Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-08-25
2001-09-04
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06284508
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and molecular structure of
Cryptococcus neoformans
. More specifically, the present invention relates to cloning, sequencing and expression of a gene encoding an enzyme which de-O-acetylates glucuronoxylomannan of
Cryptococcus neoformans.
2. Description of the Related Art
Cryptococcus neoformans
is an encapsulated yeast that exists in two varieties.
C. neoformans neoformans
has been isolated from pigeon droppings and is found worldwide in temperate climates whereas
C. neoformans gattii
has been associated with eucalyptus trees and is found in tropical or subtropical regions (13). Both varieties are pathogenic to humans and can produce fatal infections of cryptococcal meningitis (13). The yeast is most commonly pathogenic in immunosuppressed individuals, particularly those with advanced AIDS (8,49).
C. neoformans
has four distinct capsular serotypes, A through D, which are characterized by unique chemical compositions. (33). The serotype used exclusively for this study is serotype A, which comes from
C. neoformans
var.
neoformans
. This serotype is the cause of most cases of cryptococcal meningitis found in AIDS patients (8,49).
Cryptococcus neoformans
cells have very large capsules (FIG.
1
), which are composed of the polysaccharide, glucuronoxylomannan (GXM). Serotype A GXM has an &agr;-1,3-D-mannose backbone with one &bgr;-D-glucuronide and two &bgr;-D-xyloside sugars per each trimer of mannose (
FIG. 2
) (14). The backbone is also O-acetylated, ranging from approximately 3-16.5%(33). It has been shown that the O-acetylation forms part of the antigenic epitope for some monoclonal antibodies (MAbs) (46). These monoclonal antibodies can, therefore, be used for determining the presence and degree of O-acetylation on glucuronoxylomannan through ELISA antibody capture assays.
The capsule has been determined to be the single most important virulence factor for the pathogenicity of
C. neoformans
(33). Acapsular mutant strains produced by several laboratories have been found to be avirulent (12,16,35,38). Glucuronoxylomannan has been shown to affect host resistance in a number of ways including, but not limited to inhibition of phagocytosis (10,11,37), suppression of lymphocyte responses and proliferation (9,43), induction of T-cell dependent and independent immunologic tolerance (36,44,53), and even enhancement of HIV-1 infectivity in vivo (47). Treatment consists of the antimycotic agents, amphotericin B and flucytosine, or the azoles, ketoconazole and fluconazole (45). These treatments are often complicated by existing infections and their treatments as well as having some very severe side effects. The disease presents challenges on many fronts to the medical community.
Another challenge is the high viscosity caused by circulating glucuronoxylomannan, and perhaps encapsulated yeast cells, which are thought by some to lead to cerebral edema (23-27,39,40). The edema is characterized by increased intracranial pressure and has not been uniformly amenable to surgical intervention. It can evolve rapidly and be fatal. The soluble glucuronoxylomannan also presents a problem in that it is not cleared from the circulation and tissues of the host very efficiently (32a, 32b. 32c and 32d).
Prior to the advent of antibiotic and antimycotic agents, investigators experimented with enzymes that could degrade capsular polysaccharides. The first study of this type involved the use of a bacterium to degrade the polysaccharide capsule of Type III
Streptococcus pneumoniae
(4,15). The bacterium was isolated from a cranberry bog in New Jersey. Several studies followed and were expanded into in vivo studies with mice and rabbits. Enzymes were found to be effective in protection against lethal injections as well as in a curative manner when infections had been firmly established prior to treatment (5,51,52). A glucuronoxylomannan-hydrolase was discovered in a similar manner by Gadebusch in 1960. Soil samples tested for enzymatic activity led to isolation of a Gram-negative rod, designated Alcaligenes sp. S-3723, which completely degraded the capsule of
C. neoformans
(17-20). At the time of the Gadebusch report, the composition and structure of GXM was incompletely and sometimes erroneously understood. In retrospect, the Gadebusch was probably a mixture of two or more unidentified and uncharacterized enzymes. The enzyme cocktail was tested in vivo on mice infected with
C. neoformans
. The ET
50
increased from 18 days for mice with no treatment to 47 days for enzyme-treated mice.
Gadebusch's findings on this enzyme were published in 1960 and 1961 (17-20). Amphotericin B was gaining acceptance as a lifesaving treatment for cryptococcal meningitis and the disease was not very prevalent at that time. Molecular cloning had yet not been conceived, making the use of enzymic treatments tedious and costly, as enzyme had to be purified through a lengthy process from native bacteria. Today,
C. neoformans
infects 5-10% of AIDS patients in the U.S. and is the most common life threatening opportunistic fungal infection in AIDS (34). Antimycotic treatments prolong survival of these patients, but are ineffective against the cerebral edema and have little impact on high serum titers of antigen. Enzyme treatment may be the answer to this lingering problem.
The prior art is deficient in the lack of identification of specific GXM-cleaving enzymes and the lack of a gene encoding an enzyme that modifies the structure of the capsular polysaccharide of
C. neoformans
. Further, the prior art is deficient in the lack of means to block the deleterious activities of GXM that occur during the course of cryptococcosis. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention discloses the cloning, sequencing, expression, and characterization of a novel enzyme shown to de-O-acetylate glucuronoxylomannan. This novel enzyme was isolated from a mixture of microorganisms that were cultured from a sample of sewage sludge obtained from the Washoe County sewage treatment plant. The microbial culture produced a group of enzymes that fully degrade glucuronoxylomannan. The culture contains an unknown number of microbial species which have, as yet, not been identified. The culture does not grow well, if at all, when the species are separated, making identification of the different culture components difficult. The enzymatic activity appears to be the result of a minimum of four enzymes: mannosidase, xylosidase, glucuronidase. and O-acetylhydrolase (FIG.
2
). The O-acetylhydrolase was the first of the group to be purified. Following purification, the GXM-O-acetylhydrolase was subjected to peptide mapping which provided six partial amino acid sequences (Table 1): These fragments define the starting point for the cloning of a gene that encodes the enzyme.
TABLE 1
Sequences from Peptide Mapping of Purified Native GXM-O-
Acetylhydrolase and Its LysC-Cleaved Fragments
Peptide
N-Terminal Amino Acid Sequences
Whole protein
AETIYQDPVPAGANRAAVAVPRNDWYRD
VQNKFDKYSGKPADIVF (SEQ ID No. 1)
LysC-cleaved fragments
peptide 1*
YSGKPADIVFEGDSITNR (SEQ ID No. 2)
peptide 2
MIQPDGTISTDMMPDFVIIPT (SEQ ID No. 3)
peptide 3
IISRYADGDFVSFVDII (SEQ ID No. 4)
peptide 4
EHFEGRAADFGIEGDRVENAL (SEQ ID No. 5)
peptide 5
GYEIWGDAILPINN (SEQ ID No. 6)
*This sequence is a continuation of the whole protein N-terminal sequence.
The present invention is directed to DNA encoding glucuronoxylomannan (GXM)-O-acetylhydrolase, wherein the DNA is selected from the group consisting of (a) isolated DNA which encodes GXM-O-acetylhydrolase; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes GXM-O-acetylhydrolase; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes GXM-O-acetylhydrolase. Preferably, the DNA has the sequence shown in SEQ ID N
Bloomer Sherri L.
Kozel Thomas R.
Savoy Anne C.
Adler Benjamin Aaaron
Patterson Jr. Charles L.
Research Development Foundation
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