Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-06-26
1998-03-24
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 41, 435 691, 435 711, 435 91, 536 253, C12Q 168, C12N 1500, C12P 100, C12P 2106
Patent
active
057311510
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a nucleic acid that encodes a hemolysin protein and to a nucleic acid that encodes a positive regulator of hemolysis.
2. Background Art
The World Health Organization estimates that more than 2 billion persons worldwide are or have been infected with Mycobacterium tuberculosis, and tuberculosis causes more than 3.5 million deaths annually (1). The human immunodeficiency virus (HIV) epidemic has complicated the epidemiology of tuberculosis (2), and much of the recent increase in tuberculosis in developed countries can be traced to the enhanced susceptibility of AIDS patients to developing this disease (3).
M. tuberculosis is an intracellular pathogen which multiplies within cells of the host's immune system, primarily macrophages (4). Uptake of the bacillus by macrophages is thought to be mediated by complement component C3 and complement receptor (5). After entering the cell, M. tuberculosis inhibits phagosome-lysosome fusion (6,7) and the acidification of the phagosome (8). M. tuberculosis then multiplies within the unfused vacuole (9). Macrophages heavily ladened with bacilli ultimately lyse and release the bacilli.
Studies of other bacterial pathogens have shown that soluble and membrane-bound cytolysins are important virulence factors. For example, strains of Escherichia coli expressing alpha-hemolysin are 10- to 1000-fold more virulent in animal models than strains lacking alpha-hemolysin (10). Similarly, strains of Bordetella pertussis lacking adenylate cyclase/hemolysin display reduced virulence in mouse models (11), and this phenotype can be reversed by trans-complementation with a plasmid expressing the adenylate cyclase/hemolysin (12).
Cytolysins also play important roles in the ability of intracellular bacterial pathogens to enter, replicate within, and exit host cells (13,14,15). The soluble cytolysin of Listera monocytogenes, listeriolysin O, is required for the intracellular growth of this organism in macrophages (16,17,18). Expression of listeriolysin O in Bacillus subtilis allows this non-pathogen to escape from the phagosome and multiply within macrophages (19). A membrane-bound cytolysin has also been implicated in the escape from phagosomes and intracellular growth of Shigella flexneri (20), and more recently the Shigella cytolysin, but not the Listeria cytolysin, has been shown to induce macrophage cell death through apoptosis (21).
Hemolytic activity in M. tuberculosis, however, has not been studied, due in part to unique difficulties in culturing these cells. In particular, these organisms release organic molecules which remain in the culture medium and cause dumping of the cells, a phenomenon known to scientists in the field. Thus, hemolysis assays would be a practically difficult problem with this organism. Furthermore, there has been no correlation in the literature between hemolysins and virulence of M. tuberculosis.
The present invention is based in part upon the vital and unexpected discovery that virulent strains of M. tuberculosis possess hemolytic activity while avirulent strains do not. Such a discovery will help provide long-awaited understanding of the mechanisms of infection by this organism and crucial means of treating and preventing infection and death from M. tuberculosis.
The present invention is also based upon the discovery of an E. coli gene that regulates hemolysis when placed in any of several different bacterial organisms. This gene therefore, can be utilized to provide greatly improved vaccine strains against M. tuberculosis as it can aid these vaccines in causing cell-mediated immunity. Such vaccines are greatly needed in light of the large numbers of people infected with M. tuberculosis and the devastating effects of infection. Current vaccines, such as the strain M. bovis BCG, have met with only limited success, since, over time after vaccination, protection against tuberculosis declines.
SUMMARY OF THE INVENTION
The present invention provides an isolated double-strand
REFERENCES:
Kitagawa et al. "Structural analyses of the umu operon required for inducible mutagenesis in Escherichia coli." PNASUSA 82:4336-4340 1985.
C. Harold King et al., "Cloning and Expression of a Contact Hemolysin from Mycobacterium tuberculosis", Abstract, 1992 American Soc. for Microbiology General Meeting, May 1992.
Pascale Cossart et al., "Listeriolysin O is Essential for Virulence of Listeria monocylogenes: Direct Evidence Obtained by Gene Complementation," Infection and Immunity, 57(11):3629-3636 (Nov. 1989).
M. Kuhn et al., "Hemolysin Supports Survival But Not Entry of the Intracellular Bacterium Listeria monocytogenes," Infection and Immunity, 56(1):79-82 (Jan. 1988).
Philippe J. Sansonetti et al., "Multiplication of Shigella flexneri within HeLa Cells:Lysis of the Phagocytic Vacuole and Plasmid-Mediated Contact Hemolysis," Infection and Immunity, 51(2):461-469 (Feb. 1986).
Eva S.Leake et al. "Phagosomal Membranes of Mycobacterium bovis BCG-Immune Alveolar Macrophages are Resistant to Disruption by Mycobacterium tuberculosis H37Rv," Infection and Immunicy 45(2):443-446 (Aug. 1984).
Quentin N. Myrvik et al., "Disruption of Phagosomal Membranes of Normal Alveolar Macrophages by the H37Rv Strain of Mycobacterium tuberculosis," Am. Rev. Respir. Dis. 129:322-328 (1984).
Rodney A. Welch et al., "Characterization of Escherichia coli Hemolysins Conferring Quantitative Differences in Virulence," Infection and Immunity 43(1):156-160 (1984).
King C. Harold
Sathish Mundayoor
Shinnick Thomas M.
Housel James C.
Swartz Rodney P.
The United States of America as represented by the Secretary of
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