Several lines of evidence support the conclusion that scFv3 is specific for Als3p. Similar to the cellular distribution described for Als3p (Zhao et al., 2006
), this antibody fragment recognizes a protein that is surface-exposed and binds uniformly throughout the hypha length. While we could detect binding of other hypha-specific scFv (scFv5 and scFv12), binding of scFv3 was not detected to the als3
Δ strain, 1843, while binding was restored to strain 2322 in which ALS3
is reintegrated. Binding of scFv3 was also easily detectable in S. cerevisiae
expressing the C. albicans ALS3
gene. Finally, and most importantly, our results demonstrate that treatment of wild-type C. albicans
with scFv3 makes this strain behave very similarly to the als3
Δ strain in adhesion assays.
Several different attempts to demonstrate binding of scFv3 to Als3p in vitro
were unsuccessful. Although we were able to demonstrate production of full-length Als3p in E. coli
, we were unable to detect the protein with scFv3 by immunoblotting. Additionally, although the Als3p N-terminal domain is believed to confer its adhesive properties (Zhao et al., 2006
), we were unable to detect the purified N-terminal 329 amino acids by immunoblotting, and inclusion of this protein in adhesion assays could not out-compete the ability of scFv3 to reduce wild-type C. albicans
adherence. Taken together, these findings suggest that the specific epitope recognized by scFv3 is difficult to preserve in vitro
. The epitope may be discontinuous or conformational, such that expression on the cell surface is required for the protein to be in the appropriate conformation for recognition. Alternatively, specific post-translational modifications may be required that are difficult to achieve in expression systems in vitro
, or there may be carbohydrate moities within the epitope. Detection of Als3p on the surface of S. cerevisiae
constitutively expressing the C. albicans
gene not only confirms this protein as the target of scFv3, but also supports the need for post-translational modification or a specific conformational epitope for detection. ScFv specific to other antigens and isolated from the same phage display library commonly fail to recognize their target antigen in similar analyses (unpublished observation), suggesting that recapitulation of the native epitope is frequently difficult to achieve. More definitive information awaits careful epitope mapping of the reactive domain for this antibody fragment.
The binding pattern of scFv5 and scFv12 to the als3
Δ strain was markedly different from the pattern with wild-type C. albicans
. Staining of the germ tube was quite variable and binding to the mother yeast was obvious, whereas in wild-type cells, the germ tubes were stained in a uniform fashion with no detectable staining of the mother yeast. These findings suggest that in the absence of Als3p, the antigenic milieu of the cell wall is altered. Because ALS3
transcription primarily is associated with germ tubes and hyphae (Hoyer et al., 1998
), additional investigation is required to understand how this change occurs in the mother yeast. The cognate antigens of scFv5 and scFv12 have yet to be elucidated, but the obvious change in distribution of detectable antigen in the als3
Δ strain is striking. Although the role of Als3p in adhesion has been studied extensively, these data suggest that Als3p may also function in cell wall organization and structure.
Evidence that C. albicans
Als3p functions as an adhesin is compelling. Although there may be some overlap and redundancy in function among the various C. albicans
adhesins, deletion of ALS3
results in the largest effect on adhesion among the ALS gene family (Hoyer et al., 2008
). Previous adhesion studies using a 6-well format showed a reduction in adhesion to HUVEC and pharyngeal epithelial cells by the als3
Δ strain, 1843, to approximately 40% and 60% of wild-type levels respectively (Oh et al., 2005
). In the present study, we observed an even more pronounced deficit in adhesion (approximately 20% and 45%) to these same cell types. Because our assay was adapted to a 96-well format and uses fluorescence for quantification, the discrepancy is likely due to assay methodology. The finding of reduced adherence by the mutant strain and similar trends between endothelial and epithelial cells validates both methodologies. Our assay has the advantage of high reproducibility and a robust signal-to-noise ratio. Further, this fluorescence-based assay in microtiter format allows more rapid readout, application of more variables in assay conditions, and higher throughput.
The finding that scFv3 has the most pronounced effect on adhesion relative to scFv5 and scFv12, coupled with the observation that all three scFv bind to germ tubes with an identical pattern, supports the notion that the interference with adhesion is specific to blocking Als3p. The smaller molecular size of scFv relative to whole antibody and the specificity of effect of scFv3 compared to other scFv that bind with an identical pattern suggest that blocking Als3p accounts for the adhesion defect, rather than merely resulting from steric interference.
Als3p binds N-cadherin and E-cadherin on endothelial and epithelial cells respectively (Phan et al., 2007
). The capacity of scFv3 to block adhesion of wild-type C. albicans
was more robust for epithelial cells than for endothelial cells, suggesting that the epitopes of Als3p involved in binding to these 2 cadherins may be distinct and blocked to different degrees by scFv3. Further, since the als3
Δ strain also adheres more efficiently to endothelial cells than to epithelial cells (), Als3p may have a more significant role as an adhesin to epithelial surfaces than endothelial. Alternatively, there may be more redundancy among C. albicans
adhesins targeted to endothelium, such that elimination of one adhesin, Als3p, either by mutation or antibody blocking, has less of an effect on adhesion to endothelium than other host surfaces.
A monoclonal antibody, C7, which was raised against a C. albicans
stress mannoprotein that is the main target of salivary secretory immunoglobulin A (IgA) has been described (Moragues et al., 2003
). In addition to interfering with C. albicans
adhesion to HEp-2 larynx carcinoma cells and human buccal epithelial cells, C7 inhibits filamentation and has direct candidacidal activity. Subsequent study showed that this antibody reacts with Als3p after deglycosylation and with a recombinant N-terminal fragment of Als3p (Brena et al., 2007
). This antibody has several other intriguing properties, including antifungal activity against a range of other genera (Cryptococcus neoformans, Aspergillus fumigatus, Scedosporium prolificans
), direct tumoricidal activity, and recognition of the C. albicans
enolase and the nuclear pore protein Nup88 (Moragues et al., 2003
; Omaetxebarria et al., 2005
). The authors suggest that the binding of Als3p by C7 may explain the inhibition of adhesion, the inhibition of germination, and the candidacidal activity of this antibody (Brena et al., 2007
). However, this antibody binds neither to the hypha surface nor to dithiothreitol extracts of hyphae until they have been deglycosylated, suggesting that this antibody, unlike scFv3, may not bind efficiently to the Als3p antigen in its native state. This observation, coupled with the effects of C7 binding to other fungal antigens and to tumor cells, suggests that at least some of the properties attributed to this antibody relate to it binding antigens distinct from Als3p. Our observation that scFv3 has no effect on germination or on C. albicans
growth (data not shown) supports the notion that antibody binding to this protein may not affect fungal physiology beyond its ability to adhere to human cells and may therefore have narrower antigen specificity than C7. Regardless, this study validates the use of phage display technology to generate antibody fragments that can function at the host-pathogen interface, and adds to the armamentarium of immune-based reagents available for study of invasive candidiasis.