Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-29
2002-09-24
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S024300, C536S025320
Reexamination Certificate
active
06455248
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel methods for identifying fungal pathogens in a biological sample. In particular, this invention relates to methods for screening biological samples for the presence of fungal pathogens using hybridization methods and probes capable of sensitively and specifically detecting and distinguishing nucleic acid sequences unique to fungi. Also provided are antibodies capable of binding selectively to fungal proteins.
2. Background of the Invention
Candida albicans
(hereinafter referred to as “
C. albicans
”), once considered a relatively minor fungal pathogen, has recently become a particularly serious health concern as the causative agent of candidosis (also called candidiasis). The incidence of
C. albicans
infections is rising rapidly with the increase in immune deficiency diseases and immunosuppressive therapy (Bodey and Fainstein, In
Systemic Candidiasis
, pp. 135 (Eds., Raven Press, New York 1985). Candidosis is a common nosocomial infection afflicting both immunosuppressed and postoperative patients. (Holmes, A. R., et al. Yeast-
specific DNA probes and their application for the detection of Candida albicans, J. Med. Microbiol
., 37:346-351 (1992)). Although candidosis is a particular concern among immunocompromised individuals, Candida infections are not limited to this group.
C. albicans
is the major opportunistic fungal pathogen in humans (odds, F. C., In Candida and candidosis. (Ed.) Leicester University Press, Leicester, United Kingdom (1989)) and is capable of establishing infection whenever the host immune system or normal flora are perturbed.
Although the
C. albicans
species is a particular health concern, other species of the Candida genus are also pathogenic. The genus Candida is comprised of approximately 200 diverse yeast species classified together due to their lack of a sexual cycle (Meyer et al., In
Genus
4
, Candida
, pp. 1-12, (Ed.) N. J. W. Kregervan Riij, Elsevier, Amsterdam (1984)). A minority of Candida species are pathogenic and 80% of the clinical isolates are either
C. albicans
or
C. tropicalis
(Hopfer, R. L.
In Mycology of Candida Infections
, G. P. Bodey. an V. Fainstein (eds.), Raven Press, New York (1985)).
In immunocompromised hosts, candidosis is a life threatening condition. The prognosis for a patient infected with
C. albicans
can be improved markedly, however, with prompt antifungal treatment. Treatment may be delayed until a positive diagnosis of Candidosis is obtained since antifungal drugs are toxic. (See Holmes, et al., 1992.)
Diagnostic tests for the identification of
C. albicans
or other fungal pathogens in vivo often require complete cultural identification protocols (Musial et al., Fungal Infections of the Immunocompromised Host: Clinical and Laboratory Aspects,
Clin. Microbiol. Rev
. 1:349-364 (1988)). Methods currently used for the diagnosis of fungal pathogens include: cultural identification, biopsy, serodiagnosis, identification of metabolites, isoenzyme determination, pulsed field gel electrophoresis and analysis of restriction fragment length polymorphisms. Most of these methods are time consuming, laborious and provide inconclusive results.
Potential methods for diagnosing fungal infections through DNA screening have focused on detecting specific nucleotide sequences such as ribosomal DNA (Hopfer, R. L. et al., Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis,
J Med. Vet. Pharm
. 31:65-75 (1993)) and the p450 genes (Buchman, T. G. et al., Detection of surgical pathogens by in vitro DNA amplification. Part I, Rapid identification of
Candida albicans
by in vitro amplification of a fungal specific gene.
Surgery
, 108:338347 (1990)). However, no commercial diagnostic techniques embodying methods related to the identification of these genes in biological samples are known.
The sequences of approximately 1800
C. albicans
genes are available in computerized databases. The relatively small amount of fungal specific or unique genetic information available for
C. albicans
places limitations upon the number of DNA sequences that can be used as targets for screening probes and concomitantly reduces the likelihood of identifying a sequence unique to fungi and amenable to identification through DNA screening techniques. For example, very few of available sequences are from genes involved in fungal amino acid biosynthesis pathways. One impediment to developing nucleic acid based screening techniques for Candidosis is that basic information about uniquely fungal metabolic pathways and cognate genes of
C. albicans
is lacking (Kurtz et al.,
Molecular Genetics of Candida Albicans
, pp. 21-73, Kirsch, Kelly and Kurtz (eds.) CRC Press Inc. Boca Raton, Fla. (1990)).
Similar impediments exist to developing immunological methods of identifying a fungus present in a biological sample. Relatively few antigenic determinants unique to fungi are known, and none are believed to have been successfully utilized as targets for antibody binding in commercially available form.
Among the proteins that have been studied in
C. albicans
and other pathogenic fungi are the enzymes that make up the &agr;-aminoadipate pathway for the biosynthesis of lysine. This unique pathway has only been identified in
Phycomycetes, Euglenids
, yeasts and other higher fungi (Bhattacharjee, The &agr;-aminoadipate Pathway for the Biosynthesis of Lysine in Lower Eukaryotes, CRC
Critical Rev. in Microbiol
. 12:131-151 (1985); Lejohn, Enzyme Regulation. Lysine Pathways and Cell Wall Structures as Indicators of Evolution in Fungi, Nature 231:164-168 (1971); and Vogel, Two Modes of Lysine Synthesis Among Lower Fungi: Evolutionary Significance,
Biochim. Biophys. Acta
41:172-174 (1960); (Garrad, R. Masters Thesis, Miami University (1989) and, Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of
Candida albicans
, J. Bacteriol. 174:7379-7384 (1992)). Lysine biosynthesis is an example of a biochemical divergence between higher fungi, which use the &agr;-aminoadipic acid pathway (distinct from the diaminopimelic acid pathway used by bacteria and plants), and human host cells, which cannot synthesize lysine. The aminoadipate pathway for lysine biosynthesis, therefore, offers a unique opportunity to develop molecular probes for detection of fungal pathogens and as a potential drug target.
The &agr;-aminoadipate pathway consists of eight enzyme catalyzed steps; there appear to be seven free intermediates in
S. cerevisiae
(Bhattacharjee, The &agr;-aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes, CRC
Critical Review in Microbiol
. 12:131-151 (1985)). An understanding of the genetics, biochemical aspects, and regulation of the &agr;-aminoadipic acid pathway has been obtained by studies in the model organisms
Saccharomyces cerevisiae, Schizosaccizaromyces pombe
, and in the yeast
Candida maltosa
(Bhattacharjee 1992; Feller et al. 1994; Hinnebusch 1992; Schmidt et al. 1985). The final reversible step of the &agr;-aminoadipate pathway is catalyzed by saccharopine dehydrogenase, which is encoded by the LYS1 gene of
S. cerevisiae
and
C. albicans
, and the LYS5 gene of
Y. Lipolytica
(Fujioka, Chemical mechanism of saccharopine dehydrogenase (NAD, L-lysine forming) as deduced from initial rate pH studies,
Arch. Biochem. Biophys
. 230:553-559 (1984); Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of
Candida albicans, J. Bacteriol
. 174:7379-7384 (1992); and Xuan et al., Overlapping reading frames at the LYS5 locus in the yeast
Yarrowia lipolytica, Mol. Cell. Biol
. 10:47954806 (1990)).
The conversion of aminoadipic acid to [&agr;-aminoapidate] &agr;-aminoadipate semialdehyde is an obligatory step for the biosynthesis of lysine in yeast and is catalyzed by the enzyme
Bhattacharjee Jnanendra K
Bhattacherjee Vasker
Suvarna Kalavati
Horlick Kenneth R.
McDonnell Boehnen Holbert & Berghoff
Miami University
Tung Joyce
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