WO1999067419A1

WO1999067419A1 - β(1,6)-GLUCAN SYNTHESIS AND CELL WALL ASSEMBLY ASSAY

Info

Publication number: WO1999067419A1
Application number: PCT/US1999/013434
Authority: WO
Inventors: Gary R. Ostroff
Original assignee: The Collaborative Group, Ltd.
Priority date: 1998-06-25
Filing date: 1999-06-15
Publication date: 1999-12-29
Also published as: AU4566699A

Abstract

Methods are disclosed for assessing an agent for an ability to inhibit β(1,6)-glucan synthesis and/or incorporation of β(1,6)-glucan into the cell wall, comprising contacting samples of yeast cells that are sensitive to a killer toxin that targets β(1,6)-glucan with a test agent and a lethal dose of the killer toxin, and/or contacting samples of yeast cells that are sensitive to the killer toxin with a test agent and a sublethal dose of the killer toxin, and assessing the permeability of the cells in the samples, such as by using a membrane-impermeable dye that fluoresces on contact with DNA.

Description

β(l,6)-GLUCAN SYNTHESIS AND CELL WALL ASSEMBLY ASSAY

RELATED APPLICATIONS

This application claims priority to U. S. Application No. 09/104,873, filed June 25, 1998, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Immunocompromised patients are susceptible to a variety of neoplastic, protozoal, viral, bacterial and fungal diseases; of these, bacterial, viral and fungal infections result in the greatest mortality (Bartlett, M. and J. Smith, Clin. Microbiol. Rev. 4:137-149 (1991); Bodey, G. et al, Eur. J. Clin. Microbiol. Infect. Dis. 77:99- 109 (1992); Sternberg, S., Science 2df5:1632-1634(1994); Cox, G. and J. Perfect, Curr. Opin. Infect. Dis. 6:422-426 (1993); Deepe, G. and W. Bullock, Eur. J. Clin. Microbiol. Infect. Dis. 9:377-380 (1990); Fox, J.L., ASM News 59:515-518 (1993); Kujath, P., Mycoses 55:225-228 (1992); Pfaller, M. and R. Wenzel, Eur. J. Clin. Microbiol. Infect. Dis. //:287-291 (1992); and Samonis, G. and D. Bafaloukos, In vivo (5:183-194 (1992)). During the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunodeficient hosts (Odds, F., Antimicrob. Chemother. 37:463-471 (1993); Ringel, S., Mycopath. 109:15-81 (1990); Walsh, T. et al, Diagn. Microbiol. Infect. Dis. 73:37-40 (1990); Nouza, M., Infection. 20:113-117 (1992); Rhodes, J. et al, J. Med. Vet. Myc. 30:51-51 (1992); Saral, R., Rev. Infect. Dis. 73:487-492 (1991); Levitz, S., Clin. Infect.Dis. 14:31-42 (1992); Polak, A. and P. Hartman, Progress in Drug Research, Basel: Birkhauser Verlag. 37: 181-269 (1991); Matthewson, H.S., Resp. Care 35:987-989 (1990); Hoeprich, P., Prog. Drug Res. 4 :87-127 (1995)). Fungal infections in immunocompromised patients are mainly the result of opportunistic infections by normally harmless, asymptomatic commensals, which can be pathogenic under certain conditions (Odds, F., Antimicrob. Chemother. 37:463-471 (1993); Rhodes, J. et al, J. Med. Vet. Myc. 30:51-51 (1992); Saral, R., Rev. Infect. Dis. 73:487-492 (1991)). Species of Cryptococcus, Candida, Coccidioides, Histoplasma, Blastomyces, Sporothrix and Aspergillus, as well as other opportunistic fungi, are important causative agents; of these, Candida species, especially C. albicans, are the most common (Ainsworth, G., Fungal parasites of vertebrates, in The Fungi, an Advanced Treatise (Ainsworth, G., ed., New York, Academic Press, vol. 3y, 1968; Khardori, N., Eur. J. Clin. Microbiol. Infect Dis. 5:331-351 (1989). Candidemia accounts for 8-10% of all hospital-acquired bloodstream infections and Candida species are the fourth most common cause of nosocomial septicemias. Mortality rates associated with systemic Candida infections are estimated to be as high as 50% of infected patients. Infections caused by other types of fungi (e.g., Aspergillus, Cryptococcus) are also common in immunocompromised patients and result in significant treatment costs and mortality (Meunier, F., Amer. J. Med. 99 (Suppl 6A):6 S-61S (1995)).

Although the demand for effective antifungal agents continues to increase, few effective agents are available in the clinic. Available drugs for the treatment of mycotic infections include azoles and polyenes, both of which inhibit sterol biosynthesis. The polyene amphotericin B, which is a commonly prescribed antifungal drug, interacts with membrane ergosterol (Lyman, C. and T. Walsh, Drugs 44:9-35 (1993)). These drugs, however, have several drawbacks: for example, the development of resistance to azoles has been observed in C. albicans (Bartlett, M. et al, Antimicrobial Agents. Chemo. 35:1859-1861 (1994); Odds, F., Internat. J. Antimicrob. Agents. 6:145-147 (1996)). Further, amphotericin B causes toxic side effects, including renal dysfunction, fever, chills and hypotension. The development of new drugs depends upon the discovery of new therapeutic targets and new assays for assessing the desired biological activity.

SUMMARY OF THE INVENTION

The present invention is drawn to methods of identifying agents that inhibit synthesis and/or incorporation of β(l,6)-glucan into the cell wall. The invention takes advantage of the ability of certain killer toxins to render sensitive yeast cells producing β(l,6)-glucan permeable to small molecules, such as small fluorescent or chromogenic dyes. One embodiment of the invention is accordingly drawn to assessing a test agent's ability to inhibit β(l,6)-glucan synthesis, by assessing the test agent's ability to render sensitive cells producing β(l,6)-glucan resistant to a killer toxin that targets β(l,6)-glucan using a membrane-impermeable dye that fluoresces on contact with DNA. In this embodiment, a sample of sensitive yeast cells is contacted with a test agent and a lethal dose of a killer toxin, and the permeability of the is assessed cells using the membrane-impermeable dye. If cells contacted with the test agent and the lethal dose of the killer toxin are less permeable than cells exposed to the lethal dose of the killer toxin but not to the test agent, then the test agent is an agent that renders sensitive cells resistant to the killer toxin, and thus is an agent that inhibits β(l,6)-glucan synthesis. In a preferred embodiment, the agent's ability to inhibit β(l,6)-glucan synthesis is assessed by contacting a first set of samples of killer toxin-sensitive yeast with a control agent, and contacting a second set of samples of killer toxin-sensitive yeast with a test agent; incubating the samples under conditions that are sufficient to allow metabolic turnover of β(l,6)- glucan in the yeast cells; contacting a sample from the incubated, control agent- contacted samples and a sample from the set of incubated, test agent-contacted samples with control media, and contacting a sample from the set of incubated, control agent-contacted samples and a sample from the set of incubated, test agent- contacted samples with a lethal dose of killer toxin; further incubating the samples under conditions that are sufficient to allow the killer toxin to interact with the cells in those samples exposed to the killer toxin; and assessing the permeability of the cells in the samples by using a membrane-impermeable dye that fluoresces on contact with DNA. If cells exposed to the test agent and the lethal dose of the killer toxin are less permeable than cells exposed to the control agent and the lethal dose of the killer toxin, in an amount that is significant, then the test agent is an agent that renders sensitive cells resistant to the killer toxin, and thus is an agent that inhibits β(l,6)-glucan synthesis.

A second embodiment of the invention is drawn to assessing a test agent's ability to inhibit incorporation of β(l,6)-glucan into the cell wall, by assessing the test agent's ability to render sensitive cells producing β(l,6)-glucan hypersensitive to a killer toxin that targets β(l,6)-glucan. In this embodiment, a sample of sensitive yeast cells is contacted with a test agent and a sublethal dose of Kl killer toxin, and the permeability of the cells is assessed. If cells contacted with the test agent and the sublethal dose of the killer toxin are more permeable than cells exposed to the sublethal dose of the killer toxin but not to the test agent, then the test agent is an agent an agent that renders sensitive cells hypersensitive to the killer toxin, and thus is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall. In a preferred embodiment, the agent's ability to inhibit incorporation of β(l,6)-glucan into the cell wall is assessed by contacting a first set of samples of killer toxin- sensitive yeast with a control agent, and contacting a second set of samples of killer toxin-sensitive yeast with a test agent; incubating the samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells; contacting a sample from the incubated, control agent-contacted samples and a sample from the set of incubated, test agent-contacted samples with control media, and contacting a sample from the set of incubated, control agent-contacted samples and a sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin; further incubating the samples under conditions that are sufficient to allow the killer toxin to interact with the cells in those samples exposed to the killer toxin; and assessing the permeability of the cells in the samples, such as by using a membrane-impermeable fluorescent dye that fluoresces on contact with DNA. If cells exposed to the test agent and the sublethal dose of the killer toxin are more permeable than cells exposed to the control agent and the sublethal dose of the killer toxin, in an amount that is significant, then the test agent is an agent that renders sensitive cells hypersensitive to the killer toxin, and thus is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

A third embodiment of the invention combines the two methods described above into a single method for assessing a test agent's ability to inhibit β(l,6)-glucan synthesis and/or incorporation of β(l,6)-glucan into the cell wall, by contacting a sample of sensitive yeast cells with a test agent and a lethal dose of a killer toxin, contacting a sample of sensitive yeast cells with a test agent and a sublethal dose of the killer toxin, and assessing the permeability of the cells. If cells contacted with the test agent and the lethal dose of the killer toxin are less permeable than cells exposed to the lethal dose of the killer toxin but not to the test agent, then the test agent is an agent an agent that renders sensitive cells resistant to the killer toxin, and thus is an agent that inhibits β(l,6)-glucan synthesis or incorporation of β(l,6)- glucan into the cell wall. If cells contacted with the test agent and the sublethal dose of the killer toxin are more permeable than cells exposed to the sublethal dose of the killer toxin but not to the test agent, then the test agent is an agent an agent that renders sensitive cells hypersensitive to the killer toxin, and thus is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall. In a preferred embodiment, the agent's ability to inhibit β(l,6)-glucan synthesis and/or incorporation of β(l,6)-glucan into the cell wall is assessed by contacting a first set of samples of killer toxin-sensitive yeast with a control agent, and contacting a second set of samples of killer toxin-sensitive yeast with a test agent; incubating the samples under conditions that are sufficient to allow metabolic turnover of β(l,6)- glucan in the yeast cells; contacting a sample from the incubated, control agent- contacted samples and a sample from the set of incubated, test agent-contacted samples with control media, contacting a sample from the set of incubated, control agent-contacted samples and a sample from the set of incubated, test agent-contacted samples with a lethal dose of the killer toxin, and contacting a sample from the set of incubated, control agent-contacted samples and a sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin; further incubating the samples under conditions that are sufficient to allow the killer toxin to interact with the cells in those samples exposed to the killer toxin; and assessing the permeability of the cells in the samples, as described above.

The methods of the invention provide a simple, convenient means for identifying agents that interfere with β(l,6)-glucan synthesis or incorporation into the cell wall. Such agents may interfere with the normal assembly of yeast cell walls; because cell wall assembly is necessary for growth and viability not only of yeast, but of all fungi, agents which affect assembly of yeast cell walls are potential antifungal agents. Thus, the methods of the invention facilitate identification and development of novel antifungal agents.

DETAILED DESCRIPTION OF THE INVENTION

THE FUNGAL CELL WALL The fungal cell wall has a complex composition and structure (Ruiz-Herrera, J. Fungal cell wall: Structure, synthesis and assembly, CRC Press, FI, 1992; Wessels, J., New Phytol 723:397-413 (1993)). In general, the cell wall of human pathogenic fungi contains β(l,3)-glucan, β(l,6)-glucan, other α-linked glucans (including glycogen and α(l,3)-glucan), mannoproteins, and smaller amounts of peptides, chitin and lipids (Fleet, G.H., in The Yeasts (2^nd edition, Rose, A.H. and Harrison, J.S., eds) 4:199-277, Academic Press, London (1991)). Cell wall components are either structural, providing mechanical strength to the wall, or cementing, keeping structural components glued together. Structural materials are fibrillar and include chitin, β-l,3-linked-glucans and cellulose. Cementing materials are amorphous and include β(l,3)- and α-linked glucans, chitosan and glycoproteins.

Normal cell wall biosynthesis and assembly is essential for growth and viability of fungi, as shown using cell wall-acting drugs, as well as by analysis of mutants defective in key cell-wall assembly steps (Scott, W., Ann. Rev. Microbiol. 30:85-104 (1972)). Cell wall assembly is a complex, incompletely understood process (Klis, F., Yeast 70:851-569 (1994)). Nascent chitin and β(l,3)-glucan polymers of the cell wall are vectorially synthesized through the plasma membrane by transmembrane multi-subunit enzyme complexes into the periplasmic space in a random coil conformation; subsequently they undergo an ordered assembly process to form microfibrils. β(l,6)-glucan, which appears not only in the cell wall, but also in the cell membrane linked to glycosylphosphatidylinositol (GPI) (Van Der Vaart, J.M., et al, FEMS Microbiol. Let. 745:401-407 (1996)), is synthesized in the endoplasmic reticulum from UDP-glucose and transported to the cell wall through the Golgi apparatus. The β(l,3)-glucan fibrils are interwoven with chitin microfibrils in forming the fungal cell wall; β(l,6)-glucan serves to interconnect mannoproteins, β(l,3)-glucans, and chitin (Kollar, R. et al, J. Biol. Chem. 272:17762-17775 (1997)).

A potential target of antifungal agents is fungal cell wall biosynthesis and assembly. Interference with cell-wall biosynthesis by inhibitors such as cilofungin (β(l,3)-glucan synthesis), nikkomycin (chitin synthesis) and tunicamycin

(mannoprotein synthesis), interferes with fungal cell growth (Kurtz, ASM News 64(1 ):3l (1997)). Inhibition of the non-enzymatic chitin and β( l,3)-glucan microfibril assembly processes by calcofluor and congo red also interferes with fungal cell growth (Vanni, G. et al, Plant Set Letters 31 :9-17 (1983); Ilorza, M. et al, J. Gen. Microbiol. 729:1577-1582 (1983)). Assays to identify agents that inhibit β(l,3)-glucan microfibril assembly are described in detail in U.S. Patent application Serial No. 09/104,914, filed June 25, 1998, entitled "β(l,3)-GLUCAN

MICROFIBRIL ASSEMBLY ASSAY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference. Assays to identify agents that inhibit chitin microfibril assembly are described in detail in U.S. Patent application Serial No. 09/104,315, filed June 25, 1998, entitled "CHITIN MICROFIBRIL ASSEMBLY ASSAY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference.

Targeting enzymatic processes involved in cell wall biosynthesis and assembly has proven to be difficult. From genome sequencing of S. cerevisiae, it is known that the fungal genome includes redundant genes for many critical enzymatic activities. The resulting isozymes enable the fungal cell to synthesize a cell wall even if a single enzyme is inhibited or a particular gene is genetically inactivated. This redundancy complicates the drug discovery process and results in significant difficulties in the development of new antifungal drug candidates.

METHODS OF THE INVENTION The present invention provides in vitro methods for assessing the ability of an agent to inhibit (partially or totally) the synthesis of β(l,6)-glucan and/or extracellular incorporation of β(l,6)-glucan into the cell wall. Agents that inhibit the synthesis of β(l,6)-glucan or inhibit its extracellular incorporation into the cell wall thus prevent the extracellular assembly of the fungal cell wall. Such agents, which target the essential, β(l ,6)-glucan synthesis and/or assembly into the cell wall, can be useful as antifungal agents.

The methods of the invention take advantage of killer toxins that target β(l,6)-glucan. A killer toxin that "targets β(l,6)-glucan," as used herein, refers to a killer toxin whose activity is based on an interaction between the killer toxin and β(l,6)-glucan, such as by binding of the killer toxin to β(l,6)-glucan.

Representative killer toxins that target β(l,6)-glucan include Kl killer toxin and K2 killer toxin (Magliani, W. et al, Clin. Microbiol Rev. 10(3):369-400 (1997)). Kl killer toxin is a virally-coded porin protein that is secreted by certain yeast cells. Kl killer toxin destroys sensitive yeast cells by a process in which it binds to β(l,6)- glucan both in the cell wall and in the cytoplasmic membrane, and where the toxin bound to membrane β(l,6)-glucan forms a lethal pore in the yeast cell membrane (Bussey, H., Mol Microbiol. 5 (10). -2339-2343 (1991); Martinac, B. et al, Proc. Natl Acad. Sci. USA 87(16) .-6228-6232 (1990); Schmitt, M.J., and Compain, P., Arch. Microbiol. 164:435-443 (1995)). The activity of K2 killer toxin is essentially the same as that of Kl killer toxin (Magliani, W. et al, Clin. Microbiol. Rev. 10(3):369- 400 (1997)).

The step in the process in which the toxin binds to β(l,6)-glucan components of the outer surface of the yeast cell wall, is blocked in yeast mutants having mutations in KRE (killer resistant) genes whose products are involved in synthesis and/or assembly of cell wall β(l,6)-glucans (Meaden, P. et al, Mol. Cell Biol. 70f6):3013-3019 (1990); Roemer, T. et al, Mol. Cell Biol. 73( ):4039-4048 (1993); Brown, J.L. et al, Genetics 733 4J:837-849 (1993); Jiang, B. et al, J. Bacteriol 178(4):\ \62-l \l\ (1996)). Killer resistant (KRE) mutants that have been characterized confer toxin resistance by disrupting β(l,6)-glucan synthesis (Brown, J.L. et al, Molecular Genetics of Yeast (IRL Press, J.R. Johnston, Ed., A practical Approach Series, Oxford, University Press, 1994)). These KRE mutants have reduced levels of cell wall β(l,6)-glucan (Brown, J.L. et al, Genetics 133(4):831- 849 (1993); Jiang, B. et al, J. Bacteriol 178(4):\ 162-1171 (1996)).

ASSAY FOR RESISTANCE TO Kl KILLER TOXIN In one embodiment of the invention, an agent that inhibits synthesis of β(l,6)-glucan is identified by assessing whether the agent confers, to an otherwise sensitive yeast cell, resistance to a killer toxin that targets β(l,6)-glucan. Sensitive cells that have "resistance" to a killer toxin that targets β(l,6)-glucan are those cells which survive challenge with a dose of the killer toxin that would otherwise be fatal to sensitive cells. As stated above, KRE mutants confer resistance to Kl killer toxin by affecting β(l,6)-glucan synthesis; therefore, if an agent renders a sensitive yeast cell resistant to the effects of a killer toxin that targets β(l,6)-glucan, it is an agent that inhibits β(l,6)-glucan.

In these methods, a yeast strain, producing β(l,6)-glucan, that is sensitive (susceptible) to a killer toxin that targets β(l,6)-glucan (i.e., a strain that is not a killer resistant mutant) is used. Representative yeasts include Saccharomyces cerevisiae and Candida albicans. Alternatively, any other fungus that produces β(l,6)-glucan can be used. The S. cerevisiae strain TA405 (Whiteway, M. and J.W. Szostak, Cell 43:483-492 (1985)) is a representative sensitive yeast strain. The sensitive yeast strain is grown to midlog phase in a standard media. Sets of samples of the sensitive yeast cells are then established. The first set of samples is contacted with a control agent. In one embodiment, two samples are in the set; these two samples are referred to herein as samples "Cl" and "C2" (Control agent-contacted samples). A different number of samples can be used if desired. Representative control agents include sterile water, saline, DMSO, or ethanol solvents. The second set of samples is contacted with the agent to be tested for its ability to inhibit β(l,6)-glucan synthesis (the test agent). If two samples are in the set, these samples are referred to herein as samples "Tl" and "T2" (lest agent- contacted samples). Again, a different number of samples can be used if desired. The two sets of samples (control agent-contacted samples and test agent-contacted samples) are then incubated under conditions that are sufficient to allow metabolic turnover of β(l ,6)-glucan on the plasma membrane of the cells in the samples. The term, "metabolic turnover," as used herein, refers to the degradation and resynthesis of β(l,6)-glucan as the cell wall matures and the cell divides. Representative conditions include incubation at a temperature that is between about 18°C and about 37 °C, inclusive. The samples are incubated for approximately 1 to 5 hours. In a preferred embodiment, the samples are incubated at 30 °C for approximately 2.5 hours.

A first sample from the set of incubated, control agent-contacted samples, and a first sample from the set of incubated, test agent-contacted samples, is then contacted with control media. These samples (e.g., Cl and Tl) are referred to as the control/control sample, and the test agent/control sample, respectively. A second sample from the set of incubated, control agent-contacted samples, and a second sample from the set of incubated, test agent-contacted samples, is contacted with a lethal dose of the killer toxin. A "lethal dose", as used herein, is an amount of killer toxin that results in mortality of greater than 50% of susceptible cells in approximately 6 hours at 20 °C when the cells are exposed to the toxin. These samples (e.g., C2 and T2) are referred to as the control/lethal toxin sample and the test agent/lethal toxin sample, respectively. A summary of representative samples is set forth in Table 1.

The control/control sample, test agent/control sample, control/lethal toxin sample and test agent lethal toxin sample are then incubated under conditions that are sufficient to allow the killer toxin in the lethal toxin samples to interact with the cells. Representative conditions include incubation at a temperature that is between about 15 °C and about 22 °C, inclusive, for approximately 2-16 hours. The incubated control/control sample, test agent/control sample, control/lethal toxin sample and test agent/lethal toxin sample are then assessed to determine whether the test agent confers resistance to the killer toxin; a test agent that confers resistance to the killer toxin is an agent that inhibits synthesis of β(l,6)-glucan. To determine whether a test agent confers resistance to the killer toxin, the permeability of the cells is assessed. In the absence of the killer toxin, sensitive yeast cells are generally impermeable; however, cells that have formed pores following exposure to the killer toxin are highly permeable. Resistance to the killer toxin can thus be assessed by comparing the relative permeability of the cells. Cells that are less permeable are resistant to the killer toxin; therefore, if the test agent renders cells less permeable, it is an agent that confers resistance to the killer toxin, and therefore is an agent that inhibits β(l,6)-glucan synthesis. A membrane-impermeable fluorescent dye that fluoresces on contact with

DNA is used to determine the permeability of the cells. Representative membrane- impermeable fluorescent dyes that fluoresce on contact with DNA include ethidium bromide, prepidium iodide (Hoescht 33342), or Sytox Green (Molecular Probes, Eugene, Oregon). Cells which have an intact membrane (impermeable cells) do not take up the dye, and thus, the dye does not fluoresce. In contrast, those cells which have a permeable membrane take up the dye and become fluorescent when the dye contacts the cellular DNA. Since the extracellular dye itself is not fluorescent, the samples can be scored for bulk fluorescence using, for example, a microplate fluorimeter. This obviates the need to assess individual cells as taught by prior art (see, e.g., Brown, J., Mol. Genet, of Yeast (J.R. Johnston, ed.), IRL Press, Oxford, 1994, pp. 217-229; and Kurcweilova, H., et al, Yeast 9.T207-1211 (1993)); furthermore, this assay is high-throughput compatible.

The incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample and incubated test agent/lethal toxin samples are contacted with the membrane-impermeable dye that fluoresces on contact with DNA. For example, approximately 0.1 - 10 μM dye can be used. If desired, a detergent can be added with the membrane-impermeable dye, to facilitate entry of the dye into the permeable cells. Representative detergents include NP-40 and Triton X-100. In a particularly preferred embodiment, Sytox Green dye is added (approximately 0.1 - 10 μM), together with approximately 0.05-0.5% NP-40, to each sample. The dye-contacted samples are then incubated under conditions that are sufficient to allow the dye to enter permeable cells. Representative conditions include incubation at approximately room temperature for approximately 2 hours; or incubation at approximately 4°C for overnight. The fluorescence of each of the dye- contacted samples is then assessed by an appropriate means, such as by a fluorimeter. Those cells which are sensitive to the killer toxin take up the dye and become fluorescent when the dye contacts the cellular DNA; those cells which are resistant to the killer toxin do not take up dye, or take up less dye, and therefore are not fluorescent or are less fluorescent.

If the sample that has been exposed to the test agent and the lethal dose of the killer toxin (test agent/lethal toxin sample, T2) fluoresces less than the sample that has been exposed to the control agent and the lethal dose of the killer toxin (control agent/lethal toxin sample, C2), in an amount that is significant, then the test agent has rendered the sensitive cells less permeable, and thus, has conferred resistance to the killer toxin on the sensitive cells. A "significant" amount of difference in fluorescence occurs if the value of: (T2-T1)/(C2-C1) is less than or equal to approximately 2. Therefore, such an agent is an agent that inhibits synthesis of β(l,6)-glucan.

In another embodiment, the effect of the test agent on the sensitive cells can be assessed by using other physical methods, such as by measuring viscosity or polarimetry, or by fluorescent polarization. The use of fluorescence polarization is described in detail in U.S. Patent application Serial No. 09/104,560, filed on June

25, 1998, entitled "ASSAYS FOR AGENTS WHICH ALTER CELL WALL

BIOPOLYMER SYNTHESIS OR ASSEMBLY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference.

ASSAY FOR HYPERSENSITIVITY TO KILLER TOXIN

In another embodiment of the invention, an agent that inhibits incorporation of β(l,6)-glucan into the cell wall is identified by determining whether the agent confers, to an otherwise sensitive yeast cell, hypersensitivity to the killer toxin. Sensitive cells that have "hypersensitivity" to the killer toxin are those cells which do not survive challenge with a dose of the killer toxin that would not otherwise be fatal to sensitive cells. Because β(l,6)-glucan is synthesized in the endoplasmic reticulum and transported to the cell wall, an agent that inhibits incorporation of β(l,6)-glucan into the cell wall (but does not effect synthesis of β(l,6)-glucan) causes a buildup of β(l,6)-glucan that is associated with the cell membrane. Because the killer toxin interacts with membrane-associated β(l,6)-glucan in forming lethal pores, the presence of an increased amount of β(l,6)-glucan associated with the cell membrane renders the cell hypersensitive to the killer toxin: with more β(l,6)-glucan target for the the killer toxin, more lethal pores can be formed. The hypersensitive cells thus are more permeable, and will die from exposure to even a small amount of the killer toxin. As described above, a yeast strain that is sensitive (susceptible) to the killer protein is used. The sensitive yeast strain is grown to midlog phase in a standard media. In one embodiment, two sets of samples of the sensitive yeast cells are established. The first set of samples is contacted with a control agent, as described above. In one embodiment, two samples are in the set; these two samples are referred to herein as samples "Cl" and "C3" (Control agent-contacted samples). A different number of samples can be used if desired. The second set of samples is contacted with the agent to be tested for its ability to inhibit incorporation of β(l,6)- glucan into the cell wall (the test agent). In one embodiment, two samples are in the set; these two samples are referred to herein as samples "Tl" and "T3" (lest agent- contacted samples). As before, a different number of samples can be used if desired. The set of control agent-contacted samples and the set of agent-contacted samples are then incubated under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan on the plasma membrane and in the cell wall of the cells of the samples, as described above. A first sample from the set of incubated, control agent-contacted samples, and a first sample from the set of incubated, test agent-contacted samples, is then contacted with control media. These samples (e.g., Cl and Tl) are the control/control sample and the test agent control sample, respectively. A second sample from the set of incubated, control agent-contacted samples, and a second sample from the set of incubated, test agent-contacted samples is contacted with a sublethal dose of the killer toxin. A "sublethal dose", as used herein, is an amount of the killer toxin that results in mortality of less than approximately 10% of susceptible cells when the cells are exposed to the toxin. These samples (e.g., C3 and T3) are referred to as the control/sublethal toxin sample and the test agent/sublethal toxin sample, respectively. A summary of representative samples is set forth in Table 2.

The control/control sample, test agent/control sample, control/sublethal toxin sample and test agent/sublethal toxin sample are then incubated under conditions that are sufficient to allow the killer toxin in the sublethal toxin samples to interact with the cells. Representative conditions include incubation at a temperature that is between about 15 °C and about 22 °C, inclusive, for approximately 2-16 hours. The incubated control/control sample, test agent/control sample, control/lethal toxin sample and test agent/lethal toxin sample are then assessed to determine whether the test agent confers hypersensitivity to the killer toxin; a test agent that confers hypersensitivity to the killer toxin is an agent that inhibits incorporation of β(l,6)-glucans into the cell wall. Because the yeast cells are generally impermeable in the absence of the killer toxin, and are highly permeable following exposure to the killer toxin, hypersensitivity to the killer toxin can be assessed by comparing the relative permeability of the samples. Cells that are more permeable are hypersensitive to the killer toxin; therefore, if the test agent renders cells more permeable, it is an agent that confers hypersensitivity to the killer toxin, and therefore is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

To determine whether a test agent confers hypersensitivity to the killer toxin, the permeability of the cells is assessed. The permeability of the yeast cells can be determined using various methods. For example, a light microscope can be used to examine the cells. A phase bright microscopic image is indicative of permeability ("leakiness") of a cell; permeability that results in death is evidenced by phase dark, small cell microscopic images. Alternatively, a membrane-impermeable dye, such as trypan blue or a fluorescent dye, can be used. The dye can be detected using standard methods, such as a light or fluorescent microscope, respectively. In a preferred embodiment, the permeability of the cells is assessed using a membrane-impermeable dye that fluoresces on contact with DNA, in the same manner as described above. If the sample that has been exposed to the test agent and the sublethal dose of the killer toxin (test agent/sublethal toxin sample, T3) fluoresces more than the sample that has been exposed to the control agent and the sublethal dose of the killer toxin (control agent/sublethal toxin sample, C3), in an amount that is significant, then the test agent has rendered the sensitive cells more permeable, and thus, has conferred hypersensitivity to the killer toxin on the sensitive cells. A "significant" amount of difference in fluorescence occurs if the value of: (T3-T1)/(C3-C1) is greater than or equal to approximately 2.5. Such an agent is an agent that inhibits incorporation of β(l,6)-glucans into the cell wall.

COMBINATION ASSAY FOR RESISTANCE OR HYPERSENSITIVITY

In a preferred embodiment of the invention, an assay is conducted to assess an agent both for an ability to confer resistance on sensitive cells, as well as for an ability to confer hypersensitivity on sensitive cells. In this embodiment, two sets of samples of the sensitive yeast cells are established. The first set of samples is contacted with a control compound, as described above. In one embodiment, three samples are in the set; these samples are referred to herein as samples "Cl, C2, C3" (control agent-contacted samples). As before, a different number of samples can be used if desired. The second set of samples is contacted with the agent to be tested for its ability to inhibit β(l,6)-glucan synthesis or assembly (the test agent). In one embodiment, three samples are in the set; these samples are referred to herein as samples "Tl, T2, T3" (test agent-contacted samples). Again, a different number of samples can be used if desired. The two sets of samples are then incubated under conditions that are sufficient to allow metabolic turnover of β(1.6)-glucan on the plasma membrane and in the cell wall of the cells of the samples, as described above.

A first sample from the set of incubated, control agent-contacted samples, and a first sample from the set of incubated, test agent-contacted samples, is then contacted with control media. These samples (e.g., Cl and Tl) are the control/control sample and the test agent/control sample, as above. A second sample from the set of incubated, control agent-contacted samples, and a second sample from the set of incubated, test agent-contacted samples is contacted with a lethal dose of the killer toxin. These samples (e.g., C2 and T2) are referred to as the control/lethal toxin sample and the test agent/lethal toxin sample, as above. A third sample from the set of incubated, control agent-contacted samples, and a third sample from the set of incubated, test agent-contacted samples is contacted with a sublethal dose of the killer toxin. These samples (e.g., C3 and T3) are referred to as the control/sublethal toxin sample and the test agent/sublethal toxin sample, respectively. A summary of representative samples is set forth in Table 3.

Table 3 Samples in assay for agents that confer either resistance or susceptibility

The control/control sample, test agent control sample, control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample and test agent/sublethal toxin sample are then incubated under conditions that are sufficient to allow the killer toxin in the lethal and sublethal toxin samples to interact with the cells, as described above. The incubated samples (control/control sample, test agent/control sample, control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample and test agent/sublethal toxin sample) are then assessed, as described above, to determine whether the test agent confers resistance or hypersensitivity to the killer toxin, such as by assessing the permeability of the samples using a membrane-impermeable dye that fluoresces on contact with DNA. If the sample that has been exposed to the test agent and the lethal dose of the killer toxin (test agent/lethal toxin sample, T2) fluoresces less than the sample that has been exposed to the control agent and the lethal dose of the killer toxin (control agent/lethal toxin sample, C2), in an amount that is significant (as described above), then the test agent has conferred resistance to the killer toxin on the sensitive cells, and is therefore an agent that inhibits synthesis of β(l,6)-glucan. If the sample that has been exposed to the test agent and the sublethal dose of the killer toxin (test agent/sublethal toxin sample, T3) fluoresces more than the sample that has been exposed to the control agent and the sublethal dose of the killer toxin (test agent/sublethal toxin sample, C3), in an amount that is significant (as described above), then the test agent has conferred hypersensitivity to the killer toxin on the sensitive cells, and is therefore an agent that inhibits incorporation of β(l ,6)-glucans into the cell wall.

If a test agent is identified by the methods described above as an agent that inhibits β(l,6)-glucan synthesis and or assembly into the cell wall, further experiments can be conducted to assess the agent's antifungal activity. For example, experiments can be performed to confirm whether the agent inhibits fungal cell growth, or to confirm whether any fungal cell death caused by the agent is due to inhibition of β(l ,6)-glucan synthesis or assembly. The agent can also be tested for in vivo efficacy. Agents identified by the methods described herein can be used as antifungal agents that target the essential, non-enzymatic β(l,6)-glucan synthesis or assembly processes, thereby avoiding the problems associated with targeting enzymatic activities.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of an agent identified by the methods described above, and a pharmaceutically acceptable carrier or excipient. The carrier and composition can be sterile. The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyroUidone, sodium saccharine, cellulose, magnesium carbonate, etc.

The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The agent may be incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air. Agents described herein can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

The amount of agents which will be effective in the treatment of a particular disorder or condition will depend on the nature of the infection, disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection, disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. Agents assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.

The agents identified by the methods of the invention as inhibiting β(l,6)- glucan synthesis and/or incorporation of β(l,6)-glucan into the cell wall, as well as the compositions described above, can be used either in vitro or in vivo to kill fungi and/or treat fungal infection. The agent is generally administered to an animal, a human, or a location of fungal contamination or growth (e.g., an environmental location) in an amount sufficient to inhibit and/or eliminate fungal infection or growth (a "therapeutically effective amount" or an "effective amount"). The mode of administration of the agent (or composition), in the case of in vivo administration, can be oral, enteral, parenteral, intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intranasal. For in vitro administration, the agent (or composition) is administered by a means that allows contact of the agent (or composition) with the fungal growth. The form in which the composition will be administered (e.g., powder, tablet, capsule, solution, emulsion) will depend on whether it is used in vivo or in vitro, as well as (in the case of in vivo administration) the route by which it is administered. The quantity of the agent or composition to be administered in vivo will be determined on an individual basis, and will be based at least in part on consideration of the severity of infection or injury in the patient, the patient's condition or overall health, the patient's weight and gender. In general, a single dose will preferably contain approximately 0.01-100 mg per kilogram of body weight, and preferably about 1 mg/kg. The quantity of the agent or composition to be administered in vitro will also be determined on a case-by-case basis, and will be based at least in part on consideration of the type and extent of the fungal contamination or growth. In general, the agents or compositions of the present invention can be administered to an individual or applied to an in vitro fungal source as necessary to treat the fungal infection or contamination. An individual skilled in the medical arts will be able to determine the length of time during which the agent or composition is administered and the dosage.

The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incorporated by reference in their entirety.

EXAMPLES

EXAMPLE 1 Measurement of β( 1 ,6)-specific Killing by Lethal and Sublethal Concentrations of Killer Toxin

A Kl killer toxin-binding inhibitory polysaccharide (pustulan) was used to confirm that Kl killer toxin is a β(l,6)-glucan dependent toxin, and to confirm that specific killing could be identified by Sytox green fluorescence. Sets of samples of midlog S. cerevisiae (strain TA405) yeast cells (1 x 10°) were established in YPD-H media. The first set of samples (Cl, C2, C3) was contacted with a control agent (saline); the second set of samples (Clp, C2p, C3p) was contacted with pustulan at 10 μg/ml. The sets of samples were then incubated at 30°C for 2.5 hours, to allow metabolic turnover of β(l,6)-glucan on the plasma membrane in the samples. A baseline fluorescence reading of the samples was taken using a fluorimeter (485_sx/520_em).

A first sample from each of the two sets (Cl and Clp) was then contacted with approximately 10 μl of control media (MMG). A second sample from each of the two sets (C2 and C2p) was contacted with a lethal dose (12.5 μg/ml) of killer toxin, diluted in MMG. A third sample from each of the two sets (C3 and C3p) was contacted with a sublethal dose (1.25 μg/ml) of the killer toxin diluted in MMG. The contacted samples were incubated at room temperature for approximately 2-5 hours. Subsequently, the samples were centrifuged to wash out YPD. The centrifuged samples were contacted with 10 μM Sytox Green membrane- impermeable dye, and 20 μl 2% NP-40 to facilitate entry of the dye into the permeable cells. The dyed samples were then incubated for approximately 2 hours at room temperature, or at 4°C for overnight. The fluorescence of the samples was then assessed with a fluorimeter, and the percent of surviving cells was examined under a microscope. Results are shown in Table 4, below.

Table 4 Identification of Specific Killing

These results demonstrate that killing of cells by both optimal (C2/C1 = 14.8) and suboptimal (C3/C1 = 1.42) doses of Kl killer toxin can be identified using sytox green; and that Kl killing is inhibited by the β(l,6)-glucan-specific agent, pustulan (C2p/Clp = 1.03; C3p/Clp = 1.10). EXAMPLE 2 Assay for Identification of Agents That Confer Resistance or Hypersensitivity to Killer Toxin

Two test compounds (tunicamycin, a known oligosaccharide synthesis inhibitor, and nikkomycin, a known chitin synthesis inhibitor) were evaluated. Three sets of samples of S cerevisiae (strain TA405) midlog yeast cells (1 x 10⁶) were established in YPD-H media. The first set of samples was contacted with a control agent (saline), as described above. The second set of samples was contacted with tunicamycin at 10 μg/ml. The third set of samples) was contacted with nikkomycin at 10 μg/ml. The three sets of samples were then incubated at 30°C for 2.5 hours, to allow metabolic turnover of β(l ,6)-glucan on the plasma membrane in the samples. A baseline fluorescence reading of the samples was taken using a fluorimeter.

A first sample from each of the two sets (Cl and Tl) was then contacted with approximately 10 μl of control media (MMG). A second sample from each of the two sets (C2 and T2) was contacted with a lethal dose of the killer toxin, 12.5 mg/ml diluted in MMG. A third sample from each of the two sets (C3 and T3) was contacted with a sublethal dose of the killer toxin (1.25 μg/ml in MMG). The contacted samples were incubated at room temperature for approximately 2-5 hours. Subsequently, the samples were centrifuged to wash out YPD. The centrifuged filtered were contacted with 10 μM Sytox Green membrane-impermeable dye, and 20 μl 2% NP-40 to facilitate entry of the dye into the permeable cells. The dyed samples were then incubated for approximately 2 hours at room temperature, or at 4°C for overnight. The fluorescence of the samples was then assessed with a fluorimeter. Results are shown in Table 5, below.

These results demonstrate that tunicamycin renders sensitive cells resistant to the effects of the killer toxin [(T2-T1)/(C2-C1) = 0.02, which is less than 2], and that nikkomycin rendered sensitive cells hypersensitive to the effects of the killer toxin [(T3-T1)/(C3-C1) = 42, which is greater than 2.5].

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims..

Claims

CLAIMS What is claimed is:

1. A method of assessing a test agent for an ability to inhibit β(l,6)-glucan synthesis, comprising contacting a sample of yeast cells that are sensitive to a killer toxin that targets β(l ,6)-glucan with a test agent and a lethal dose of the killer toxin, and assessing the permeability of the cells using a membrane-impermeable dye that fluoresces on contact with DNA, wherein if the cells that are contacted with the test agent and the lethal dose of the killer toxin are less permeable than cells that are contacted with a lethal dose of the killer toxin but not with the test agent, then the test agent is an agent that inhibits β(l,6)-glucan synthesis.

2. A method of assessing a test agent for an ability to inhibit β(l,6)-glucan synthesis, comprising the steps of: a) providing sets of samples of yeast cells that are sensitive to a killer toxin that targets β( 1 ,6)-glucan; b) contacting a first set of samples with a control agent and contacting a second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media, and contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a lethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/lethal toxin sample, and a test agent/lethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/lethal toxin sample and test agent/lethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/lethal toxin sample and the test agent/lethal toxin sample, thereby generating incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample and incubated test agent/lethal toxin sample; and f) assessing the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control lethal toxin sample, and incubated test agent/lethal toxin sample, using a membrane-impermeable dye that fluoresces on contact with DNA, wherein if cells in the incubated test agent/lethal toxin sample are less permeable than cells in the incubated control agent lethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits β(l,6)- glucan synthesis.

->. The method of Claim 2, wherein the yeast cells are Saccharomyces cerevisiae cells.

4. The method of Claim 3, wherein the Saccharomyces cerevisiae cells are from the strain TA405.

5. The method of Claim 2, wherein the control agent is selected from the group consisting of: water, saline, DMSO, and an ethanol solvent.

6. The method of Claim 2. wherein the set of control agent-contacted samples and the set of test agent-contacted samples are incubated in step (c) at a temperature between about 18°C and 37 °C, inclusive, for approximately 1 to 5 hours.

7. The method of Claim 2, wherein the control/control sample, test agent/control sample, control/lethal toxin sample and test agent/lethal toxin sample are incubated in step (e) at room temperature between about 15°C and 22 °C, inclusive, for approximately 2 to 16 hours.

8. The method of Claim 2, wherein the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, and incubated test agent/lethal toxin sample is assessed in step (f) using a membrane-impermeable fluorescent dye that fluoresces on contact with DNA.

9. The method of Claim 8, wherein the membrane-impermeable fluorescent dye that fluoresces on contact with DNA is selected from the group consisting of: ethidium bromide and Sytox Green.

10. The method of Claim 8, wherein the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, and incubated test agent/lethal toxin sample is assessed in step (f) by a method comprising contacting the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, and incubated test agent/lethal toxin sample with the membrane-impermeable fluorescent dye that fluoresces on contact with DNA, generating dye-contacted samples; incubating the dye- contacted samples under conditions which allow the dye to enter permeable cells; and detecting fluorescence of the cells in each of the dye-contacted samples, wherein fluorescence is indicative of permeability of the cells.

11. A method of assessing a test agent for an ability to inhibit incorporation of β( 1 ,6)-glucan into the cell wall, comprising contacting a sample of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan with a test agent and a sublethal dose of the killer toxm, and assessing the permeability of the cells, wherein if the cells that are contacted with the test agent and the sublethal dose of the killer toxin are more permeable than cells that are contacted with a lethal dose of the killer toxin but not with the test agent, then the test agent is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

12. A method of assessing an agent for an ability to inhibit incorporation of β(l,6)-glucan into the cell wall, comprising the steps of: a) providing sets of samples of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan; b) contacting a first set of samples with a control agent and contacting a second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media, and contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/sublethal toxin sample, and a test agent/sublethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/sublethal toxin sample and test agent/sublethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/sublethal toxin sample and the test agent/sublethal toxin sample, thereby generating incubated control/control sample, incubate test agent/control sample, incubated control/sublethal toxin sample and incubated test agent/sublethal toxin sample; and f) assessing the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control sublethal toxin sample, and incubated test agent/sublethal toxin sample, wherein if cells in the incubated test agent/sublethal toxin sample are more permeable than cells in the incubated control agent/sublethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

13. The method of Claim 12, wherein the yeast cells are Saccharomyces cerevisiae cells.

14. The method of Claim 14, wherein the Saccharomyces cerevisiae cells are from the strain TA405.

15. The method of Claim 12, wherein the control agent is selected from the group consisting of: water, saline, DMSO, and an ethanol solvent.

16. The method of Claim 12, wherein the set of control agent-contacted samples and the set of test agent-contacted samples are incubated in step (c) at a temperature between about 18°C and 37°C, inclusive, for approximately 1 to 5 hours.

17. The method of Claim 12, wherein the control/control sample, test agent/control sample, control/sublethal toxin sample and test agent/sublethal toxin sample are incubated in step (e) a temperature between about 15°C and

22 °C, inclusive, for approximately 2 to 16 hours.

18. The method of Claim 12, wherein the permeability of the cells in the incubated control control sample, incubated test agent/control sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample is assessed in step (f) using a membrane-impermeable fluorescent dye that fluoresces on contact with DNA.

19. The method of Claim 18, wherein the membrane-impermeable fluorescent dye that fluoresces on contact with DNA is selected from the group consisting of: ethidium bromide and Sytox Green.

20. The method of Claim 18, wherein the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control sublethal toxin sample, and incubated test agent/sublethal toxin sample is assessed in step (f) by a method comprising contacting the incubated control/control sample, incubated test agent/control sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample with the membrane-impermeable fluorescent dye that fluoresces on contact with DNA, generating dye-contacted samples; incubating the dye-contacted samples under conditions which allow the dye to enter permeable cells; and detecting fluorescence of the cells in each of the dye-contacted samples, wherein fluorescence is indicative of permeability of the cells.

21. A method of assessing a test agent for an ability to inhibit β(l,6)-glucan synthesis and/or incorporation of β(l,6)-glucan into the cell wall, comprising contacting a sample of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan with a test agent and a lethal dose of the killer toxin; contacting a sample of yeast cells that are sensitive to the killer toxin with a test agent and a sublethal dose of the killer toxin; and assessing the permeability of the cells in the samples, wherein if the cells that are contacted with the test agent and the lethal dose of the killer toxin are less permeable than cells that are contacted with a lethal dose of the killer toxin but not with the test agent, then the test agent is an agent that inhibits β(l,6)- glucan synthesis, and wherein if the cells that are contacted with the test agent and the sublethal dose of the killer toxin are more permeable than cells that are contacted with a sublethal dose of the killer toxin but not with the test agent, then the test agent is an agent that inhibits incorporation of β(l,6)- glucan into the cell wall.

22. A method of assessing an agent for an ability to inhibit synthesis of β( 1 ,6)- glucan and/or inhibit incorporation of β(l,6)-glucan into the cell wall, comprising the steps of: a) providing two sets of samples of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan; b) contacting the first set of samples with a control agent and contacting the second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media; contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a lethal dose of the killer toxin; and contacting a third sample from the set of incubated, control agent-contacted samples and a third sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/lethal toxin sample, a test agent/lethal toxin sample, a control/sublethal toxin sample, and a test agent/sublethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample, and test agent/sublethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample, and test agent/sublethal toxin sample, thereby generating incubated control control sample, incubated test agent/control sample, incubated control/lethal toxin sample, incubated test agent/lethal toxin sample, incubated control/sublethal toxin sample and incubated test agent/sublethal toxin sample; and f) assessing the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, incubated test agent lethal toxin sample, incubated control/sublethal toxin sample, and incubated test agent sublethal toxin sample, wherein if cells in the incubated test agent/lethal toxin sample are less permeable than cells in the incubated control agent lethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits β(l,6)- glucan synthesis; and wherein if cells in the incubated test agent/sublethal toxin sample are more permeable than cells in the incubated control agent/sublethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

23. The method of Claim 22, wherein the yeast cells are Saccharomyces cerevisiae cells.

24. The method of Claim 23, wherein the Saccharomyces cerevisiae cells are from the strain TA405.

25. The method of Claim 22, wherein the control agent is selected from the group consisting of: water, saline, DMSO, and an ethanol solvent.

26. The method of Claim 22, wherein the set of control agent-contacted samples and the set of test agent-contacted samples are incubated in step (c) at a temperature between about 18°C and 37°C, inclusive, for approximately 1 to

5 hours.

27. The method of Claim 22, wherein the control/control sample, test agent/control sample, control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample and test agent/sublethal toxin sample are incubated in step (e) at a temperature between about 15°C and 22 °C, inclusive, for approximately 2 to 16 hours.

28. The method of Claim 22, wherein the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample is assessed in step (f) using a membrane-impermeable fluorescent dye that fluoresces on contact with DNA.

29. The method of Claim 28, wherein the membrane-impermeable fluorescent dye that fluoresces on contact with DNA is selected from the group consisting of: ethidium bromide and Sytox Green.

30. The method of Claim 28, wherein the permeability of the cells in the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, incubated test agent/lethal toxin sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample is assessed in step (f) by a method comprising contacting the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, incubated test agent/lethal toxin sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample with the membrane-impermeable fluorescent dye that fluoresces on contact with DNA, generating dye-contacted samples; incubating the dye-contacted samples under conditions which allow the dye to enter permeable cells; and detecting fluorescence of the cells in each of the dye-contacted samples, wherein fluorescence is indicative of permeability of the cells.

31. A method of assessing a test agent for an ability to inhibit β(l ,6)-glucan synthesis, comprising the steps of: a) providing two sets of samples of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan; b) contacting the first set of samples with a control agent and contacting the second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media, and contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a lethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/lethal toxin sample, and a test agent/lethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/lethal toxin sample and test agent/lethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/lethal toxin sample and the test agent/lethal toxin sample, thereby generating incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample and incubated test agent/lethal toxin sample; f) contacting the incubated control/control sample, incubated test agent/control sample, incubated control/lethal toxin sample, and incubated test agent/lethal toxin sample with a membrane- impermeable fluorescent dye that fluoresces on contact with DNA, thereby generating dye-contacted samples; g) incubating the dye-contacted samples under conditions which allow the dye to enter permeable cells; and h) detecting fluorescence of the cells in each of the dye-contacted samples, wherein if cells in the dye-contacted, incubated test agent/lethal toxin sample are less fluorescent than cells in the dye-contacted, incubated control agent/lethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits β(l,6)-glucan synthesis.

32. A method of assessing a test agent for an ability to inhibit incorporation of β(l,6)-glucan into the cell wall, comprising the steps of: a) providing two sets of samples of yeast cells that are sensitive to a killer toxin that targets β(l,6)-glucan; b) contacting the first set of samples with a control agent and contacting the second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media, and contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/sublethal toxin sample, and a test agent/sublethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/sublethal toxin sample and test agent/sublethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/sublethal toxin sample and the test agent/sublethal toxin sample, thereby generating incubated control/control sample, incubated test agent/control sample, incubated control/sublethal toxin sample and incubated test agent/sublethal toxin sample; f) contacting the incubated control/control sample, incubated test agent/control sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample with a membrane- impermeable fluorescent dye that fluoresces on contact with DNA, thereby generating dye-contacted samples; g) incubating the dye-contacted samples under conditions which allow the dye to enter permeable cells; and h) detecting fluorescence of the cells in each of the dye-contacted samples, wherein if cells in the dye-contacted, incubated test agent/sublethal toxin sample are more fluorescent than cells in the dye-contacted, incubated control agent/sublethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

3. A method of assessing an agent for an ability to inhibit synthesis of β(l ,6)- glucan and/or inhibit incorporation of β(l,6)-glucan into the cell wall, comprising the steps of: a) providing two sets of samples of yeast cells that are sensitive to a killer toxin that targets β(l ,6)-glucan; b) contacting the first set of samples with a control agent and contacting the second set of samples with a test agent, thereby generating a set of control agent-contacted samples and a set of test agent-contacted samples; c) incubating the set of control agent-contacted samples and the set of test agent-contacted samples under conditions that are sufficient to allow metabolic turnover of β(l,6)-glucan in the yeast cells, thereby generating a set of incubated, control agent-contacted samples and a set of incubated, test agent-contacted samples; d) contacting a first sample from the set of incubated, control agent- contacted samples and a first sample from the set of incubated, test agent-contacted samples with control media; contacting a second sample from the set of incubated, control agent-contacted samples and a second sample from the set of incubated, test agent-contacted samples with a lethal dose of the killer toxin; and contacting a third sample from the set of incubated, control agent-contacted samples and a third sample from the set of incubated, test agent-contacted samples with a sublethal dose of the killer toxin, thereby generating a control/control sample, a test agent/control sample, a control/lethal toxin sample, a test agent/lethal toxin sample, a control/sublethal toxin sample, and a test agent/sublethal toxin sample, respectively; e) incubating the control/control sample, test agent/control sample, control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample, and test agent/sublethal toxin sample under conditions that are sufficient to allow the killer toxin to interact with the cells in the control/lethal toxin sample, test agent/lethal toxin sample, control/sublethal toxin sample, and test agent/sublethal toxin sample, thereby generating incubated control/control sample, incubated test agent control sample, incubated control lethal toxin sample, incubated test agent/lethal toxin sample, incubated control/sublethal toxin sample and incubated test agent/sublethal toxin sample; and f) contacting the incubated control/control sample, incubated test agent control sample, incubated control/lethal toxin sample, incubated test agent/lethal toxin sample, incubated control/sublethal toxin sample, and incubated test agent/sublethal toxin sample, with a membrane-impermeable fluorescent dye that fluoresces on contact with DNA, thereby generating dye-contacted samples; g) incubating the dye-contacted samples under conditions which allow the dye to enter permeable cells; and h) detecting fluorescence of the cells in each of the dye-contacted samples, wherein if cells in the dye-contacted, incubated test agent/lethal toxin sample are less flourescent than cells in the dye-contacted, incubated control agent/lethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits β(l,6)-glucan synthesis; and wherein if cells in the dye-contacted, incubated test agent/sublethal toxin sample are more flourescent than cells in the dye-contacted, incubated control agent/sublethal toxin sample, in an amount that is significant, then the test agent is an agent that inhibits incorporation of β(l,6)-glucan into the cell wall.

34. An agent, having the ability to inhibit synthesis of β(l ,6)-glucan, identifiable by the method of Claim 1.

35. An agent, having the ability to inhibit incorporation of β(l ,6)-glucan into the cell wall, identifiable by the method of Claim 10.

36. An agent, having the ability to synthesis of β( 1 ,6)-glucan or inhibit incorporation of β(l,6)-glucan into the cell wall, identifiable by the method of Claim 21.

37. A pharmaceutical composition comprising an agent of Claim 34.

38. A pharmaceutical composition comprising an agent of Claim 35.

39. A method of treating a fungal infection in an individual, comprising administering to the individual an agent having the ability to inhibit synthesis of β(l,6)-glucan, identifiable by the method of Claim 1, in a therapeutically effective amount.

40. A method of treating a fungal growth, comprising administering to the growth an agent having the ability to inhibit synthesis of β(l,6)-glucan, identifiable by the method of Claim 1 , in an effective amount.

41. A method of treating a fungal infection in an individual, comprising administering to the individual an agent having the ability to inhibit incorporation of β(l,6)-glucan into the cell wall, identifiable by the method of Claim 10, in a therapeutically effective amount.

42. A method of treating a fungal growth, comprising administering to the growth an agent having the ability to inhibit incorporation of β(l,6)-glucan into the cell wall, identifiable by the method of Claim 10, in an effective amount.

43. A method of assessing an agent of interest for antifungal activity, comprising assessing the ability of the agent to inhibit synthesis of β(l,6)-glucan or incorporation of β(l,6)-glucan into the cell wall, wherein if the agent of interest inhibits synthesis of β(l,6)-glucan or inhibits incorporation of β(l,6)-glucan into the cell wall, it is an agent that has antifungal activity.