Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-07-01
2002-08-13
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S300000, C530S387200, C530S371000, C435S069100, C435S254200, C435S483000, C435S006120, C435S252300, C435S320100, C435S325000, C536S023200
Reexamination Certificate
active
06433137
ABSTRACT:
TECHNICAL FIELD
This invention relates to the field of polynucleotides and polypeptides. More specifically, this invention relates to TUP1 polynucleotides from
Candida albicans
, Tup1 polypeptides, and methods using these polynucleotides and polypeptides, especially for screening candidate anti-fungal agents.
BACKGROUND OF THE INVENTION
The yeast Candida is a ubiquitous human commensal, known as the causative agent of candidiasis. The majority of the diseases are caused by the species
Candida albicans
. It is the most prevalent commensal and opportunistic fungal pathogen of humans, causing common superficial infections as well as more serious systemic and organ infections. Cannon et al. (1995)
J. Dental Research
. 74:1152-1161. Exposure to
C. albicans
at or shortly after birth results in lifelong colonization in the host tissues, such as the gastrointestinal tract, oral cavity and genital area. It has been noted that approximately 75% of women would suffer from vaginal candidiasis at some stage in their lifetime. Bossche et al. (1993)
Fungal Dimorphism
3-10; Fidel et al. (1996)
Clin. Micro. Rev
. 9(3):335-348. Whereas
C. albicans
infection often remains localized to the initial sites of contact in healthy individuals,
C. albicans
cells can invade submucosal vessels, disseminate hematogenously and become life-threatening, especially to immunocompromised patients. The invasive forms of
C. albicans
infection are not only dangerous in their own right, but they are believed to facilitate infections by other opportunistic pathogens.
In the last decades, the incidence of severe and systemic candidiasis has increased dramatically because of the growing number of immunocompromised patients suffering from AIDS, diabetes, cancer and other conditions. In addition, the widespread use of immunosuppressants for organ transplant patients, the common practice of radiation and chemotherapy for treating malignancies, as well as the growing size of the aging population have increased the morbidity of this opportunistic pathogen. For reviews, see Rubin et al. (1993)
Eur. J Clin. Microbiol. Infect. Dis
. 12 Suppl. 1, 542; Dudley et al. (1990)
Pharmacotherapy
10:133; Paya (1993)
Clin. Infect. Dis
. 16:677-688; Rubin (1993)
Eur. J Clin. Micro. Infect. Dis
. 12 Suppl. 1: S42-S48.
Despite decades of intensive study, the properties of
C. albicans
that contribute to its virulence are only beginning to be understood. Among the most investigated virulence factors are adherence, production of hydrolytic enzymes and adoption of various cell morphologies. Odds et al. (1994)
Am. Soc. Microbiol. News
60:313-318. The ability of
C. albicans
to adhere to the host surfaces probably allows initial colonization and infection of the host tissues. Secretion of a variety of hydrolytic enzymes which are capable of degrading proteins and lipids is thought to generate tissue cavitation and thereby facilitate deeper penetration. The morphological transition between various forms of
C. albicans
is also considered a key determinant of virulence.
C. albicans
cells can exist in a variety of shapes, ranging from elipsoidal budding yeast cells (also known as blastopores) to cylindrical hyphae (also known as filaments) in which cells remain attached to each other after dividing and thereby form long branched strings of connected cells (FIG.
1
). Transitions between these forms take place by outgrowth of new cells with the altered morphology, rather than remodeling of pre-existing cells. The ability of
C. albicans
to adopt these different morphologies is thought to allow the fungus to adapt to, and possibly travel to, different host micro-environments. Odds et al. (1988) Candida and Candidosis (Bailliere Tindall, London, 2nd ed.); Odds et al. (1994); Odds et al. (1994)
J. Am. Acad. Dermatol
. 31:52. The regulation of cellular morphology is in response to environmental conditions. In vitro studies have shown that most
C. albicans
strains assume filamentous forms when they are subjected to either unfavorable growth conditions, such as nutrient-poor media and high CO
2
:O
2
ratio, or host-related conditions, such as high temperature (37° C.) and mammalian serum (10%). Conversely, rich media, low temperatures and aerated conditions promote blastospore growth. Intermediate conditions can induce various pseudohyphal forms as well as true hyphae. For reviews, see Odds et al. (1988) Candida and Candidosis, Bailliere Tindall, London, ed. 2nd; Odds et al
Crit. Rev Microbiol
. (1985) 12:45; Gow et al. (1984)
Sabouraudia
22:137. The pseudohyphal cells are elongated but still elipsoidal in shape, whereas the true hyphal cells are cylindrical and separated by perpendicular septal walls. Very little is known about the genetic identity of regulators controlling the morphological transition of
C. albicans.
The ability of
C. albicans
to adopt these different morphologies is thought to contribute to colonization and dissemination within host tissues and thereby to promote infection. Odds (1988); Odds (1994)
J. Am. Acad. Dermatol
. 31:S2. It has been commonly suggested that the hyphal form is invasive and pathogenic, while the blastospore is the commensal non-pathogenic form. However, all morphological forms have been found within infected tissues. Histopathological examination of candidiasis lesions indicates that hyphae are not always present. More recent studies have shown that commensal
C. albicans
does not exist uniquely in the blastopore form. In fact, sometimes invading
C. albicans
cells are seen exclusively as the budding yeast form. Odds et al. (1994)
Am. Soc. Microbiol. News
60:313-318. Despite the uncertainty with regard to the relative roles these two distinct forms of
C. albicans
have in fungal virulence, phenotypic switching represents a remarkable adaptation that
C. albicans
has acquired to cope with different host microenvironments. Identifying the genetic components that regulate the morphological transition are therefore of great significance for identifying the role of this transition in pathogenesis and developing potential therapeutic agents of candidiasis.
Current therapy available for systemic candidiasis is limited to the use of anti-fungal agents. In practice, the arsenal of anti-fungal drugs is based on a few antimycotics, such as flucytosine, amphotericin B and azole derivatives. Many of these antimycotics are somewhat water insoluble which restrict their bioavailability and present problems in intravenous formulation. In addition, they cause serious and often difficult side effects, such as renal toxicity, bone marrow destruction, as well as unpleasant symptoms such as fever and shivering. Furthermore, the chronic use of these anti-fungal agents has led to the emergence of drug-resistant strains of Candida, which can cause fatal relapse of the disease. Dupont et al. (1995)
J. Am. Podiatric Med. Assn
. 85:104-115; Fox et al. (1991)
J. Infect. Dis
. 22:201-204; Scheife (1990)
Pharmacotherapy
10:S133-S183. Taken together, anti-fungal therapy alone is inadequate for treating chronic candiasis. The availability of recombinant cytokines, such as interleukin-2, provides an alternative way to stimulate the cell-mediated immunity of infected individuals. However, this type of cytokine replacement therapy for fungal infections remains highly experimental. Weinberg et al. (1990)
N. Eng. J. Med
. 332: 1718.
S. cerevisiae
Tup1 encoded by the TUP1 gene is a member of a family of WD repeat containing proteins. Tup1, along with the SSN6 protein, represses sets of genes involved in a variety of cellular processes, including glucose repression, mating, sporulation and flocculation. The gene targets of TUP1 regulation are each regulated by a distinct upstream DNA-binding protein, and each DNA-binding protein recruits to the promoter a complex containing the TUP1 protein. The biochemical mechanisms by which TUP1 in
S. cerevisiae
mediates transcriptional repression are yet not well understood. Tzamarias et al. (1994)
Nature
369: 758; Komachi et al.
Genes Dev
. 8: 2857; Wahi et al.
Braun Burkhard
Johnson Alexander D.
Carlson Karen Cochrane
Morrison & Foerster / LLP
Robinson Hope A.
The Regents of the University of California
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