This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Staib, P.
Right arrow Articles by Morschhäuser, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Staib, P.
Right arrow Articles by Morschhäuser, J.

 Previous Article  |  Next Article 

Infection and Immunity, April 2004, p. 2386-2389, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2386-2389.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Tec1p-Independent Activation of a Hypha-Associated Candida albicans Virulence Gene during Infection

Peter Staib,1 Ayfer Binder,1 Marianne Kretschmar,2 Thomas Nichterlein,2 Klaus Schröppel,3 and Joachim Morschhäuser1*

Institut für Molekulare Infektionsbiologie, Universität Würzburg, D-97070 Würzburg,1 Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, D-68167 Mannheim,2 Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universität Erlangen, D-91054 Erlangen, Germany3

Received 27 October 2003/ Returned for modification 10 December 2003/ Accepted 22 December 2003


arrow
ABSTRACT
 
The Tec1p transcription factor is involved in the expression of hypha-specific genes in Candida albicans. Although the induction of the hypha-associated SAP5 gene by serum in vitro depends on Tec1p, deletion of all Tec1p binding site consensus sequences from the SAP5 promoter did not affect its activation. In two different animal models of candidiasis, the SAP5 promoter was induced even in a {Delta}tec1 deletion mutant, demonstrating that the requirement for Tec1p in gene expression in C. albicans depends on the environmental conditions within the host.


arrow
TEXT
 
The ability to switch from a yeast to a filamentous, hyphal form, together with the expression of hypha-associated genes, is important for virulence of the opportunistic fungal pathogen Candida albicans (4). Morphogenesis and expression of hypha-associated genes are regulated in response to environmental cues by several signal transduction pathways, including a mitogen-activated protein kinase pathway that ends in the transcription factor Cph1p and a cyclic AMP-dependent pathway involving the Efg1p transcription factor (3, 5, 12, 15, 24). Depending on the growth conditions used to induce hyphal formation, mutants that are defective in these signaling pathways are unable to form filaments and do not express hypha-associated genes (2, 10, 13, 14, 23).

The TEA/ATTS transcription factor Tec1p also has been shown to be necessary for proper hyphal formation in C. albicans in vitro (19). C. albicans mutants with a deletion of the TEC1 gene displayed attenuated virulence in a mouse model of systemic candidiasis, although they formed hyphae in the infected tissue (19). It has been proposed that Tec1p regulates hypha-associated genes in C. albicans by binding to the sequence 5'-CATTCY-3', which is the consensus binding sequence for fungal TEA/ATTS transcription factors (1, 6, 19). This sequence is found in the promoter region of hypha-associated genes, and recent evidence suggests that different signal transduction pathways involved in hyphal growth of C. albicans converge on the Tec1p transcription factor, which then mediates the expression of hypha-associated genes (10).

The SAP5 gene encodes one of the secreted aspartic proteinases of C. albicans which have been shown to contribute to virulence of the fungus (9, 17). Its expression is induced upon hyphal formation in vitro and during experimental infection in vivo, and full SAP5 induction by host signals depends on Cph1p and Efg1p (7, 20, 21). The SAP5 promoter contains three direct repeats of a 25-bp sequence in which a Tec1p consensus binding sequence is present, suggesting that Tec1p might induce SAP5 expression by binding to this sequence. Therefore, using a recently developed in vivo expression technology that is based on FLP-mediated deletion of a mycophenolic acid resistance marker (MPAR) from the genome as a reporter of gene activation (20, 22), we investigated whether SAP5 induction within the host is dependent on Tec1p and whether the putative Tec1p binding sites mediate this activation.

To delete the three 25-mer repeats containing the putative Tec1p binding sites from PSAP5, a 0.8-kb fragment containing the SAP5 promoter was PCR amplified with the primer pair SAP5P3 (5'-GTATAAATGCTCtaGAATTCTGTTTGGCG-3') and SAP5P6 (5'-ACATTgtcgacTTGAGCTTAACTTTGGATTAGTTATAAAGGAGTGAAT*GACTATTTGAATG-3'). The lowercase letters represent nucleotide exchanges introduced to create an upstream XbaI site and a SalI site in front of the start codon (restriction sites are underlined, and the reverse sequence of the start codon is shown in bold); the position of the 75-bp deletion is indicated by an asterisk. The PCR product was digested with XbaI and SalI, and the mutated SAP5 promoter (PSAP5{Delta}3) was substituted for the wild-type SAP5 promoter of plasmid pSFL53 (20) to generate pSFL56. Strain CFI1, which contains the FLP-deletable MPAR marker, was transformed by electroporation (8) with the insert from this plasmid. The two SAP5 alleles of the parental strain can be distinguished by a BglII restriction site polymorphism (20), and two independent transformants which contained the PSAP5{Delta}3-ecaFLP fusion integrated into the SAP5-1 (strain S5FI5A) or the SAP5-2 allele (strain S5FI5B) were selected. Replacement of the original SAP5 promoter by the mutated version was confirmed by reamplification and sequencing.

To detect activation of the wild-type SAP5 promoter in the absence of the Tec1p transcription factor, the FRT-MPAR-FRT cassette from plasmid pAFI3 containing the FLP-deletable MPAR marker between ACT1 flanking sequences (22) was first integrated into one of the ACT1 alleles of the {Delta}tec1 mutant strain CaAS15 (19). The resulting strain, CFI6, was then transformed with the PSAP5-ecaFLP reporter gene fusion from pSFL53, and two independent transformants in which integration had occurred into the SAP5-1 (strain C6S5F1D) or the SAP5-2 allele (strain C6S5F1E) were selected. All integration events were confirmed by Southern hybridization (data not shown).

We first assessed activation of the SAP5 promoter during serum-induced hyphal growth in vitro. Five microliters of an overnight culture of each reporter strain was inoculated in 250 µl of fresh serum from a healthy human donor and incubated for 4 h at 37°C. Appropriate dilutions were then spread on MPA indicator plates to determine the percentage of MPAS cells in which induction of the SAP5 promoter had resulted in FLP-mediated deletion of the MPAR marker. As shown in Fig. 1A, activation of the SAP5 promoter in a wild-type background (strains S5FI2A and S5FI2B [20]) during serum-induced hyphal growth was readily detected with this reporter system, whereas virtually no SAP5 induction was seen in the {Delta}tec1 mutant, confirming previous results from Northern hybridization experiments (19). Surprisingly, the deletion of the putative Tec1p binding sites did not affect the inducibility of the SAP5 promoter by serum. Since there is no other Tec1p binding consensus sequence within 2 kb upstream of the SAP5 coding region, this result suggests that Tec1p binding sites in C. albicans may differ from those in other organisms, as has recently been reported for the Rim101p transcription factor (16), and Tec1p might bind to sites other than the consensus sequence in the SAP5 promoter to mediate activation of this gene. Alternatively, the involvement of Tec1p in the transcriptional induction of SAP5 may be indirect.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1. Activation of the wild-type (SAP5) and mutated (SAP5{Delta}3) SAP5 promoters in a wild-type background and of the wild-type SAP5 promoter in a {Delta}tec1 mutant (SAP5{Delta}tec1) by serum in vitro (A) and after intraperitoneal (B) or intravenous (C) infection of mice. Fungal cells were recovered from the liver at 24 h after intraperitoneal infection and from the kidneys at 4 days after intravenous infection. Each bar shows the percentage of MPAS cells in one infected animal. The light grey bars show the results obtained with strains S5FI2A, S5FI5A, and C6S5F1D, and the dark grey bars show the results obtained with strains S5FI2B, S5FI5B, and C6S5F1E.

To analyze the dependence of SAP5 induction on the Tec1p transcription factor during infection of an animal host, we used a mouse model of Candida peritonitis as described previously (20-22). We observed that in this model hyphal formation within the host was impaired in the {Delta}tec1 mutant strains. In contrast to the wild-type strains, which formed hyphae and invaded into the liver, the {Delta}tec1 mutants attached to the liver surface but did not invade and were surrounded by inflammatory cells (Fig. 2, left panels). Despite the failure of the {Delta}tec1 mutants to invade the liver, activation of the SAP5 promoter was clearly detected in the cells remaining at the organ surface, although the average percentage of MPAS cells was reduced (20% compared with 43% in the wild-type controls) (Fig. 1B). These results demonstrate that SAP5 activation also occurred in the absence of the Tec1p transcription factor within an infected host. No significant differences were observed when the activations of the wild-type SAP5 promoter and the mutated version with a deletion of the putative Tec1p binding sites were compared in a wild-type background (43 and 36% MPAS cells, respectively) (Fig. 1B), demonstrating that the Tec1p binding consensus sequences are also not essential for activation of the SAP5 promoter within an infected host. The lower level of detectable activation of the SAP5 promoter in the {Delta}tec1 mutants may be due to Tec1p binding to sites other than the consensus sequence in C. albicans or to indirect activation, as discussed above. However, since the {Delta}tec1 mutants resided in host niches other than the wild-type strains, it seems possible that stronger host signals, and not Tec1p-mediated transcriptional activation, account for the increased SAP5 induction seen in the wild-type strains compared with that in the {Delta}tec1 mutants.



View larger version (144K):
[in this window]
[in a new window]
  		FIG. 2. Hyphal formation and tissue invasion of wild-type strain SC5314 (TEC1) and reporter strain C6S5F1E ({Delta}tec1). Both {Delta}tec1 strains behaved identically, and all reporter strains carrying the PSAP5-ecaFLP fusion in a wild-type background were indistinguishable from SC5314. (Left panels) Microscopic appearance of the strains at 24 h after intraperitoneal infection. (Right panels) Microscopic appearance of the strains in invaded kidneys at 4 days after intravenous infection.

To investigate whether the Tec1p-independent SAP5 activation within the host was a peculiarity of the intraperitoneal infection model, we also analyzed the behavior of our reporter strains in mice after intravenous infection. Histological examination showed that, as previously observed (19), the {Delta}tec1 mutants formed hyphae in infected kidneys which were indistinguishable from those formed by wild-type strains, demonstrating that the defect in hyphal formation of a {Delta}tec1 mutant depends on the host niche (Fig. 2, right panels). Importantly, deletion of the putative Tec1p binding sites had no effect on the inducibility of the SAP5 promoter and SAP5 activation was also observed in the absence of TEC1 in this infection model (Fig. 1C). These results demonstrate that the roles of the Tec1p transcription factor in the induction of hypha-associated genes can differ in vitro and in vivo and that SAP5 can be activated within the host by Tec1p-independent signaling mechanisms. Staib et al. have shown previously that SAP5 activation during infection depends on the transcriptional regulators Cph1p and Efg1p (21). In addition, no SAP5 mRNA was detected in vitro in {Delta}efg1 or {Delta}tec1 mutants (18, 19). Therefore, the in vitro expression of the SAP5 gene might require Efg1p-dependent activation by the Tec1p transcription factor, whereas SAP5 induction by host signals involves Tec1p-independent mechanisms, possibly including direct activation of the SAP5 promoter by Cph1p and Efg1p (10, 11). The results of our study clearly demonstrate that the importance of specific signaling pathways for the induction of C. albicans virulence-associated genes depends on the environmental conditions encountered within the host during an infection.


arrow
ACKNOWLEDGMENTS
 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG grants MO 846/1-3 and KR 2002/1-1). Joachim Morschhäuser was the recipient of a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-31 21 52. Fax: 49-931-31 25 78. E-mail: joachim.morschhaeuser{at}mail.uni-wuerzburg.de. Back

Editor: T. R. Kozel


arrow
REFERENCES
 
    1
  1. Andrianopoulos, A., and W. E. Timberlake. 1994. The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol. Cell. Biol. 14:2503-2515.[Abstract/Free Full Text]
    2. 2
  2. Braun, B. R., W. S. Head, M. X. Wang, and A. D. Johnson. 2000. Identification and characterization of TUP1-regulated genes in Candida albicans. Genetics 156:31-44.[Abstract/Free Full Text]
    3. 3
  3. Brown, A. J., and N. A. Gow. 1999. Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol. 7:333-338.[CrossRef][Medline]
    4. 4
  4. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candida albicans. Trends Microbiol. 9:327-335.[CrossRef][Medline]
    5. 5
  5. Ernst, J. F. 2000. Transcription factors in Candida albicans—environmental control of morphogenesis. Microbiology 146:1763-1774.[Free Full Text]
    6. 6
  6. Gavrias, V., A. Andrianopoulos, C. J. Gimeno, and W. E. Timberlake. 1996. Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth. Mol. Microbiol. 19:1255-1263.[Medline]
    7. 7
  7. 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.[CrossRef][Medline]
    8. 8
  8. Köhler, G. A., T. C. White, and N. Agabian. 1997. Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid. J. Bacteriol. 179:2331-2338.[Abstract/Free Full Text]
    9. 9
  9. Kretschmar, M., B. Hube, T. Bertsch, D. Sanglard, R. Merker, M. Schröder, 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.[Abstract/Free Full Text]
    10. 10
  10. Lane, S., C. Birse, S. Zhou, R. Matson, and H. Liu. 2001. DNA array studies demonstrate convergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans. J. Biol. Chem. 276:48988-48996.[Abstract/Free Full Text]
    11. 11
  11. Leng, P., P. R. Lee, H. Wu, and A. J. Brown. 2001. Efg1, a morphogenetic regulator in Candida albicans, is a sequence-specific DNA binding protein. J. Bacteriol. 183:4090-4093.[Abstract/Free Full Text]
    12. 12
  12. Liu, H. 2001. Transcriptional control of dimorphism in Candida albicans. Curr. Opin. Microbiol. 4:728-735.[CrossRef][Medline]
    13. 13
  13. Liu, H., J. Köhler, and G. R. Fink. 1994. Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266:1723-1726.[Abstract/Free Full Text]
    14. 14
  14. Lo, H. J., J. R. Köhler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G. R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell 90:939-949.[CrossRef][Medline]
    15. 15
  15. Mitchell, A. P. 1998. Dimorphism and virulence in Candida albicans. Curr. Opin. Microbiol. 1:687-692.[CrossRef][Medline]
    16. 16
  16. Ramon, A. M., and W. A. Fonzi. 2003. Diverged binding specificity of Rim101p, the Candida albicans ortholog of PacC. Eukaryot. Cell 2:718-728.[Abstract/Free Full Text]
    17. 17
  17. 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.[Abstract]
    18. 18
  18. Schröppel, K., K. Sprößer, M. Whiteway, D. Y. Thomas, M. Röllinghoff, and C. Csank. 2000. Repression of hyphal proteinase expression by the mitogen-activated protein (MAP) kinase phosphatase Cpp1p of Candida albicans is independent of the MAP kinase Cek1p. Infect. Immun. 68:7159-7161.[Abstract/Free Full Text]
    19. 19
  19. Schweizer, A., S. Rupp, B. N. Taylor, M. Röllinghoff, and K. Schröppel. 2000. The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol. Microbiol. 38:435-445.[CrossRef][Medline]
    20. 20
  20. Staib, P., M. Kretschmar, T. Nichterlein, H. Hof, and J. Morschhäuser. 2000. Differential activation of a Candida albicans virulence gene family during infection. Proc. Natl. Acad. Sci. USA 97:6102-6107.[Abstract/Free Full Text]
    21. 21
  21. Staib, P., M. Kretschmar, T. Nichterlein, H. Hof, and J. Morschhäuser. 2002. Transcriptional regulators Cph1p and Efg1p mediate activation of the Candida albicans virulence gene SAP5 during infection. Infect. Immun. 70:921-927.[Abstract/Free Full Text]
    22. 22
  22. Staib, P., M. Kretschmar, T. Nichterlein, G. Köhler, S. Michel, H. Hof, J. Hacker, and J. Morschhäuser. 1999. Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol. Microbiol. 32:533-546.[CrossRef][Medline]
    23. 23
  23. Stoldt, V. R., A. Sonneborn, C. E. Leuker, and J. F. Ernst. 1997. Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J. 16:1982-1991.[CrossRef][Medline]
    24. 24
  24. Whiteway, M. 2000. Transcriptional control of cell type and morphogenesis in Candida albicans. Curr. Opin. Microbiol. 3:582-588.[CrossRef][Medline]

Infection and Immunity, April 2004, p. 2386-2389, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2386-2389.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Staib, P.
Right arrow Articles by Morschhäuser, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Staib, P.
Right arrow Articles by Morschhäuser, J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»