1 Research Service, University of Arizona, Tucson, AZ, USA
2 Southern AZ VA Healthcare System and Department of Medicine, University of Arizona, Tucson, AZ, USA
Correspondence
Stephen A. Klotz
sklotz{at}u.arizona.edu
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ABSTRACT |
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INTRODUCTION |
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The ability of C. albicans to establish and maintain residency at mucosal surfaces in the host is likely to be governed by mechanisms allowing it to adhere rapidly in a stable but reversible manner to host surfaces. A number of mechanisms of adherence have been described in C. albicans, although most involve relatively weak interactions and thus are less likely to be primary mechanisms of adherence in vivo (Calderone & Braun, 1991; Pendrak & Klotz, 1995
). One stable adherence mechanism is the covalent coupling of a fungal cell surface protein, Hwp1, to host cells by the host enzyme transglutaminase (Stabb et al., 1999
; Sundstrom, 2002
). This mechanism provides for lasting adherence but it is not clear that it can be reversed. Furthermore, the expression of Hwp1 is restricted to the hyphal form, whereas both hyphal and yeast forms of C. albicans are known to adhere in vivo and are found in infected tissues. We have previously characterized a mechanism of adherence in C. albicans which displays properties such as stability to shear forces, adherence in acidic and neutral pH, resistance to the presence of various biological molecules and sensitivity to reagents known to disrupt hydrogen bonds (Gaur et al., 1999
). To distinguish this mechanism of adherence from other previously described C. albicans adherence mechanisms we will henceforth refer to it as SRS (stable, reversible and specific) adherence. In contrast to Hwp1-mediated adherence, all morphological forms of C. albicans exhibit SRS adherence (Gaur et al., 1999
, 2002
).
A large gene family, the agglutinin-like sequence (ALS) family, has been described in C. albicans, where all members share similarities in the amino acid sequence at their N-termini and have similar predicted structural motifs (Hoyer, 2001). At least two members of the ALS family, Als5p and Als1p, have been shown to confer upon Saccharomyces cerevisiae properties similar to that of C. albicans SRS adherence (Gaur & Klotz, 1997
; Fu et al., 1998
; Gaur et al., 1999
). It is expected that other Als proteins will have similar properties as they conserve all the structural features of Als5p.
It is widely accepted that the characterization of molecular changes occurring in the host as well as in C. albicans during the transition from commensal to pathogenic relationship is essential for the complete understanding of Candida pathogenesis (Casadevall & Pirofski, 2000; Van Burik & Magee, 2001
; Soll, 2002
). Accordingly, we have pursued a host-centred view in which the susceptible host may acquire new functions that are not prominent in the normal host. In this view of Candida pathogenesis, the generation of new SRS adherence target sites in the susceptible host may in part be responsible for the increased proliferation of C. albicans during candidiasis. This assumption necessitates the characterization of molecular features of target recognition in SRS adherence for the understanding of Candida pathogenesis. We have developed an adherence assay in which small synthetic peptides are immobilized in a controlled manner to orient them in specific configurations on the surface of magnetic beads (Gaur et al., 2002
). These studies have suggested that in addition to chemical composition of the ligand, the accessibility of patches of certain amino acids is also important.
We continue to define the molecular features of SRS adherence targets in this study and report on the nature of the interaction of C. albicans and S. cerevisiae expressing Als5p with protein and peptide ligands. These studies have suggested functional similarities between Als5p interactions in SRS adherence and those of the molecular chaperone Hsp70, with the peptide backbone of protein ligands. We discuss the significance of the accessibility of the peptide backbone of protein ligands as an important specificity determinant in defining SRS adherence targets.
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METHODS |
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Peptides and magnetic beads.
Peptides were chemically synthesized and purity determined by mass spectroscopy (Research Genetics, Huntsville, AL, USA). Tosyl-activated magnetic beads were purchased from Dynal and were coupled with fibronectin that had been denatured by boiling for 10 min (Gaur & Klotz, 1997). Carboxylate-modified magnetic beads were purchased from Seradyne and peptides were coupled to them using the carbodiimide coupling method as described before (Gaur et al., 2002
).
Adherence assay.
C. albicans yeast cells were grown in YPD and harvested from the late exponential phase of growth and S. cerevisiae harbouring pGK114 were grown in YPRG and harvested from the stationary phase for the adherence assay as described before (Gaur et al., 1999). Both yeast cells were washed and stored in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7·0). The quantitative adherence assay using fibronectin (FN)-coated tosyl-activated (TA) magnetic beads and the qualitative adherence assay using peptide-coated carboxylate-modified (CM) magnetic beads were performed as described before (Gaur et al., 2002
). Briefly, an excess of yeast cells was mixed with magnetic beads and incubated with shaking at room temperature for 30 min. Magnetic beads and adhered yeast cells were collected by a magnet and washed three times each with 1 ml TE buffer. For microscopic observation, adherent yeast cells were suspended in 0·1 ml TE buffer. Adherent yeast cells were dissociated from magnetic beads by suspending in an appropriate amount of 0·1 M NaOH, and beads and cells were counted using a haemocytometer. Competition experiments consisted of peptides or proteins incubated with yeast cells for 30 min prior to adding FN-coated TA magnetic beads. Denatured proteins were prepared just before use by incubating the required amount in a tube in a boiling water bath for 10 min. The stability of adherence was determined by preparing a microscope slide with cells adherent to polythreonine- or polyalanine-coated CM magnetic beads; the cover slip was sealed and the preparation observed under the light microscope. The slide was left at 4 °C or 25 °C for 24 h before observing it again under the microscope. Magnetic beads remained bound to yeast cells after 24 h incubation when the interaction was stable. When the interaction was less stable, the magnetic beads separated from yeast cell aggregates in less than 24 h, resulting in an abundance of free beads and yeast cells.
Statistical analysis.
The adherence assay for each experiment was performed a minimum of three times. Results are presented as means and standard deviations and were analysed by using a paired Student t test with a P value of <0·05 considered significant.
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RESULTS |
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Adherence to threonine peptides is more stable than to alanine peptides
Since threonine and alanine side chains are chemically different and adherence occurring to peptides containing six residues of either threonine or alanine is phenotypically indistinguishable, we compared the stability of adherent yeast cells to threonine and alanine peptides. As shown in Fig. 1, the polyalanine-coated CM beads dissociated from C. albicans yeast cells within 24 h, whereas polythreonine-coated CM beads remained attached to the yeast cells. It is interesting to note that the dissociation of polyalanine-coated CM beads from the yeast cells also caused aggregates to disintegrate, resulting in free beads and cells. Similar results were obtained with S. cerevisiae expressing Als5p (results not shown). These results suggest that although the primary interaction occurs with the peptide backbone in alanine and threonine peptides, the interaction with the side chain of threonine and not alanine provides stability to the primary interaction.
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DISCUSSION |
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Using peptides containing varying numbers of threonine or alanine residues, we have shown that peptides of a minimum of six amino acid residues must be present to serve as adherence targets. The side chain of threonine has a hydroxyl group capable of forming a hydrogen bond, whereas the alanine side chain contains a methyl group, which cannot form a hydrogen bond. Since six residues are required for both threonine and alanine peptides for maximal adherence to occur, we propose that the primary interaction occurs to the backbone of a minimum five peptide bonds. As demonstrated, adherence to threonine peptides is more stable than to alanine peptides, presumably because the threonine side chain has the potential to form a hydrogen bond that would provide stability to this interaction, whereas the side chain of alanine cannot form a hydrogen bond.
The secondary and tertiary structures of protein ligands prevent interactions with C. albicans and S. cerevisiae expressing Als5p. The discussion above suggesting the initial interaction of C. albicans Als proteins with the peptide backbone of a specific amino acid sequence is further supported by results obtained from the competition of adherence experiments using free peptides and proteins. Peptides that serve as targets of adherence when immobilized compete with the adherence of C. albicans and S. cerevisiae expressing Als5p to FN-coated TA magnetic beads when added exogenously to the assay. In contrast, when soluble native proteins are added to the assay, these proteins rarely have any measurable effect on adherence of C. albicans or S. cerevisiae expressing Als5p. However, if these same proteins are denatured and added to the assay they paradoxically stimulate adherence by promoting yeast cell aggregation. Denaturation of proteins breaks the secondary and tertiary structures and, as shown in this work, only denatured proteins and not native proteins bind to C. albicans and S. cerevisiae expressing Als5p. Many peptide groups in native proteins are involved in the formation of hydrogen bonds that maintain the secondary and tertiary structure and thus are not available to interact with C. albicans Als proteins. These peptide groups of protein ligands probably become accessible for the interaction with Als proteins following denaturation.
The mechanism of interaction in SRS adherence is similar to binding of the molecular chaperone Hsp70 to nascent polypeptides and small peptides
Molecular chaperones are a group of proteins involved in protecting nascent polypeptides from misfolding and aggregation (Hartl & Hayer-Hartl, 2002). There are different classes of molecular chaperones; however, all of them function by their unique ability to interact with non-native proteins (nascent and unfolded polypeptides) and not with native proteins. One of these classes is the Hsp70 family, which is found in all living organisms. Hsp70 or DnaK has two functional domains: the N-terminus is an ATP-dependent regulatory domain that controls the activity of the C-terminal peptide-binding domain. It is estimated that in an average protein the Hsp70 binding sites occur approximately every 40 residues and typically are seven residues long with hydrophobic amino acids in their central region. The cocrystal structure of DnaK with a 7-mer peptide substrate has been solved and the primary interactions determined (Zhu et al., 1996
). The structure which has been determined at 2·0 Å resolution clearly identifies the hydrogen bond interactions with the peptide backbone and hydrophobic interactions with the amino acid side chain. These interactions are strikingly similar to what we are proposing in SRS adherence, in which primary interactions are with the peptide backbone and secondary interactions are with the side chain through hydrogen bonds. The peptide backbone in the DnaKpeptide cocrystal structure has an extended conformation. Our results support a similar extended polypeptide structure of protein ligands in which the peptide backbone is accessible. The peptide-binding domain of DnaK is folded into a compact
-sandwich of two sheets with four antiparallel strands in each. Circular dichroism analysis of the N-terminal portion (Ig-like domain) of Als5p suggested an abundance of
-strands and very low helical content (Hoyer, 2001
). As it is expected that the Ig-like domain of Als proteins is involved in the interaction with SRS adherence targets, these adhesins may also have structural similarities with Hsp70. Furthermore, C. albicans Als proteins are resistant to denaturation as is Hsp70. Thus, Als proteins may have similarities with Hsp70 in the way they interact with protein ligands as well as similarities in physical properties. To our knowledge this is the first example of receptorligand interactions in which the specificity is achieved by the lack of secondary structure in the protein ligands.
This work is the continuation of our efforts to characterize molecular features of protein ligands in defining SRS adherence targets that may be generated in the susceptible host during candidiasis. We have demonstrated in this work that the accessibility of the peptide backbone is an important specificity determinant in the interaction of C. albicans Als adhesins with protein ligands. In the susceptible host, SRS adherence targets with their accessible peptide backbone might be generated in response to the host inflammatory reaction or actions of the micro-organism. One such mechanism could involve C. albicans-secreted hydrolytic enzymes such as aspartic proteases and lipases. Both classes of enzymes are encoded by genes belonging to large gene families (Hube et al., 2000; Hube & Naglik, 2001
; Naglik et al., 2003
). The hydrolytic activity of these enzymes has the potential to modify host surfaces in ways that may make them more adhesive for C. albicans. The complete understanding of these mechanisms may provide an opportunity to design strategies to develop novel therapeutic approaches for treating candidiasis.
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ACKNOWLEDGEMENTS |
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Received 28 August 2003;
revised 23 October 2003;
accepted 27 October 2003.