Induction of SAP7 Correlates with Virulence in an Intravenous Infection Model of Candidiasis but Not in a Vaginal Infection Model in Mice
http://www.100md.com
感染与免疫杂志 2005年第10期
Institute of Clinical Microbiology, Immunology, and Hygiene, Friedrich-Alexander University Erlangen-Nürnberg, Wasserturmstrasse 3/5, D-91054 Erlangen, Germany
ABSTRACT
SAP7 of Candida albicans is induced after vaginal infection of mice. Conversely, virulence during vaginal infection was not affected in a sap7/sap7 mutant strain. Only a partial virulence phenotype was detectable after intravenous injection. In conclusion, SAP7 expression does not correlate with C. albicans virulence in mice.
TEXT
The secretions of hydrolytic enzymes, specifically, the 10 secreted aspartic proteinases Sap1p through Sap10p have been comprehensively studied as key virulence determinants of Candida albicans (1, 2, 13, 14, 17, 18, 24). Previous studies utilizing reverse transcription-PCR and gene deficient mutant strains have highlighted the importance of certain SAP genes in relation to various models of infection (12). Thereby, individual members of the SAP gene family might have their own roles in the infectious process because environmental conditions have been shown to affect production of Sap proteins differentially, suggesting that Sap proteins may contribute to the development of active Candida infection in different tissues and at certain stages of infection (7, 9-11, 27).
Sap7p shares at most 27% similarity with other Sap proteins (18), and investigation of SAP7 should focus on its genetic characteristics in vivo, because in vitro induction of SAP7 mRNA expression has not yet been detected. SAP7 transcript was detected in 60% of oral candidiasis patients as opposed to 25% of Candida carriers by means of reverse transcription-PCR (19). Felk et al. found that SAP7 transcript was not induced in response to an intraperitoneal model of C. albicans infection that examines invasion of parenchymal organs (4). Vaginal samples examined for the presence of SAP7 transcript showed that strains collected from patients with vaginal candidiasis induced SAP7 significantly compared to samples collected from carriers (20). Furthermore, a study examining infection of reconstituted human vaginal epithelium found induction of SAP7 transcript after 24 h of infection (25).
Due to our own results showing that SAP7 is up-regulated in response to vaginal candidiasis in mice, we postulated that SAP7 plays a role in the pathogenesis of candidiasis. Therefore, we generated a homozygous sap7/sap7 mutant, which we examined in vitro for growth, stress response, and morphogenetic development as well as in vivo for invasion, dissemination, and survival in host tissue.
Genetically engineered C. albicans strains created and used in this study are summarized in Table 1. C. albicans strains were cultured on agar plates with complete supplement medium without uridine or in liquid medium, respectively (28). Nucleic acid techniques and microscopic analysis were performed as described previously (26). The vaginal, intravenous, intraperitoneal, and pulmonary models of C. albicans infection were performed as described previously (3, 5, 14, 15, 26). Survival curves were generated according to the Kaplan-Meier method by using the PRISM program (GraphPad Software) and compared using the log rank test (16). Total RNA from the lavage samples containing mucus, epithelial cells, and C. albicans isolates was isolated by hot acid phenol extraction. A FastStart DNA Master SYBR green I kit (Roche, Mannheim, Germany) was used for real-time PCR analysis using the LightCycler system (Roche, Mannheim, Germany). Oligonucleotide primers SAP7.01 (5'-GAAATGCAAAGAGTATTAGAGTTATTAC-3') and SAP7.02 (5'-GAATGATTTGGTTTACATCATCTTCAACTG-3') were used for detection of SAP7 cDNA. Primers EFB1.03 (5'-AACGAATTCTTGGCTGACA-3') and EFB1.04 (5'-GCGGCTGGGGCTTTACC-3') were used to amplify EFB1 that encodes elongation factor-1. Copy numbers were calculated, and values represented are normalized (number of copies of SAP7 cDNA/number of copies of EFB1 cDNA). Sequential homologous recombination (6) was used to delete both SAP7 alleles in C. albicans strain CAI-4 to generate the homozygous ura3/ura3 sap7/sap7 mutant BNT16. This strain was then transformed with pVEC (21) to make BNT18 [sap7/sap7(pVEC)] or plasmid p348 (pVEC-SAP7) containing the SAP7 wild-type gene to generate the revertant strain BNT19 [sap7/sap7(pVEC-SAP7)]. Appropriate genotypes and presence or absence of SAP7 expression of BNT16, BNT18, and BNT19 were confirmed by PCR and Southern blot analysis in vitro and in vivo.
We infected mice intravaginally with the SC5314 wild-type strain and performed lavages on days 1, 7, and 17. SAP7 levels were detectable by real-time PCR after overnight growth in complete supplement medium without uridine (inocula) at very low levels (between 6.0 x 10–6 and 1.6 x 10–5 copies SAP7/copy EFB1) but rose at least 30-fold after 24 h of infection (Fig. 1). By day 7 postinfection, levels had risen over 1,000-fold and remained high through day 17 of infection. This suggested that induction of SAP7 occurred as a consequence of murine vaginal infection and is consistent with results seen with vaginal samples from patients.
The deletion of SAP7 had no effect on cellular replication, colony phenotype, anaerobic growth, resistance to hydrogen peroxide, or the ability of C. albicans to form hyphae at 37°C in medium containing 10% fetal calf serum (data not shown). Furthermore, deletion of SAP7 had no effect on the number of C. albicans CFU recovered from the vaginal canal, as illustrated in Fig. 2A. Vaginal fungal burdens of mice infected with either the sap7/sap7 mutant or the control increased after 24 h but then dropped and remained stable (between [5.3 ± 3.3] x 103 and [1.3 ± 0.7] x 104) throughout the 28 days of observation.
In the intravenous model of systemic candidiasis (15, 23, 26), injection of the control BNT19 resulted in the death of 80% of the mice by day 12 and 100% by day 24 postinfection (Fig. 2B). In contrast, 60% of mice survived up to 56 days after infection with the sap7/sap7 mutant BNT18, and the remaining animals showed no clinical signs of disease (P < 0.02). Although previous work had suggested a role of proteinases in a pulmonary model of candidiasis (3), no significant differences between mutant and control strains during short-term pulmonary infection of immunosuppressed mice were observed (data not shown). Moreover, invasiveness of the sap7/sap7 mutant was only slightly but not significantly reduced as measured by organ fungal burdens after intravenous infection.
SAP7 has provided difficulty for researchers due to the futile attempts to identify conditions that induce SAP7 in vitro. By use of sensitive techniques, trace amounts can be found in overnight cultures; however, no tested conditions seem to significantly up-regulate SAP7 transcription (11, 22). However, the observation that SAP7 is strictly an in vivo factor signifies that it may be required during the pathogenesis of candidiasis. In our vaginal model of murine candidiasis, SAP7 expression in SC5314 rose after one day of infection and remained highly induced compared to inoculum throughout the 17 days of observation. This confirms findings from previous studies, including those involving patient samples, mouse models, and the examination of C. albicans by using an in vitro reconstituted human vaginal epithelium model of disease (19, 20, 22, 25). Surprisingly, in the vaginal model in which SAP7 had been induced, no significant differences between the SAP7-deficient strain and the control organisms were observed over 28 days of infection. Although the observed up-regulation of SAP7 is a consequence of this type of superficial infection (20, 25), it could well be that C. albicans expresses at least one or even several redundant genes which compensate for the SAP7 defect, and therefore, the sap7/sap7 mutant does not display an obvious phenotype in virulence in this model of candidiasis. It underscores the fact that induction or lack thereof is not a predictor of involvement in a process. Genes induced under a set of conditions may not be essential to the process being observed. The increase in the survival time of mice after intravenous infection with the sap7/sap7 mutant was very clear, while the number of fungal CFU recovered from the kidneys indicated only a trend towards a reduced invasiveness. The reasons for this type of selective virulence attenuation of the sap7/sap7 mutant only after intravenous infection may be due to niche specificity which has been reported for other members of the SAP gene family (27, 28).
ACKNOWLEDGMENTS
We thank B. Bodendorfer and H. Arnold for skillful technical assistance. We thank B. Hube and J. Naglik for fruitful discussions during early stages of the project. Sequence data for C. albicans were obtained from the Stanford Genome Technology Center website (http://www-sequence.stanford.edu/group/candida).
Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. This work was supported by Deutsche Forschungsgemeinschaft grants Schr 450/4-1 and/5-1 (K.S.), the Interdisciplinary Research Center (IZKF) at the University of Erlangen (TP.A15/A2; to B.N.T. and M.S.), and NRC-HGF Science and Technology Fund (01SF0201/2.2).
REFERENCES
1. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candida albicans. Trends Microbiol. 9:327-335.
2. De Bernardis, F., S. Arancia, L. Morelli, B. Hube, D. Sanglard, W. Schafer, and A. Cassone. 1999. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis. J. Infect. Dis. 179:201-208.
3. Fallon, K., K. Bausch, J. Noonan, E. Huguenel, and P. Tamburini. 1997. Role of aspartic proteases in disseminated Candida albicans infection in mice. Infect. Immun. 65:551-556.
4. Felk, A., M. Kretschmar, A. Albrecht, M. Schaller, S. Beinhauer, T. Nichterlein, D. Sanglard, H. C. Korting, W. Schafer, and B. Hube. 2002. Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs. Infect. Immun. 70:3689-3700.
5. Fidel, P. L., Jr., M. E. Lynch, and J. D. Sobel. 1993. Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis. Infect. Immun. 61:1990-1995.
6. Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717-728.
7. Ghannoum, M. A. 2000. Potential role of phospholipases in virulence and fungal pathogenesis. Clin. Microbiol. Rev. 13:122-143.
8. Gillum, A. M., E. Y. Tsay, and D. R. Kirsch. 1984. Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol. Gen. Genet. 198:179-182.
9. Gow, N. A. 1997. Germ tube growth of Candida albicans. Curr. Top. Med. Mycol. 8:43-55.
10. Hube, B. 1998. Possible role of secreted proteinases in Candida albicans infection. Rev. Iberoam. Micol. 15:65-68.
11. Hube, B., M. Monod, D. A. Schofield, A. J. Brown, and N. A. Gow. 1994. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol. Microbiol. 14:87-99.
12. Hube, B., and J. Naglik. 2001. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147:1997-2005.
13. Hube, B., D. Sanglard, F. C. Odds, D. Hess, M. Monod, W. Schafer, A. J. Brown, and N. A. Gow. 1997. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect. Immun. 65:3529-3538.
14. Kretschmar, M., B. Hube, T. Bertsch, D. Sanglard, R. Merker, M. Schroder, H. Hof, and T. Nichterlein. 1999. Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis. Infect. Immun. 67:6637-6642.
15. Leberer, E., D. Harcus, I. D. Broadbent, K. L. Clark, D. Dignard, K. Ziegelbauer, A. Schmidt, N. A. Gow, A. J. Brown, and D. Y. Thomas. 1996. Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci. USA 93:13217-13222.
16. Mantel, N., and W. Haenszel. 1958. Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl. Cancer Inst. 22:719-748.
17. Monod, M., B. Hube, D. Hess, and D. Sanglard. 1998. Differential regulation of SAP8 and SAP9, which encode two new members of the secreted aspartic proteinase family in Candida albicans. Microbiology 144:2731-2737.
18. Monod, M., G. Togni, B. Hube, and D. Sanglard. 1994. Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol. Microbiol. 13:357-368.
19. Naglik, J. R., G. Newport, T. C. White, L. L. Fernandes-Naglik, J. S. Greenspan, D. Greenspan, S. P. Sweet, S. J. Challacombe, and N. Agabian. 1999. In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis. Infect. Immun. 67:2482-2490.
20. Naglik, J. R., C. A. Rodgers, P. J. Shirlaw, J. L. Dobbie, L. L. Fernandes-Naglik, D. Greenspan, N. Agabian, and S. J. Challacombe. 2003. Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections. J Infect. Dis. 188:469-479.
21. Navarro-Garcia, F., R. M. Perez-Diaz, B. B. Magee, J. Pla, C. Nombela, and P. Magee. 1995. Chromosome reorganization in Candida albicans 1001 strain. J. Med. Vet. Mycol. 33:361-366.
22. Ripeau, J. S., M. Fiorillo, F. Aumont, P. Belhumeur, and L. de Repentigny. 2002. Evidence for differential expression of Candida albicans virulence genes during oral infection in intact and human immunodeficiency virus type 1-transgenic mice. J. Infect. Dis. 185:1094-1102.
23. Rocha, C. R., K. Schrppel, D. Harcus, A. Marcil, D. Dignard, B. N. Taylor, D. Y. Thomas, M. Whiteway, and E. Leberer. 2001. Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol. Biol. Cell 12:3631-3643.
24. Sanglard, D., B. Hube, M. Monod, F. C. Odds, and N. A. Gow. 1997. A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect. Immun. 65:3539-3546.
25. Schaller, M., M. Bein, H. C. Korting, S. Baur, G. Hamm, M. Monod, S. Beinhauer, and B. Hube. 2003. The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium. Infect. Immun. 71:3227-3234.
26. Schweizer, A., S. Rupp, B. N. Taylor, M. Rllinghoff, and K. Schrppel. 2000. The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol. Microbiol. 38:435-445.
27. Staib, P., M. Kretschmar, T. Nichterlein, H. Hof, and J. Morschhuser. 2000. Differential activation of a Candida albicans virulence gene family during infection. Proc. Natl. Acad. Sci. USA 97:6102-6107.
28. Taylor, B. N., P. Staib, A. Binder, A. Biesemeier, M. Sehnal, M. Rllinghoff, J. Morschhuser, and K. Schrppel. 2005. Profile of Candida albicans-secreted aspartic proteinase elicited during vaginal infection. Infect. Immun. 73:1828-1835.(Brad N. Taylor, Holger Ha)
ABSTRACT
SAP7 of Candida albicans is induced after vaginal infection of mice. Conversely, virulence during vaginal infection was not affected in a sap7/sap7 mutant strain. Only a partial virulence phenotype was detectable after intravenous injection. In conclusion, SAP7 expression does not correlate with C. albicans virulence in mice.
TEXT
The secretions of hydrolytic enzymes, specifically, the 10 secreted aspartic proteinases Sap1p through Sap10p have been comprehensively studied as key virulence determinants of Candida albicans (1, 2, 13, 14, 17, 18, 24). Previous studies utilizing reverse transcription-PCR and gene deficient mutant strains have highlighted the importance of certain SAP genes in relation to various models of infection (12). Thereby, individual members of the SAP gene family might have their own roles in the infectious process because environmental conditions have been shown to affect production of Sap proteins differentially, suggesting that Sap proteins may contribute to the development of active Candida infection in different tissues and at certain stages of infection (7, 9-11, 27).
Sap7p shares at most 27% similarity with other Sap proteins (18), and investigation of SAP7 should focus on its genetic characteristics in vivo, because in vitro induction of SAP7 mRNA expression has not yet been detected. SAP7 transcript was detected in 60% of oral candidiasis patients as opposed to 25% of Candida carriers by means of reverse transcription-PCR (19). Felk et al. found that SAP7 transcript was not induced in response to an intraperitoneal model of C. albicans infection that examines invasion of parenchymal organs (4). Vaginal samples examined for the presence of SAP7 transcript showed that strains collected from patients with vaginal candidiasis induced SAP7 significantly compared to samples collected from carriers (20). Furthermore, a study examining infection of reconstituted human vaginal epithelium found induction of SAP7 transcript after 24 h of infection (25).
Due to our own results showing that SAP7 is up-regulated in response to vaginal candidiasis in mice, we postulated that SAP7 plays a role in the pathogenesis of candidiasis. Therefore, we generated a homozygous sap7/sap7 mutant, which we examined in vitro for growth, stress response, and morphogenetic development as well as in vivo for invasion, dissemination, and survival in host tissue.
Genetically engineered C. albicans strains created and used in this study are summarized in Table 1. C. albicans strains were cultured on agar plates with complete supplement medium without uridine or in liquid medium, respectively (28). Nucleic acid techniques and microscopic analysis were performed as described previously (26). The vaginal, intravenous, intraperitoneal, and pulmonary models of C. albicans infection were performed as described previously (3, 5, 14, 15, 26). Survival curves were generated according to the Kaplan-Meier method by using the PRISM program (GraphPad Software) and compared using the log rank test (16). Total RNA from the lavage samples containing mucus, epithelial cells, and C. albicans isolates was isolated by hot acid phenol extraction. A FastStart DNA Master SYBR green I kit (Roche, Mannheim, Germany) was used for real-time PCR analysis using the LightCycler system (Roche, Mannheim, Germany). Oligonucleotide primers SAP7.01 (5'-GAAATGCAAAGAGTATTAGAGTTATTAC-3') and SAP7.02 (5'-GAATGATTTGGTTTACATCATCTTCAACTG-3') were used for detection of SAP7 cDNA. Primers EFB1.03 (5'-AACGAATTCTTGGCTGACA-3') and EFB1.04 (5'-GCGGCTGGGGCTTTACC-3') were used to amplify EFB1 that encodes elongation factor-1. Copy numbers were calculated, and values represented are normalized (number of copies of SAP7 cDNA/number of copies of EFB1 cDNA). Sequential homologous recombination (6) was used to delete both SAP7 alleles in C. albicans strain CAI-4 to generate the homozygous ura3/ura3 sap7/sap7 mutant BNT16. This strain was then transformed with pVEC (21) to make BNT18 [sap7/sap7(pVEC)] or plasmid p348 (pVEC-SAP7) containing the SAP7 wild-type gene to generate the revertant strain BNT19 [sap7/sap7(pVEC-SAP7)]. Appropriate genotypes and presence or absence of SAP7 expression of BNT16, BNT18, and BNT19 were confirmed by PCR and Southern blot analysis in vitro and in vivo.
We infected mice intravaginally with the SC5314 wild-type strain and performed lavages on days 1, 7, and 17. SAP7 levels were detectable by real-time PCR after overnight growth in complete supplement medium without uridine (inocula) at very low levels (between 6.0 x 10–6 and 1.6 x 10–5 copies SAP7/copy EFB1) but rose at least 30-fold after 24 h of infection (Fig. 1). By day 7 postinfection, levels had risen over 1,000-fold and remained high through day 17 of infection. This suggested that induction of SAP7 occurred as a consequence of murine vaginal infection and is consistent with results seen with vaginal samples from patients.
The deletion of SAP7 had no effect on cellular replication, colony phenotype, anaerobic growth, resistance to hydrogen peroxide, or the ability of C. albicans to form hyphae at 37°C in medium containing 10% fetal calf serum (data not shown). Furthermore, deletion of SAP7 had no effect on the number of C. albicans CFU recovered from the vaginal canal, as illustrated in Fig. 2A. Vaginal fungal burdens of mice infected with either the sap7/sap7 mutant or the control increased after 24 h but then dropped and remained stable (between [5.3 ± 3.3] x 103 and [1.3 ± 0.7] x 104) throughout the 28 days of observation.
In the intravenous model of systemic candidiasis (15, 23, 26), injection of the control BNT19 resulted in the death of 80% of the mice by day 12 and 100% by day 24 postinfection (Fig. 2B). In contrast, 60% of mice survived up to 56 days after infection with the sap7/sap7 mutant BNT18, and the remaining animals showed no clinical signs of disease (P < 0.02). Although previous work had suggested a role of proteinases in a pulmonary model of candidiasis (3), no significant differences between mutant and control strains during short-term pulmonary infection of immunosuppressed mice were observed (data not shown). Moreover, invasiveness of the sap7/sap7 mutant was only slightly but not significantly reduced as measured by organ fungal burdens after intravenous infection.
SAP7 has provided difficulty for researchers due to the futile attempts to identify conditions that induce SAP7 in vitro. By use of sensitive techniques, trace amounts can be found in overnight cultures; however, no tested conditions seem to significantly up-regulate SAP7 transcription (11, 22). However, the observation that SAP7 is strictly an in vivo factor signifies that it may be required during the pathogenesis of candidiasis. In our vaginal model of murine candidiasis, SAP7 expression in SC5314 rose after one day of infection and remained highly induced compared to inoculum throughout the 17 days of observation. This confirms findings from previous studies, including those involving patient samples, mouse models, and the examination of C. albicans by using an in vitro reconstituted human vaginal epithelium model of disease (19, 20, 22, 25). Surprisingly, in the vaginal model in which SAP7 had been induced, no significant differences between the SAP7-deficient strain and the control organisms were observed over 28 days of infection. Although the observed up-regulation of SAP7 is a consequence of this type of superficial infection (20, 25), it could well be that C. albicans expresses at least one or even several redundant genes which compensate for the SAP7 defect, and therefore, the sap7/sap7 mutant does not display an obvious phenotype in virulence in this model of candidiasis. It underscores the fact that induction or lack thereof is not a predictor of involvement in a process. Genes induced under a set of conditions may not be essential to the process being observed. The increase in the survival time of mice after intravenous infection with the sap7/sap7 mutant was very clear, while the number of fungal CFU recovered from the kidneys indicated only a trend towards a reduced invasiveness. The reasons for this type of selective virulence attenuation of the sap7/sap7 mutant only after intravenous infection may be due to niche specificity which has been reported for other members of the SAP gene family (27, 28).
ACKNOWLEDGMENTS
We thank B. Bodendorfer and H. Arnold for skillful technical assistance. We thank B. Hube and J. Naglik for fruitful discussions during early stages of the project. Sequence data for C. albicans were obtained from the Stanford Genome Technology Center website (http://www-sequence.stanford.edu/group/candida).
Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. This work was supported by Deutsche Forschungsgemeinschaft grants Schr 450/4-1 and/5-1 (K.S.), the Interdisciplinary Research Center (IZKF) at the University of Erlangen (TP.A15/A2; to B.N.T. and M.S.), and NRC-HGF Science and Technology Fund (01SF0201/2.2).
REFERENCES
1. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candida albicans. Trends Microbiol. 9:327-335.
2. De Bernardis, F., S. Arancia, L. Morelli, B. Hube, D. Sanglard, W. Schafer, and A. Cassone. 1999. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis. J. Infect. Dis. 179:201-208.
3. Fallon, K., K. Bausch, J. Noonan, E. Huguenel, and P. Tamburini. 1997. Role of aspartic proteases in disseminated Candida albicans infection in mice. Infect. Immun. 65:551-556.
4. Felk, A., M. Kretschmar, A. Albrecht, M. Schaller, S. Beinhauer, T. Nichterlein, D. Sanglard, H. C. Korting, W. Schafer, and B. Hube. 2002. Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs. Infect. Immun. 70:3689-3700.
5. Fidel, P. L., Jr., M. E. Lynch, and J. D. Sobel. 1993. Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis. Infect. Immun. 61:1990-1995.
6. Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717-728.
7. Ghannoum, M. A. 2000. Potential role of phospholipases in virulence and fungal pathogenesis. Clin. Microbiol. Rev. 13:122-143.
8. Gillum, A. M., E. Y. Tsay, and D. R. Kirsch. 1984. Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol. Gen. Genet. 198:179-182.
9. Gow, N. A. 1997. Germ tube growth of Candida albicans. Curr. Top. Med. Mycol. 8:43-55.
10. Hube, B. 1998. Possible role of secreted proteinases in Candida albicans infection. Rev. Iberoam. Micol. 15:65-68.
11. Hube, B., M. Monod, D. A. Schofield, A. J. Brown, and N. A. Gow. 1994. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol. Microbiol. 14:87-99.
12. Hube, B., and J. Naglik. 2001. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147:1997-2005.
13. Hube, B., D. Sanglard, F. C. Odds, D. Hess, M. Monod, W. Schafer, A. J. Brown, and N. A. Gow. 1997. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect. Immun. 65:3529-3538.
14. Kretschmar, M., B. Hube, T. Bertsch, D. Sanglard, R. Merker, M. Schroder, H. Hof, and T. Nichterlein. 1999. Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis. Infect. Immun. 67:6637-6642.
15. Leberer, E., D. Harcus, I. D. Broadbent, K. L. Clark, D. Dignard, K. Ziegelbauer, A. Schmidt, N. A. Gow, A. J. Brown, and D. Y. Thomas. 1996. Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci. USA 93:13217-13222.
16. Mantel, N., and W. Haenszel. 1958. Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl. Cancer Inst. 22:719-748.
17. Monod, M., B. Hube, D. Hess, and D. Sanglard. 1998. Differential regulation of SAP8 and SAP9, which encode two new members of the secreted aspartic proteinase family in Candida albicans. Microbiology 144:2731-2737.
18. Monod, M., G. Togni, B. Hube, and D. Sanglard. 1994. Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol. Microbiol. 13:357-368.
19. Naglik, J. R., G. Newport, T. C. White, L. L. Fernandes-Naglik, J. S. Greenspan, D. Greenspan, S. P. Sweet, S. J. Challacombe, and N. Agabian. 1999. In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis. Infect. Immun. 67:2482-2490.
20. Naglik, J. R., C. A. Rodgers, P. J. Shirlaw, J. L. Dobbie, L. L. Fernandes-Naglik, D. Greenspan, N. Agabian, and S. J. Challacombe. 2003. Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections. J Infect. Dis. 188:469-479.
21. Navarro-Garcia, F., R. M. Perez-Diaz, B. B. Magee, J. Pla, C. Nombela, and P. Magee. 1995. Chromosome reorganization in Candida albicans 1001 strain. J. Med. Vet. Mycol. 33:361-366.
22. Ripeau, J. S., M. Fiorillo, F. Aumont, P. Belhumeur, and L. de Repentigny. 2002. Evidence for differential expression of Candida albicans virulence genes during oral infection in intact and human immunodeficiency virus type 1-transgenic mice. J. Infect. Dis. 185:1094-1102.
23. Rocha, C. R., K. Schrppel, D. Harcus, A. Marcil, D. Dignard, B. N. Taylor, D. Y. Thomas, M. Whiteway, and E. Leberer. 2001. Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol. Biol. Cell 12:3631-3643.
24. Sanglard, D., B. Hube, M. Monod, F. C. Odds, and N. A. Gow. 1997. A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect. Immun. 65:3539-3546.
25. Schaller, M., M. Bein, H. C. Korting, S. Baur, G. Hamm, M. Monod, S. Beinhauer, and B. Hube. 2003. The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium. Infect. Immun. 71:3227-3234.
26. Schweizer, A., S. Rupp, B. N. Taylor, M. Rllinghoff, and K. Schrppel. 2000. The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol. Microbiol. 38:435-445.
27. Staib, P., M. Kretschmar, T. Nichterlein, H. Hof, and J. Morschhuser. 2000. Differential activation of a Candida albicans virulence gene family during infection. Proc. Natl. Acad. Sci. USA 97:6102-6107.
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