Abstract
The invention provides a peptide obtainable from C. albicans as well as variants and fragments thereof, and labelled forms of these. The peptide is immunogenic and specific binding partners for the peptide and labelled forms of these specific binding partners form a further aspect of the invention. The peptide is a fragment of the ECE1 protein and has been found to be both immunogenic and act as a pore-forming toxin. A range of therapeutic and diagnostic applications for the peptide and the specific binding partners for it form further aspects of the invention. In addition, the peptide may be used in screens for identifying compounds having useful anti-fungal activity.
Claims
1. A specific binding partner for a peptide comprising SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (SEQ ID NO:1) or a labelled form thereof.
2. The specific binding partner of claim 1 which is an immunoglobulin.
3. The specific binding partner of claim 2 which is an antibody or a binding fragment thereof.
4. The specific binding partner of claim 3 which is a monoclonal antibody.
5. A pharmaceutical composition comprising the specific binding partner of claim 1 and a pharmaceutically acceptable carrier.
6. A method for prophylaxis or treatment of an infection by Candida species, said method comprising administering to a subject in need thereof an effective amount of the specific binding partner of claim 1.
7. The method of claim 6 wherein the infection is an oral or mucosal infection by Candida albicans.
8. A nucleic acid that encodes the specific binding partner of claim 1.
9. A recombinant cell that has been transformed with the nucleic acid of claim 8.
10. A method for producing the specific binding partner encoded by the nucleic acid that transformed the recombinant cell of claim 9, which method comprises culturing the recombinant cell and recovering said specific binding partner.
11. A method for diagnosis of an infection by Candida albicans, said method comprising contacting a sample from an individual with the specific binding partner of claim 1, and detecting whether the sample contains moieties that interact with said specific binding partner at a sufficient level to indicate a pathogenic invasive infection.
12. A kit for diagnosing an infection by Candida albicans, said kit comprising the specific binding partner of claim 1, and means for detecting complexes formed with said specific binding partner.
13. A kit for diagnosing an infection by Candida albicans, said kit comprising a peptide comprising SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (SEQ ID NO:1), and means for detecting complexes formed with said peptide
14. A method for diagnosis of an infection by Candida albicans, said method comprising contacting a sample from an individual with the peptide from the kit of claim 13, and detecting whether the sample contains moieties that interact with said peptide at a sufficient level to indicate a pathogenic invasive infection.
15. A method for screening for compounds having anti-fungal activity, said method comprising determining whether a test compound will interact with a peptide comprising SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (SEQ ID NO:1).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1A-C is a series of graph showing the results of an experiment to determine whether ECE1 is required for damage of a variety of host cells. Oral TR146 (FIG. 1A), gastrointestinal Caco (FIG. 1B) and vaginal (FIG. 1C) epithelial cells were infected with C. albicans wild type (wt), ece1 or ece1/-ECE1 strains for 24 hours and damage assayed by LDH release as described below.
[0064] FIG. 2 shows the structure of Ece1 and phylogeny with other species. It shows the alignment of Ece1 from C. albicans (SEQ ID NO 13) and orthologues in C. dubliniensis (SEQ ID NO 14) and C. tropicalis (SEQ ID NO 15) (which are the only three sequenced fungal species that encode Ece1). The bars below the alignment indicate that the peptides resulted from Kex2 digestion of the CaEce1. Only peptide 3 of C. albicans and C. dubliniensis form transmembrane -helical structures.
[0065] FIG. 3A-C is a series of graphs showing the results of an experiment to demonstrate that a peptide of the invention is sufficient to lyse host epithelial cells. Mixtures of Ece1 peptides at different concentrations or the peptide of SEQ ID NO 1 alone were added to TR146 oral epithelial cells in the presence of the ece1 mutant, incubated for 24 hours and damage assessed by measuring LDH release (FIG. 3A). Subsequently the different Ece1 peptides were added alone (without C. albicans cells) to TR146 (FIG. 3B) and vaginal A431 (FIG. 3C) epithelia, incubated for 24 hours and damage assessed by measuring LDH release as described below.
[0066] FIG. 4 is a graph showing the results of an experiment to determine which of the Ece1 peptides are haemolytic toxins where the peptide of SEQ ID NO 1 is peptide 3. Individual peptides were incubated with human erythrocytes for 1 hour and heamolysis assayed by measuring haemoglobin release (as measured by absorbance at 541 nm, normalised to a 100% lysis control).
[0067] FIG. 5A,B shows the results obtained by scanning electron microscopy of an experiment to determine whether the peptide of SEQ ID NO 1 forms pores in epithelial membranes. Oral epithelial (TR146) monolayers were incubated with 5 g/ml peptide 3 (SEQ ID NO 1) for 5 hours and pore formation assessed by scanning electron microscopy. FIG. 5B shows a further magnified view of a region indicated in FIG. 5A.
[0068] FIG. 6A,B is a series of graphs showing the results of an experiment to demonstrate that the haemolytic activity of the Ece1 peptide 3 of SEQ ID NO 1 is ion-dependent. The peptide was incubated with human erythrocytes with increasing concentrations of the divalent cation chelator EDTA (FIG. 6A) and haemolysis assayed by measuring haemoglobin release (absorbance at 541 nm, normalised to a 100% lysis control). The inhibitory effect of 50 mM EDTA was reversed by the addition of 50 or 100 mM magnesium (FIG. 6B).
[0069] FIG. 7A,B is a series of graphs showing the results of experiments to investigate whether the peptide of the invention activates epithelial immunity and cFos. (FIG. 7A) Induction of c-Fos DNA binding activity in TR146 epithelial cells after 3 hours infection with ece1/ null mutant, ece1/-ECE1 revertant and the BWP17 parent strain. (FIG. 7B) Production of cytokines by TR146 epithelial cells after 24 hour infection with ece1/ null mutant, ece1/-ECE1 revertant, the BWP17 parent strain or uninfected cells. Data shown are the mean of 3 independent experiments. Error bars show SEM. *=p<0.05, **=p<0.01, ***=p<0.001.
[0070] FIG. 8A,B shows the amino acid sequences of and WTSA peptides where FIG. 8A shows the amino acid sequence of (SEQ ID NO 1). Amyloidogenic regions are shown in bold type; and FIG. 8B shows the amino acid sequences of WTSA peptides. Each individual WTSA peptide constitutes a partial fragment of full length Ece1-III. Amyloidogenic regions are shown in bold type.
[0071] FIG. 9 is a graph showing the results of analysis of WTSA peptide-induced damage to TR146 oral epithelial cells. Individual WTSA peptides (250 g/ml) and WTSA 3 in combination with WTSA 6 (both peptides at 250 g/ml) were added to confluent monolayers of TR146 oral epithelial cells and incubated for 24 hours at 37 C., 5% CO.sub.2. Following incubation, growth medium was removed and assayed for the presence of lactate dehydrogenase (LDH), a surrogate marker of cellular lysis. Dimethyl sulfoxide (DMSO) was used as a vehicle only (negative control). Full length Ece1-III (250 g/mL) was used as the positive control.
[0072] FIG. 10A-E shows WTSA peptide-mediated activation of immune responses in TR146 oral epithelial cells. WTSA peptides 1-6 were added to confluent monolayers of TR146 oral epithelial cells at a final concentration of 250 g/mL and incubated for 24 hours at 37 C., 5% CO.sub.2. Following incubation, growth medium was removed and secreted immunostimulatory cytokines were quantified by multiplex bead assay. Concentration of detected cytokines was expressed as picograms per millilitre (pg/mL). ND=not detected.
[0073] FIG. 11A-C is a series of graphs showing the results of experiments to investigate whether the peptide of the invention binds EGFR and activates c-Fos. (FIG. 11A) Induction of c-Fos DNA binding activity in TR146 epithelial cells after 3 hours infection with wild-type C. albicans cells, which were pre-treated for 1 hour with 4 M GW2974 (EGFR/Her2 inhibitor), 1 M PD153035 (EGFR inhibitor) or DMSO (vehicle control). (FIG. 11B) Sensorgram of fluid phase EGFR binding to immobilised Ece1-III of SEQ ID NO 1 demonstrating high affinity binding. Injection of fluid phase components was at point A. (FIG. 11C) Induction of c-Fos DNA binding activity in TR146 epithelial cells after 3 hours infection with Ece1-III, which were pre-treated for 1 hour with 4 M GW2974 (EGRF/Her2 inhibitor), 1 M PD153035 (EGFR inhibitor) or DMSO (vehicle control). Data shown are the mean of 2(A) or 3 (C) independent experiments. Error bars show SEM. *=p<0.05, **=p<0.01, ***=p<0.001.
[0074] FIG. 12A-C is a set of graphs showing the effect of EGFR inhibition on epithelial cell signalling. Blocking EGFR signalling in oral epithelial cells (TR146) with Gefitnib (10 M) inhibits the activation of MAPK intracellular signalling in cells treated with Ece1-III (50 g/mL) for 2 hours as measured by decreases in levels of phosphorylated p38, JNK and ERK1/2 relative to the vehicle control (DMSO).
[0075] FIG. 13 shows the results of an assay to detect Ece1-III-phospholipid interactions. Ece1-III was used to probe a phospholipid array (PIP Strip membrane). Binding was assessed by hybridisation with anti-Ece1-III antibody, followed by detection with anti-rabbit antibody conjugated to horse radish peroxidase.
[0076] FIG. 14A-C is a series of graphs showing the results of an experiment to determine C. albicans-macrophage interactions. C. albicans strains were incubated with RAW264.7 macrophages for indicated times and the following parameters determined: (FIG. 14A) The number of phagocytosed fungal cells as a percentage of total number of fungal cells; (FIG. 14B) Hyphal length; (FIG. 14C) The percentage of outgrowing hyphae compared to the total number of internalised yeast mother cells.
[0077] FIG. 15 is a graph showing the results of an experiment to show that ECE1 is required for damage of macrophages. Indicated macrophage cell types were co-incubated with C. albicans cells for 24 hours (MOI of 1 for Raws, MOI of 10 for MDMs) and macrophage damage assessed by measuring lactate dehydrogenase release into the supernatant.
[0078] FIG. 16A-E shows the results of experiments showing the role of Ece1 in C. albicans mucosal pathogenesis. Infection of cortisone acetate-treated mice with wild-type (BWP17), ece1 null mutant or ece1/-ECE1 reverted strain (FIG. 16A) numbers of C. albicans fungi recovered per gram of kidney 3 days post-infection. Fungal invasion, tissue damage and inflammatory cell infiltration of tongue tissue 3 days post-infection by (FIG. 16B) BWP17, (FIG. 16C) ece1/-ECE1 and (FIG. 16D) ece1 null mutant, indicating that the ece1 null mutant does not invade, damage or recruit inflammatory cells. (FIG. 16E) tongue section 3 days post-infection with BWP17 immunostained with MPO and CD15 markers of neutrophils. **=P<0.01.
[0079] FIG. 17 is a graph showing the results of an experiment to show Drosophila killing by Ece1-III. The percentage of Drosophila death, 24 hours post-infection of 250 g/ml His-tagged full length Ece1p, Ece1-II (peptide 2) (FIG. 2), and Ece1-III (peptide 3), into a cohort of 20 age-matched flies.
EXAMPLE 1
The Role of Ece1 in Host Cell Damage
[0080] To investigate the role of Ece1 in host cell damage, a series of C. albicans gene deletion mutants lacking ECE1, as well as revertant mutant strains were created. Specifically, C. albicans gene deletion cassettes were generated using the PCR-based method described by Dalle F et al. Cellular Microbiology 2010; 12:248-71. Primers ECE1-FG and ECE1-RG as shown below as SEQ ID NO 4 and 5 respectively, were used to amplify the HIS1 and ARG4 markers from plasmids pFA-HIS1 and pFA-ARG4, respectively. The C. albicans strain BWP17 (Wachtler B, et al. Antimicrobial Agents and Chemotherapy 2011; 55:4436-9) was sequentially transformed as described by Wachtler B, et al. PloS one 2011; 6:e17046, with the ECE1-ARG4 and ECE1-HIS1 deletion cassettes and then transformed with the Candida integrating plasmid 10 or CIp10 (Murad et al. Yeast (2000) 16, 4, p 325-327), yielding the ece1 deletion strain. For generation of the complemented strain, the ECE1 open reading frame, plus upstream and downstream intergenic regions were amplified with primers ECE1-RecF3k (SEQ ID NO 6) and ECE1-RecR (SEQ ID NO 7) and cloned into plasmid CIp10 at MluI and SalI sites, yielding plasmid CIp10-ECE1. This plasmid was transformed into the uridine auxotrophic ece1-deletion strain, yielding the ece1/-ECE1 complemented strain.
Primers:
[0081]
TABLE-US-00004 ECE1-FG (SEQIDNO4) ATCAAATAACCCACCTATTTCAAAATTGTTTTATTTTTGTTTATCTCTAC AACAAACAACTTTCCTTTATTTTACTACCAACTATTTTCCATTCGTTAAA gaagcttcgtacgctgcaggtc ECE1-RG (SEQIDNO5) CACAAAAAACAACAATTAAAAAAATCAGTTACAGCAAAAGTGTCACAAGA CTTATGGAATAAAAGATTAAGCTTGTGGAAAACAAATTTTTATCTGCTGA GCATtctgatatcatcgatgaattcgag ECE1-RecF3k (SEQIDNO6) GCACGCGTCTAAAGTGGAGTAACAAC ECE1-RecR (SEQIDNO7) GGTCGACCCCAGACGTTGGTTGC
[0082] Three strains, wild type C. albicans (wt), the ECE1 deletion mutant (ece1) and the revertant mutant strain (ece1/-ECE1) were tested. They were applied to three independent human epithelial cell types: oral, gastrointestinal and vaginal. TR146, Caco-2 and A431 epithelial cell lines were maintained as previously described in the Dalle and Wachtler references given above. Epithelial damage assays were performed as previously described by Dalle et al. (supra.) and Wachtler et al. (2011) (supra.) with the following modifications: monolayers in 96 well plates were infected with 210.sup.4 C. albicans cells; infections were performed in cell culture media without fetal bovine serum.
[0083] After incubation for 24 hours, the damage to the cells was assessed by measuring lactate dehydrogenase (LDH) release using a conventional assay method.
[0084] The results are shown in FIG. 1A-C. They show that deletion of ECE1, prevented or reduced C. albicans damage in all three cell types. As C. albicans is often found associated with the oral, gastrointestinal and urogenitary sites as a member of the natural microbiome, but can also cause infections at such sites, these data suggest that the ECE1 gene product is involved C. albicans pathogenicity.
EXAMPLE 2
Role of Peptides
[0085] It has been demonstrated that Ece1 can be proteolytically processed by Kex2 into eight peptide fragments (O. Bader et al. BMC Microbiology 2008, 8:115). FIG. 2 illustrates the structure of Ece1 and the proposed resultant peptide fragments following proteolytic processing as well as its phylogenic relationship with orthologues in C. dubliniensis and C. tropicalis (note that ECE1 orthologues are only found in these two fungal species). Each of the individual C. albicans peptides were synthesised by ProteoGenix using fmoc solid phase peptide synthesis technology.
[0086] The method of Example 1 was then repeated using a 1:1 mixture of peptides (1-7) of FIG. 2 on human TR146 oral epithelial cells at various concentrations (250 g/ml, 125 g/ml, 62.5 g/ml, 31.25 g/ml, 15.625 g/ml and 7.8 g/ml of each individual peptide within the mixture) or peptide 3 (SEQ ID NO 1) alone in the presence of the ece1 mutant C. albicans. Peptide 8 could not be included, as this peptide was insoluble. After incubations for 24 hours, the damage was assessed by measuring LDH release as before. The results are shown in FIG. 3A. This shows that peptide 3 (designated Ece1-III in the Figure) produced similar levels of damage to the combination at a concentration of 250 g/ml.
[0087] The test was then repeated using each peptide individually without C. albicans cells. The results are shown in FIGS. 3B and 3C. They show that whilst peptide 3 was capable of damaging both oral epithelial and vaginal cells, the other peptides displayed essentially no cytolytic activity. Therefore, peptide 3 of processed Ece1 (Ece1-III of SEQ ID NO 1) is sufficient to cause lysis of human epithelial cells.
EXAMPLE 3
Cytolytic Activity of Peptides
[0088] In order to investigate the cytolytic activities of Ece1-III in greater detail, a haemolysis assay was carried out. Human blood was collected with the SARSTEDT blood collection system and EDTA-coated tubes, washed once with HBSS, resuspended in HBSS and stored at 4 C. for up to one week for experiments. Blood was obtained from healthy human donors.
[0089] Each individual peptide (at a final concentration of 30 g/ml) was incubated with 110.sup.7 human erythrocytes in a volume of 150 l. After incubation for 1 hour at 37 C., haemolysis was assayed by measuring haemoglobin release, as indicated by absorbance at 541 nm, normalised to a 100% lysis-water control.
[0090] The results are shown in FIG. 4. Exposure of human erythrocytes to Ece1-III, but not to the other peptides, resulted in lysis of these cells. It should be noted that, in contrast to epithelial cells, erythrocytes do not contain nuclei and cannot transcriptionally respond to stimuli. Therefore, lysis of erythrocytes by Ece1-III was due to direct cytolysis.
EXAMPLE 4
Pore Formation
[0091] Many peptide toxins elicit cytotoxicity via pore formation and ion influx across membranes. Whether addition of Ece1-III to epithelial cells was able to directly induce pore formation was then tested. Oral epithelial (TR-146) monolayers were incubated with 5 g/ml Ece1-III of SEQ ID NO 1 for 5 hours and pore formation assessed by scanning electron microscopy. The results are shown in FIG. 5A,B where FIG. 5B shows a further magnified view of the region shown in FIG. 5A. Electron microscopy suggests that Ece1-III can directly induce pores (FIG. 5 A,B), which is in agreement with the view that Ece1-III can act as a pore-forming toxin.
EXAMPLE 5
Ion-Dependency of Haemolytic Activity
[0092] Since the mode of action of many peptide and pore-forming toxins is ion-dependent, Ece1-III and erythrocytes were co-incubated in the presence of increasing concentrations of the divalent cation chelator, ethylenediaminetetraacetic acid (EDTA), which restricts the bioavailability of divalent cations. After 1 hour, haemolysis was measured as described in Example 3 above. The results, shown in FIG. 6A, indicate that increasing concentrations of EDTA blocked the haemolytic activity of Ece1-III. This effect was reversed by supplementation with magnesium (FIG. 6B). Therefore, like previously described peptide toxins, the haemolytic activity of Ece1-III appears to be ion-dependent.
EXAMPLE 6
Activation of Immune Responses
[0093] Pore-forming toxins are generally strong activators of inflammatory responses. Previously it has been shown that recognition of C. albicans hyphae resulted in the activation of the mitogen-activated protein kinase (MAPK) p38 signalling pathway and the c-Fos transcription factor, which in turn activates proinflammatory cytokine production (Moyes D L, et al.: Cell Host Microbe 2010, 8:225-235,). An extensive screen of >100 C. albicans mutants was carried out using protocols described in this reference and also Moyes D L, et al. PLoS ONE 2011, 6:e26580; Murciano C. et al. Infect. Immun 2011, 79:4902-4911; Moyes D L, et al. Med Microbiol Immunol 2012, 201:93-101; Moyes D L, et al. Methods Mol Biol 2012, 845:345-360 and Murciano C, et al. PLoS ONE 2012, 7:e33362.
[0094] The results demonstrated that only a strain deleted in ECE1 was unable to activate the c-Fos pathway (FIG. 7A) or proinflammatory cytokines (FIG. 7B) from oral epithelial cells whilst still producing hyphae. Therefore, activation of epithelial immune responses via c-Fos against C. albicans is the result of the action and/or recognition of Ece1.
EXAMPLE 7
Functional Requirements of Ece1-III
[0095] Having identified Ece1-III as the region of Ece1 responsible for cellular lysis and the activation of immune responses, the applicants next investigated the precise functional requirements of the Ece1-III amino acid sequence (FIG. 8A). Accordingly, individual peptide fragments corresponding to internal regions of Ece1-III were constructed (WTSA peptides 1-6 (SEQ ID NOS 8-11, 3 and 12 respectively: FIG. 8B). The sequence of each WTSA peptide was chosen to facilitate a detailed examination of each of the internal amyloidogenic regions of Ece1-III, either alone (WTSA peptides 1, 2, 4 and 5), or in combination (WTSA 3). An additional fragment of Ece1-III lacking both amyloidogenic regions was also created (WTSA 6: FIG. 8B). The WTSA peptides were analysed for their ability to cause epithelial cell lysis. However, none of the individual WTSA peptide fragments were observed to induce cell lysis (FIG. 9). Notably, when WTSA 3 and 6 were applied to epithelial cells in combination, (which together constitute the entire Ece1-III peptide sequence) the ability to induce cell lysis was not restored. The WTSA peptides were then analysed for their ability to stimulate secretion of pro-inflammatory cytokines from oral epithelial cells. None of the WTSA peptides stimulated significant levels of cytokine secretion (FIG. 10A-E). In contrast, a potent induction of all cytokines was observed following exposure to full length Ece1-III. Taken together, these data indicate that only intact, full length Ece1-III is capable of inducing cellular lysis and stimulation of the inflammatory response in epithelial cells.
[0096] The p38/c-Fos pathway is activated via the interaction of C. albicans hyphae with the surface receptor EGFR (epidermal growth factor receptor), as blocking EGFR activation also blocks c-Fos activation (FIG. 11A). Biacore binding assays show that Ece1-III (of SEQ ID NO 1), but not other parts of the Ece1 protein, directly interact with EGFR with high affinity (FIG. 11B) and that blocking EGFR abolishes the ability of Ece1-III to activate c-Fos (FIG. 11C). Furthermore, blocking EGFR also reduced/abolished activation of all three MAPK signalling pathways (p38, JNK and ERK1/2) (FIG. 12A-C) This demonstrates that activation of mucosal immune responses via the p38/c-Fos pathway is the result of Ece1-III/EGFR interactions and that EGFR plays a primary role in the activation of cell signalling by Ece1-III.
EXAMPLE 8
[0097] Interaction of Ece1-III with Phospholipids
[0098] In order to insert into a target membrane, a pore-forming peptide toxin must interact with components of the membrane. Because Ece1-III consists of a predicted alpha helix with a dibasic (lysine, arginine) C-terminal head, the applicants predicted that these cationic, positively charged residues may interact with negatively charged phospholipids present in target membranes. Ece1-III was used to probe a phospholipid array (PIP Strip membrane). Binding was assessed by hybridisation with anti-Ece1-III antibody, followed by detection with anti-rabbit antibody conjugated to horse radish peroxidase. The results are shown in FIG. 13. These show that Ece1-III interacts with Phosphatidylinositol-3-phosphate, Phosphatidylinositol-4-phosphate, Phosphatidylinositol-5-phosphate, Phosphatidylinositol-3,4-phosphate, Phosphatidylinositol-3,5-phosphate, Phosphatidylinositol-4,5-phosphate, Phosphatidylinositol-3,4,5-phosphate, Phosphatidic acid and Phosphatidylserine, with binding to Phosphatidylinositol-3-phosphate being particularly robust (FIG. 13). Ece1-III did not interact with Lysophosphatidic acid, Lysophosphatidylcholine, Phosphatidylinositol, Phosphatidylethanolamine, Phosphatidylcholine or Sphingosine-1-phosphate. Therefore, insertion of Ece1-III into target membranes (pore formation) may be mediated by interactions with phospholipids.
EXAMPLE 9
Ece1 Facilitates Immune Cell Killing
[0099] Given the importance of Ece1-III in killing epithelial cells, the applicants examined whether it also plays a role in immune escape of C. albicans from macrophages. In vitro, C. albicans yeast cells are phagocytosed by macrophages, but rapidly germinate and escape the macrophage via hypha formation. To examine whether Ece1 is involved in this process, wild type, ece1, and ece1/-ECE1 cells were coincubated with macrophages and the following parameters measured: phagocytosis rates, length of hyphae, escape from macrophages, and damage of macrophages. Interestingly, ece1 cells were phagocytosed by macrophages at the same rate, formed hyphae within macrophages of the same length, and even escaped from macrophages at the same rates as the wild type, but were unable to damage these immune cells (FIGS. 14A-C and 15). These data demonstrate that C. albicans hyphae can non-lytically escape from macrophages, and indicate a specific role for Ece1 in host cell damage.
EXAMPLE 10
[0100] Effect of Ece1 on Mucosal pathogenicity In Vivo
[0101] The applicants also tested whether Ece1 was required for mucosal pathogenciity in vivo. To test this, they utilised a murine model of oropharyngeal candidiasis. In this model, mice were first immunosuppressed with cortisone acetate prior to administration of wild-type (parental) C. albicans (BWP17), a ece1 null mutant and ece1/-ECE1 revertant strain into the oral cavity. After four days, fungal burdens, fungal invasion, tissue damage and immune cell recruitment was determined in tongue tissues. Significantly lower fungal burdens were found in mice infected with the ece1 null mutant as compared with the wild-type (BWP17) or the ece1/-ECE1 revertant strains, and in many cases the ece1 null mutant could not be recovered from mice (FIG. 16A). Histological analysis of mouse tongues in both the wild-type (BWP17) and ece1/-ECE1 revertant strain showed multiple foci of infection that were associated with inflammatory immune cells (neutrophils) and extensive local tissue damage (FIG. 16B-D). In contrast, mice infected with the ece1 null mutant showed no evidence of any foci of invasion, tissue damage or inflammation and no evidence of C. albicans was detectable (FIG. 16E). Therefore, Ece1 is essential for successful mucosal infection, damage induction and immune activation in this model.
EXAMPLE 11
[0102] Effect of Ece1 and Ece1-III on Drosophila melanogaster
[0103] The applicants also determined the effect of administering full length His-tagged Ece1p and Ece1-III in a Drosophila melanogaster (fruit fly) model. All flies injected with full length His-tagged Ece1p and Ece1-III displayed signs of paralysis, with only 17.5% and 10% recovering after 24 hours, as compared with the negative control Ece1-II (peptide 2) which showed no signs of paralysis (FIG. 17). This indicates that Ece1-III may have a direct or indirect neurotoxic effect.
CONCLUSION
[0104] These data provide evidence that Ece1 (and its active component Ece1-III) has a dual functional role, by acting both as a pore-forming toxin that damages host cells and as an activator of immune responses. Damage of host cells and uncontrolled immune activation are the hallmarks of several diseases caused by C. albicans, which can often lead to death (45-76% mortality) during systemic infections. Therefore, neutralisation of Ece1-III will prevent not only host damage during C. albicans infections but also deleterious inflammatory responses. This indicates that Ece1-III represents a new therapeutic target to treat C. albicans infections.
