Although Candida albicans is frequently associated with the human host as a commensal, reductions in host immune defense or microbiota can result in the fungus overgrowing its niche and causing disease. The ability of the fungal cell to adhere to host surfaces is a key feature that promotes the opportunity for subsequent disease. Considerable effort has been dedicated to characterization of C. albicans cell-surface proteins that are involved in adhesive interactions with host cells, as well as interactions with other microbial cells to form specialized structures such as biofilms (Chaffin, 2008; Ponde et al., 2021).

One notable example of a C. albicans cell-surface adhesion protein is Hyphal Wall Protein 1 (Hwp1; Staab and Sundstrom, 1998) that mediates stabilized adhesion between C. albicans and human buccal epithelial cells (BECs; Staab et al., 1999). A Gln-Pro-rich region in the N-terminal end of Hwp1 serves as the substrate for host transglutaminases that catalyze the adhesive interaction. Hwp1 is also required for C. albicans catheter biofilm formation in a rat model (Nobile et al., 2006) where it is suggested to function in concert with other cell-surface adhesins from the agglutinin-like sequence (Als) family (Nobile et al., 2008).

Development of new tools and reagents advances our understanding of host-pathogen interactions. In that regard, we previously produced MAbs specific for individual proteins in the Als family (Coleman et al., 2009; Coleman et al., 2010; Coleman et al., 2011; Zhao et al., 2011). The theoretically unlimited supply of MAbs provides a considerable advantage over the use of polyclonal antiserum for characterization of protein structure and function. Here, we describe production of an anti-Hwp1 MAb raised against a peptide immunogen from the Hwp1 Gln-Pro-rich adhesive region. We provide a comparative genomics analysis of Hwp1 and related proteins to demonstrate that the Hwp1 Gln-Pro-rich adhesive sequences are only found in C. albicans and Candida dubliniensis. We also highlight key questions about Hwp1 structure and function that can be addressed using the new anti-Hwp1 MAb.

Specificity of anti-Hwp1 MAb 2-E8 for Hwp1 on the C. albicans cell surface was demonstrated by immunolabeling wild-type C. albicans cells, a hwp1/hwp1 mutant (CAH7-1A1E2), and hwp1/hwp1::HWP1 reintegrant strain (CAHR3; Figure 1 ). MAb 2-E8 labeled the germ tube of strain CAI12 (HWP1/HWP1), but not strain CAH7-1A1E2 (hwp1/hwp1). Reintegration of a wild-type HWP1 allele in strain CAHR3 restored MAb 2-E8 immunolabeling, consistent with the conclusion that the MAb is specific for Hwp1 in C. albicans cells. Use of anti-Hwp1 MAb 2-E8 for immunogold electron microscopy showed gold particles distributed across the germ tube surface, associated with the flocculant, outermost layer of the cell ( Figure 2 ).

MAb 2-E8 was also used for atomic force microscopy (AFM) detection of C. albicans cell-surface Hwp1 ( Figure 3 ). Single Molecule Force Spectroscopy experiments, conducted with functionalized AFM tips, measured specific interaction forces between one molecule on the functionalized tip and one molecule on the sample surface. Specific molecular interactions were recorded during retraction of the AFM tip from the sample. The surface of the germ tube was scanned with either a bare AFM tip composed of silicon nitride (Si₃N₄) ( Figure 3C ) or functionalized tip coated with purified MAb 2-E8 ( Figure 3E ). The same technique was applied to the yeast cell (bare AFM tip in Figure 3G and anti-Hwp1 functionalized tip in Figure 3I ). The adhesion force was determined by measuring the piezo-retraction required to break the interaction between the MAb attached to the tip and the cell surface. This process was repeated while moving the cantilever between each measurement, thus creating a map of the interactions that revealed how Hwp1 was distributed on the cell surface. Histograms representing the repartition of recorded interaction forces are shown in Figures 3D, F, H, J , respectively. The intensity and frequency of adhesive events recorded with a bare AFM tip on the surface of the germ tube or yeast cell were very low ( Figures 3D, H ). In contrast, specificity of the antibody-antigen interaction was visible from the remarkably high frequency and intensity signals observed from scanning germ tubes with the anti-Hwp1 functionalized tip compared to those recorded on the yeast cell ( Figures 3F vs. 3J ). Low signals on the yeast cell were consistent with fluorescence microscopy images that suggested abundant representation of Hwp1 only on the germ tube ( Figure 1 ).

Genome database searching was used to predict the potential for the anti-Hwp1 MAb 2-E8 to recognize the surface of other fungal species that are closely related to C. albicans. C. albicans HWP1 (C4_03570W_A) is located on Chromosome 4 near two other genes with which it shares sequence similarity: RBT1 (C4_03520C_A) and HWP2 (C4_03510C_A). The Candida Gene Order Browser (CGOB; Maguire et al., 2013) identified orthologs of all three loci, based on synteny; these results are displayed as vertical alignments of genes with conserved chromosomal location, called pillars. Table 1 shows the pillars corresponding to HWP1, RBT1, and HWP2. BLAST search results with an E value less than 1.0 were retained in the list. The list also includes loci beyond those identified in the CGOB; those sequences are shown in the far-right column of Table 1 . Amino acid sequences translated from the Table 1 loci are included in Supplementary Table S1 to facilitate examination and construction of sequence comparisons by the reader.

The peptide immunogen used to raise anti-Hwp1 MAb 2-E8 was in the N-terminal region of repeated Gln/Pro-rich sequences that serve as a substrate for mammalian transglutaminases and facilitate stabilized adhesion of C. albicans to BECs (Staab and Sundstrom, 1998; Staab et al., 1999). Sequence comparisons and alignments among the Hwp1/Rbt1/Hwp2 orthologs ( Supplementary Table S1 ) indicated that the Gln/Pro-rich sequences were unique to Hwp1 in C. albicans and C. dubliniensis although the C. dubliniensis Cd36_43360 sequence that best matched the peptide immunogen was imperfect ( Table 2 ). The 13-mer immunogen peptide sequence was used as the query for a tblastn search of the genomes for species listed in Table 1 . Additional partial sequence matches were revealed ( Table 2 ) although the resulting proteins were unlikely to be present on the fungal cell surface.

C. albicans Hwp1 was downloaded from the AlphaFold database of predicted protein structures (https://www.alphafold.ebi.ac.uk) to visualize the position of the 13-mer immunogen; AlphaFold Colab was used to predict a structure from the C. dubliniensis Hwp1 sequence ( Figure 4 ). AlphaFold predictions showed largely unstructured molecules although the predictions did not have a high level of confidence. However, both structures suggested surface-exposure of the immunogen.

C. dubliniensis CD36, C. tropicalis MYA-3404, and C. parapsilosis CDC 317 were tested using the same growth conditions and immunolabeling methods that were successful for C. albicans; no anti-Hwp1 signals were observed (data not shown). Negative results for C. tropicalis and C. parapsilsosis were consistent with genomic information that suggested lack of an epitope that could be recognized by anti-Hwp1 MAb 2-E8 ( Tables 1 , 2 ; Supplementary Table S1 ). The potential for a Hwp1 epitope to exist in C. dubliniensis ( Table 2 ) prompted literature searches for alternative growth conditions that might produce sufficient Hwp1 protein for immunolabeling detection. Caplice and Moran (2015) demonstrated a difference in EFG1 regulation of genes between C. albicans and C. dubliniensis with C. dubliniensis genes showing a lag in serum-induced expression and a requirement for nutrient depletion for maximum expression. C. dubliniensis CD36 grown in 10% serum produced visible anti-Hwp1 immunolabeling signal on some germ tubes ( Figure 5 ). Immunolabeling was observed primarily toward the germ-tube tip in the longer germ tubes shown.

The localization of Hwp1 on C. dubliniensis germ tubes varied from C. albicans Hwp1 which was distributed over most of the germ tube length ( Figure 6 ). C. albicans Als1 and Als3 are also known for abundant presence on the germ tube. Previous work showed that ALS1 expression was detectable before ALS3 when C. albicans yeast cells were placed into RPMI medium to promote germ tube formation (Zhao et al., 2004). That result was replicated here using MAbs specific for each protein ( Figure 7 ). Immunolabeling with anti-Hwp1 MAb 2-E8 demonstrated that the kinetics of Hwp1 production were more similar to Als1 than Als3 ( Figure 7 ). Although there are regions of the germ tube where all three proteins were present, immunolabeling of longer germ tubes ( Figure 6 ), showed obvious Als1 blue false coloring closest to the junction between the mother yeast and germ tube. Therefore, despite the appearance of Als1 before Als3, there is greater overlap between Hwp1 and Als3 on the lengthening germ tube than overlap of either protein with Als1.

The Gln-Pro-rich region against which the anti-Hwp1 MAb 2-E8 was raised mediates stabilized adhesion of C. albicans to human BECs (Staab and Sundstrom, 1998; Staab et al., 1999). Assays were conducted to test whether MAb 2-E8 could block the adhesive interaction. Anti-Als1 MAb 1-B2 was demonstrated previously to block adhesion of C. albicans to BECs and was included as an assay control (Coleman et al., 2010). Anti-Als6 MAb 6-A1, which does not have detectable anti-adhesive activity, was included as a negative control for the effect of adding protein to the assay (Coleman et al., 2009). Anti-Hwp1 MAb 2-E8 at a concentration of 20 µg/ml was sufficient to block adhesion, similar to anti-Als1. ( Figure 8 ).

Immunization of mice with a peptide derived from the Gln-Pro-rich adhesive domain of C. albicans Hwp1 was used to create MAb 2-E8, which is specific for Hwp1 in C. albicans ( Figures 1 – 3 ). MAb 2-E8 also recognizes the surface of C. dubliniensis germ tubes ( Figure 5 ), suggesting cross-reactivity with a similar sequence in C. dubliniensis Hwp1 ( Table 2 ). Previous literature reports described a polyclonal rabbit antiserum raised against an Hwp1 fragment heterologously expressed in Pichia pastoris (Staab et al., 1996). The antiserum was used to demonstrate Hwp1 features such as its selective detection on C. albicans germ tubes (Staab et al., 1996), a result confirmed here using MAb 2-E8 for immunolabeling and AFM analysis ( Figures 1 , 3 ).

AFM analysis demonstrated highly specific mapping of a native cell wall protein on the C. albicans surface using tips functionalized with a MAb. Such measurements are more specific than use of tagged proteins (Formosa et al., 2015) or polyclonal antibodies (Beaussart et al., 2012) that are likely to cross react with other surface proteins. Non-specific events that were recorded in experiments reported here are mediated by other C. albicans surface proteins that display different profiles from the specific antigen-antibody interactions ( Figure 3 ). For example, the highly adhesive C. albicans surface that creates non-specific interactions with a bare AFM tip were observed for Pga proteins (Cabral et al., 2014). Another notable outcome of this work is that Hwp1 is observed earlier during germ tube formation using immunofluorescence than by AFM. Hwp1 was observed on the C. albicans surface using immunofluorescence ( Figure 7 ) but required longer germ tubes (approximately 90 to 120 min in inducing medium) for AFM observations. This difference may be due to MAb ability to access Hwp1 in solution for immunofluorescence compared to attachment to the dendrimer linker structure on the AFM tip, which is a very large structure compared to the cell wall network.

Examination of Hwp1 ortholog sequences among species closely related to C. albicans suggested that the Gln-Pro-rich repeated region that is responsible for stabilized adhesion between C. albicans and human BECs is only present in C. albicans and C. dubliniensis ( Supplementary Table S1 ). In C. albicans, HWP1 is located on chromosome 4 near other similar genes named RBT1 and HWP2. Similarity among C. albicans Hw1p, Rbt1, and Hwp2 is found in the signal peptide and C-terminal region. Sequence alignments suggest that the Gln-Pro region is essentially a little adhesive cassette dropped into C. albicans Hwp1 and preserved in C. dubliniensis Hwp1. Orthologs of Hwp1 in other species, as well as Rbt1, Hwp2 and their orthologs do not have that cassette. The presence of Hwp1 clearly contributes to the greater adhesive capacity of C. albicans compared to other closely related species (Calderone and Braun, 1991). Indeed, blocking the adhesive domain with MAb 2-E8 resulted in a significant reduction in C. albicans adhesion to BECs ( Figure 8 ).

Despite the close relationship of the species C. albicans and C. dubliniensis, differences in transcriptional regulation of germ tube/hypha growth are documented between them. For example, the regulation of HWP1 transcription by Efg1 differs between the species (Caplice and Moran, 2015). In C. dubliniensis, HWP1 expression lagged behind the kinetics of HWP1 expression in C. albicans. Approximately 2 h of growth induction in nutrient-limited conditions were required for maximum HWP1 expression levels to be reached. Anti-Hwp1 MAb 2-E8 detected protein at the tips of growing hyphae ( Figure 5 ), rather than along the full length of the germ tube ( Figure 6 ). Additionally, not all C. dubliniensis germ tubes produced a positive signal with anti-Hwp1 MAb 2-E8, another difference between the species. Availability of MAb 2-E8 can be leveraged to better understand how germ tube formation varies between the species and how differential protein abundance and localization may affect pathogenesis.

The Hwp1 Gln-Pro-rich sequences are present in multiple repeated copies (Staab et al., 1999), presumably each with potential to participate in adhesive interactions with BECs. Other C. albicans repeat-containing adhesins can have often-extreme allelic variation (Zhang et al., 2003; Hoyer et al., 2008; Oh et al., 2021), leading to questions about diversity of HWP1 alleles across C. albicans isolates. Analysis of HWP1 restriction fragment length polymorphisms was proposed as a diagnostic assay to distinguish C. albicans from C. dubliniensis, suggesting stability of fragment size, and therefore repeat copy number across isolates in the same species. Romeo et al. (2006) described PCR primers that amplified the 5’ end of HWP1 (encompassing the entire Gln-Pro-repeat-containing region) to distinguish between C. albicans and C. dubliniensis and tested them on approximately 20 isolates of each species. Size of the amplified fragments was consistent within each species with C. dubliniensis HWP1 having a considerably shorter Gln-Pro-repeat-encoding region than C. albicans HWP1. Padovan et al. (2009) described a novel C. albicans HWP1 allele (called HWP1-2) that lacked copies of sequences encoding the Gln-Pro repeat as well as a portion of the Ser-Thr-rich C-terminal region of the protein. A C. albicans strain that had two HWP1-2 alleles was defective in biofilm formation. Although variant HWP1 alleles have been described, selection seems predominantly for a consistent HWP1 allele size both in C. albicans and C. dubliniensis. Hwp1 adhesive function would require sufficient Gln-Pro copies, as well as an overall protein length to display the region on the cell surface. It seems highly likely that anti-Hwp1 MAb 2-E8 should recognize all C. albicans and C. dubliniensis isolates. Immunofluorescence, immunogold electron microscopy, and AFM techniques used here support the idea that the Gln-Pro region is displayed prominently on the C. albicans cell surface ( Figures 1 – 3 ).

The AlphaFold algorithm uses deep multiple sequence alignments and information from Protein Database structure templates to arrive at structural predictions that have unprecedented accuracy (Jumper et al., 2021). The AlphaFold database (www.alphafold.ebi.ac.uk) includes the entire C. albicans proteome; the user-friendly AlphaFold Google Colab (see Materials and Methods) can predict structures from proteins yet to appear in the AlphaFold database. While AlphaFold is viewed as game-changing in its ability to shed light on structural questions, the algorithm did not arrive at the same conclusions for Hwp1 as previous biochemical analysis. AlphaFold predicted Hwp1 to be largely unstructured ( Figure 4 ; https://www.alphafold.ebi.ac.uk/entry/P46593) but assigned a low or very low per-residue confidence score to the prediction. Staab et al. (2004) used traditional biochemical analysis of Hwp1 to infer structural properties. Circular dichroism spectra and titration of thiols led to prediction of a disulfide-cross-linked coiled-coil structure for the N-terminal adhesive portion of the protein (amino acids 40-187). In contrast, AlphaFold did not predict disulfide bonds among the 11 Cys residues in this portion of the Hwp1 sequence. Additional biochemical characterization by Staab et al. (2004) demonstrated Hwp1 modification by N- and O-glycosylation, as well as transient addition of a GPI anchor that directed Hwp1 linkage to β-glucan in the C. albicans cell wall (Lu et al., 1994).

In addition to its function in stabilized adhesion of C. albicans to human BECs, Hwp1 plays other key roles that support colonization and pathogenic potential in C. albicans. For example, Hwp1 is required for in vivo catheter biofilm formation using a rat model (Nobile et al., 2006). Subsequent investigation led to the hypothesis that Hwp1 promotes C. albicans biofilm formation through interaction with Als1 and/or Als3 (Nobile et al., 2008). Although Als1 and Hwp1 can be detected first on the C. albicans surface at the site of germ tube emergence in vitro ( Figure 7 ), the eventual protein distribution on the germ tube shows Als1 closest to the mother yeast and fading as the germ tube elongates ( Figure 6 ). Immunolabeling of hyphae isolated from a mouse oral candidiasis model indicated that the in vivo pattern of Als1 distribution is more homogeneous over the germ-tube length, likely from differential regulation of transcription in the animal (Coleman et al., 2011). It is reasonable to assume a similar Als1 distribution would occur in the rat catheter biofilm model. In cultured cells, Hwp1 is present strongly along the C. albicans germ tube length, similar to Als3 ( Figure 6 ). In vitro or in vivo, there is a sizable length of the germ tube on which Hwp1, Als1, and Als3 are co-localized in abundance with obvious opportunity to contact each other.

The literature contains speculation about the nature of the contact between Hwp1 and the Als proteins, suggesting that the interaction may have evolved from that between alpha- and a-agglutinin, the mating agglutinins in S. cerevisiae (Nobile et al., 2008; Soll, 2008). In S. cerevisiae, the monomeric, cell-wall-localized alpha-agglutinin binds with high affinity to the flexible C-terminal end of the small subunit (Aga2; 69 amino acids total in the mature protein) of the heterodimeric a-agglutinin (Dranginis et al., 2007). Aga2 is disulfide bonded to the Aga1 anchor protein that displays Aga2 on the cell surface. The idea that Als proteins are orthologs of S. cerevisiae alpha-agglutinin has gained support as the number of genomic sequences continues to grow (Oh et al., 2021). For example, CGOB predicts that S. cerevisiae SAG1 is syntenic with a C. dubliniensis ALS locus. Also, Sag1 and Als proteins appear to share a similar structure of the N-terminal domain that is responsible for adhesive (ligand-binding) function (Oh et al., 2021). In contrast, the idea that Hwp1 is an ortholog of S. cerevisiae a-agglutinin (Aga1) seems prompted by the observation that Hwp1 production and localization during C. albicans mating is specific to the a/a portion of the conjugation bridge (Daniels et al., 2003). Table 1 demonstrates that the HWP1 locus has a rather evolutionarily limited synteny pillar and database searching suggests that there does not appear to be a homolog of S. cerevisiae AGA1 in C. albicans. Alternatively, the nature of the interaction between Hwp1 and Als proteins may not involve ligand-binding but other mechanisms such as aggregation. Hwp1 contains sequences with amyloid-forming potential that could catalyze aggregative interactions with other abundant cell-surface proteins (Ramsook et al., 2010). It remains to be demonstrated whether Hwp1 glycosylation (Staab et al., 2004) obscures potential amyloid-forming sequences or if they are available to access similar sequences in other proteins and contribute to aggregative interactions.

Several tools and reagents have been developed recently that make it possible to test the hypotheses presented above. For example, the nature of the Als peptide-binding activity is characterized at the molecular level (Salgado et al., 2011). C. albicans strains that display functionally mutant versions of Als3 on the surface under control of the native promoter were described (Lin et al., 2014). One strain incorporates mutations in the peptide-binding cavity that abolish ligand-binding function while still displaying the structurally correct full-length, cell-surface Als3 under control of its native promoter. Another strain displays an Als3 molecule in which the amyloid-forming (aggregative) function is disrupted, but ligand-binding (peptide-binding cavity) function remains. These strains, in combination with theoretically unlimited quantities of MAbs specific for Hwp1, Als1, and Als3 can be used to better understand dynamics of the C. albicans cell wall and the role of Hwp1 in host-pathogen interactions and biofilm formation.

The anti-Hwp1 2-E8 hybridoma was deposited in the Developmental Studies Hybridoma Bank, University of Iowa (https://dshb.biology.uiowa.edu). Other data described in this study can be found in online repositories as specified in the article and Supplementary Material .

The studies involving human participants were reviewed and approved by University of Illinois at Urbana-Champaign Office for the Protection of Research Subjects. The patients/participants provided their written informed consent to participate in this study.

LH conceptualized the study. LH and ED acquired funding and were responsible for project administration. All authors developed the study methodology, performed the investigation, and conducted formal analysis. LH wrote the original draft. All authors reviewed and edited the manuscript and approved the submitted version.

This work was supported by grant R01 DE14158 from the National Institute of Dental and Craniofacial Research, National Institutes of Health (to LH) and IDEX Transversality UFTMIP: 2016-106-CIF-D-DRDV (to HM-Y, ED).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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