically identical. An individual subspecies-specific antigen has not been identified and serological reactions demonstrate immunological relatedness. Both Wassermann and anti-
T. pallidum
subsp.
pallidum
antibodies develop in response to each treponemal disease, and known protective immunogens are also related, as shown by cross-resistance (T. B. Turner, et al.,
Biology of the Treponematoses. W.H.O Monogr. Ser
. 35:1-277, 1957). Therefore, the geographical location together with the clinical manifestations of the patient have been considered the key to diagnosis (Manual of Clinical Microbiology, 5th Ed., A. Balows, et al., Eds., p 567, 1991).
Freeze-fracture electron microscopy of outer membranes from pathogenic spirochetes has revealed that their integral transmembrane outer membrane protein density is one to two orders of magnitude less than that of typical gram negative bacterial pathogens. It has been proposed that this low outer membrane composition, and thus low surface exposure of antigenic target molecules, allows these organisms to effectively evade the host immune response, contributing to the chronic nature of infection exhibited by all spirochetal pathogens.
As is well known, the outer membranes of spirochetes, including that of
Treponema pallidum
subsp.
pallidum
, the agent of syphilis, are fragile structures as compared to those of typical gram negative bacteria. Consequently, separation of the outer membrane from the inner membrane has proven extremely difficult. Certain other medically relevant spirochetal bacteria with outer membrane structure and, hence, protein structure, similar to those of
T. pallidum
include Borrelia burgdorferi (Lyme Disease), Borrelial species (relapsing fever), and Leptospiral species (leptospirosis).
The outer membrane of
T. pallidum
has been found to be antigenically inert and resistant to specific treponemidical antibody (Radolf, et al.,
Infect. Immun
., 52:579, 1986; Hovind-Hougen, et al.,
Acta Pathol. Microbiol. Scand
., 87:263, 1979; Nelson, et al.,
J. Exp. Med
., 89:369, 1949). Yet freeze-fracture electron microscopy has shown that certain rare outer membrane protein (tromp) molecules of
T. pallidum
have surface exposed antigenic sites that bind antibody present in the serum of challenge immune animals (Blanco, et al.,
J. Immunol
., 14:1914-1921, 1990). Taken together the spirochetal bacterial pathogens imperil the health of a considerable portion of the human population, yet development of effective and specific vaccines and isolation of antigens capable of generating protective immune response has been hampered by a considerable number of problems associated with this organism: the impossibility of culturing the
T. pallidum
in vitro, the limited numbers of organisms that can be obtained from infected animals, the contamination of treponemes by host tissue components, the fragility of the treponemal outer membrane, and the difficulty of isolating and identifying the outer membrane proteins of pathogenic spirochetes.
Previous studies attempting to identify transmembrane outer membrane proteins of pathogenic spirochetes have utilized various detergent solubilization approaches. Spirochetal outer membranes bleb form the underlying protoplasmic cylinder under relatively mild conditions, including dilute detergents and hypotonic environments. However, such apporaches have identified only abundant subsurface located proteins, including various lipoproteins which by definition are not transmembrane molecules and do not form particles viewed by freeze-fracture analysis.
Therefore, the need exists for new and better vaccines based upon the identification of virulence related outer membrane molecules to be used in diagnosis and for prophylaxis of diseases related to the pathogenic spirochetal bacteria, especially the genus
T. pallidum.
SUMMARY OF THE INVENTION
A novel method is provided for isolating outer membrane of pathogenic Spirochaetacae family without use of detergents. The pathogen is purified from contaminating host components using a density gradient centrifugation medium that is stable at pH from about 3.2 to about 3.0, preferably a Ficoll step gradient. The purified pathogen is treated with a lipid soluble chromophore that intercalates into outer membrane to provide a visual marker of membrane matter. Outer membrane is released from protoplasmic cylinders using a hypotonic, low pH buffer, preferably citrate, followed by density gradient centrifugation, which yields, for example, the chromophore labeled bands at 7% and 35% sucrose (wt/vol) for
T. pallidum
and
T. vincentii
, respectively.
Freeze-fracture electron microscopy of membrane vesicles from the spirochete reveals an extremely low density of protein particles. For instance, the particle density of
T. pallidum
is approximately six times less than that of
T. vincentii
. Comparative immunoblot analysis of the
T. vincentii
membrane material to that of whole organisms showed a lipopolysaccharide (LPS) ladder consistent with 20% recovery of the outer membrane. Immunoblots of
T. vincentii
outer membrane also showed two antigenic proteins at 55- and 65-kDa.
125
I-penicillin, which binds only to inner membrane and not to outer membrane, was used to detect the presence of any inner membrane in the outer membrane preparation isolated according to the method of the invention. No penicillin binding proteins in the
T. pallidum
membrane material were detected, indicating the absence of inner membrane contamination.
Immunoblot analysis of
T. pallidum
outer membrane using antibodies specific for periplasmic associated proteins showed no detection of known 19-kDa “4D” protein or the 47-kDa lipoprotein and only trace amounts of endoflagellar protein. Rare outer membrane proteins associated with the
T. pallidum
were detected by one and two dimensional reducing SDS-PAGE separation and immunoblot analysis, using gold staining and serum from infected and challenge immune animals. As compared to whole organism preparations, four of the isolated proteins were obtained in significantly enriched amounts from the outer membrane preparation.
Methods are provided for the use of outer membrane proteins of pathogenic Spirochaetacae family for detection and amelioration of associated disease states.
The isolation of the
T. pallidum
outer membrane and identification of its protein constituents has been complicated by the fragility of this structure, the limited number of treponemes that can be acquired by rabbit infection, and the significant amount of
Blanco David R.
Champion Cheryl I.
Lovett Michael A.
Miller James N.
Gray Cary Ware & Freidenrich LLP
Robinson Hope A.
The Regents of the University of California
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