Progress 10/01/96 to 06/30/11
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project ended about 10 years ago. Please end this project in the REEport system.
Publications
|
Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: In the past year, we continued to build our understanding of the abundance and distribution of Als proteins on the C. albicans cell surface. Through a collaborative interaction, we contributed to work that solved the structure of the Als N-terminal domain, a much sought-after piece to the puzzle of understanding Als protein function. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work provides new insights into the function of proteins that play a key role in the biology and pathogenesis of Candida albicans, the most common fungal pathogen of humans.
Publications
- Coleman, D.C., S.-H. Oh, X. Zhao, J.T. Hutchins, J.H. Vernachio, J.M. Patti, and L.L. Hoyer. 2009. Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces. J. Microbiol. Meth. 78:71-78.
- Coleman, D.A., S.-H. Oh, X. Zhao, L.L. Hoyer. 2010. Heterogenous distributions of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody. Microbiology 156:3645-3659.
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: During the past year, we continued our analysis of the function of proteins encoded by the ALS genes in the pathogenic fungus Candida albicans. Historically, our approach to studying protein function has involved deletion of each of the eight ALS genes individually and phenotypic characterization of the resulting strain. These studies suggest a role for some of the Als proteins in adhesion to host cells and molecules and also suggest roles for Als proteins outside of adhesion. The results of these studies provide a picture of Als protein function that is different from that observed using other approaches such as heterologous expression of the ALS genes in Saccharomyces cerevisiae. Within the past year, we have developed monoclonal antibodies specific for various Als proteins. Immunostaining of C. albicans cells with the monoclonal antibodies has shown that in several cases, the Als proteins have a specialized location rather than a diffuse presence. This information is valuable as we continue our studies to understand the scope of function of this family of proteins. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses uses gene families in disease processes.
Publications
- Beucher, B., A. Marot-LeBlond, S. Billaud-Nail, S.-H. Oh, L.L. Hoyer, and R. Robert. 2008. Recognition of Candida albicans Als3p by the germ-tube-specific monoclonal antibody 3D9.3. FEMS Immunology and Medical Microbiology 55:314-23.
- Coleman, D.A., S.-H. Oh, X. Zhao, H. Zhao, J.T. Hutchins, J.H. Vernachio, J.M. Patti, and L.L. Hoyer. 2009. Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces. Journal of Microbiological Methods 78:71-8.
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: During the past year, we continued our analysis of the function of proteins encoded by the ALS genes in the pathogenic fungus Candida albicans. Historically, our approach to studying protein function has involved deletion of each of the eight ALS genes individually and phenotypic characterization of the resulting strain. These studies suggest a role for some of the Als proteins in adhesion to host cells and molecules and also suggest roles for Als proteins outside of adhesion. The results of these studies provide a picture of Als protein function that is different from that observed using other approaches such as heterologous expression of the ALS genes in Saccharomyces cerevisiae. Within the past year, we have developed monoclonal antibodies specific for various Als proteins. Immunostaining of C. albicans cells with the monoclonal antibodies has shown that in several cases, the Als proteins have a specialized location rather than a diffuse presence. This information is valuable as we continue our studies to understand the scope of function of this family of proteins. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses uses gene families in disease processes.
Publications
- Hoyer, L.L., C.B. Green, S.-H. Oh, and X. Zhao. 2007. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family: a sticky pursuit. Medical Mycology 46:1-15.
- Laforce-Nesbitt, S.S., M.A. Sullivan, L.L. Hoyer, and J.M. Bliss. 2008. Inhibition of Candida albicans adhesion by recombinant human antibody single-chain variable fragment specific or Als3p. FEMS Immunology and Medical Microbiology 54:195-202.
- Beucher, B. A. Marot-LeBlond, S. Billaud-Nail, S.-H. Oh, L.L. Hoyer, and R. Robert. 2008. Recognition of Candida albicans Als3p by the germ-tube-specific monoclonal antibody 3D9.3. FEMS Immunology and Medical Microbiology (In press).
|
Progress 10/01/06 to 09/30/07
Outputs
During the past year, we continued our analysis of the function of proteins encoded by the ALS genes in the pathogenic fungus Candida albicans. Historically, our approach to studying protein function has involved deletion of each of the eight ALS genes individually and phenotypic characterization of the resulting strain. These studies suggest a role for some of the Als proteins in adhesion to host cells and molecules and also suggest roles for Als proteins outside of adhesion. The results of these studies provide a picture of Als protein function that is different from that observed using other approaches such as heterologous expression of the ALS genes in Saccharomyces cerevisiae. Within the past year, we have developed monoclonal antibodies specific for various Als proteins. Immunostaining of C. albicans cells with the monoclonal antibodies has shown that in several cases, the Als proteins have a specialized location rather than a diffuse presence. This information is
valuable as we continue our studies to understand the scope of function of this family of proteins.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses uses gene families in disease processes.
Publications
- Zhao, X., S.-H. Oh, and L.L. Hoyer. 2007. Unequal contribution of ALS9 alleles to adhesion between Candida albicans and human vascular endothelial cells. Microbiology 153:2342-2350.
- Zhao, X., S.-H. Oh, and L.L. Hoyer. 2007. Deletion of ALS5, ALS6 or ALS7 increases adhesion of Candida albicans to human vascular endothelial and buccal epithelial cells. Medical Mycology 45:429-434.
- Jajko, R., S.-H. Oh, C. Pujol, D.R. Soll, and L.L. Hoyer. 2007. Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates. Fungal Genetics and Biology 45:429-434.
- Hoyer, L.L., C.B. Green, S.-H. Oh, and X. Zhao. 2007. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family: a sticky pursuit. Medical Mycology (Epub ahead of print).
|
Progress 10/01/05 to 09/30/06
Outputs
During the past year, we continued our analysis of the function of proteins encoded by the ALS genes in the pathogenic fungus Candida albicans. Our approach to studying protein function involves deletion of each of the eight ALS genes individually and phenotypic characterization of the resulting strain. These studies suggest a role for some of the Als proteins in adhesion to host cells and molecules and also suggest roles for Als proteins outside of adhesion. The results of these studies provide a picture of Als protein function that is different from that observed using other approaches such as heterologous expression of the ALS genes in Saccharomyces cerevisiae. Microarray analysis of the C. albicans als/als mutant strains suggested mechanisms by which the proteins function in their non-adhesive roles. Additional experimentation is underway to investigate these mechanisms and understand the scope of function of this family of proteins.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses uses gene families in disease processes.
Publications
- Green, C.B., S.L. Manfra, G. Cheng, F.F. Faddoul, E.J. Ehrhart, and L.L. Hoyer. 2006. RT-PCR analysis of Candida albicans ALS gene expression in a hyposalivatory rat model of oral candidiasis and in HIV-positive human patients. Medical Mycology 44:103-111.
- Zhao, X., K. Daniels, C.B. Green, S.-H. Oh, D.R. Soll, and L.L. Hoyer. 2006. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology 152:2287-2299.
|
Progress 10/01/03 to 09/30/04
Outputs
During the past year, we continued our analysis of gene expression for the Candida albicans ALS family. We found that expression of some genes in the family is characterized by large increases and decreases in transcript level while others are expressed at a relatively low level. Analysis of ALS gene expression in clinical specimens and disease models showed host-site-specific effects. For example, in clinical material from the human oral cavity, expression of ALS6 and ALS7 was most difficult to detect. This conclusion held true in the model systems or oral disease that were investigated. In clinical material from the human vagina, expression of ALS4 and ALS5 were most difficult to detect, a trend that was also preserved in two vaginal disease models. We extended our work on ALS gene expression by also developing a real-time RT-PCR assay to quantitate gene expression levels and further characterize the trends that were identified using a non-quantitative approach.
Analysis of C. albicans strains, each mutant in one of the ALS genes, included phenotypic assays to evaluate hyphal formation, adhesion to various host cell types and molecules, virulence in disease models, and biofilm formation. Mutant strains were also characterized using microarray analysis. The adhesive function of the highly expressed genes was demonstrated easily while function of proteins from genes with lower expression levels required alternative approaches. These phenotypic assays also suggested novel roles for Als proteins that will be investigated in the coming year.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses gene families in disease processes.
Publications
- Green, C.B., G. Cheng, J. Chandra, P.K. Mukherjee, M.A. Ghannoum, and L.L. Hoyer. 2004. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms. Microbiology 150:267-275.
- Zhao, X., S. -H. Oh, G. Cheng, C.B. Green, J.A. Nuessen, K. Yeater, R.P. Leng, A.J.P. Brown and L.L. Hoyer. 2004. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p. Microbiology 150:2415-2428.
- Cheng, G., K. Wozniak, M.A. Wallig, P.L. Fidel Jr., S.R. Trupin, and L.L. Hoyer. 2004. Comparison of Candida albicans ALS gene expression in human clinical specimens and models of vaginal candidiasis. Infection and Immunity (In press).
- Green, C.B., X. Zhao, and L.L. Hoyer. 2004. Use of green fluorescent protein and RT-PCR to monitor Candida albicans ALS gene expression in a murine model of disseminated candidiasis. Infection and Immunity (In press).
- Oh, S.- H., G. Cheng, J.A. Nuessen, R. Jajko, K.M. Yeater, X. Zhao, C. Pujol, D.R. Soll, and L.L. Hoyer. 2004. Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology (In press).
- Green, C.B., X. Zhao, and L.L. Hoyer. 2004. Construction and real-time RT-PCR validation of Candida albicans PALS-GFP reporter strains and their use in flow cytometry analysis of ALS gene expression in budding and filamenting Candida albicans GFP reporter strains. Microbiology (In press).
|
Progress 10/01/02 to 09/30/03
Outputs
During the past year, we continued our functional analysis of proteins produced by Candida albicans ALS genes. Our main approach to this analysis was creation of a set of mutant strains, each lacking one ALS gene. Functional analyses revealed differences between the various Als proteins, even those with more closely related sequences. We also determined that alleles of the same ALS gene can produce proteins that have a different readout in functional assays. This difference appears to be due to the overall length of the protein produced. Because of this allele effect, we determined the allelic genotype of each ALS gene in a large collection of C. albicans isolates that represent the various geographic regions of the world. This analysis indicated which alleles are representative of the overall population of C. albicans strains and which are more rare. From this information, we are selecting specific alleles for further analysis.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses gene families in disease processes.
Publications
- No publications reported this period
|
Progress 10/01/01 to 09/30/02
Outputs
During the past year, we have optimized a reverse transcriptase polymerase chain reaction (RT-PCR) assay to assess expression of genes in the Candida albicans ALS family. The assay was applied to cultured cells, clinical specimens and organisms derived from disease models. We found that a subset of genes in the family is more readily detected than others, but that under most conditions, all of the genes in the family are expressed. We have also constructed strains mutant in specific ALS genes and began our phenotypic analysis of these organisms. These studies are beginning to reveal variation in function between the different Als proteins. Our mutational analysis has also confirmed that there are eight genes in the ALS family, rather than the nine loci that had been implicated previously from results obtained by other groups.
Impacts
This work provides more insight into a set of genes that is involved in the pathogenesis of the fungal pathogen, Candida albicans. This work is also providing a comparative analysis of how C. albicans uses gene families in disease processes.
Publications
- No publications reported this period
|
Progress 10/01/00 to 09/30/01
Outputs
For reason of confidentiality associated with this work, significant findings and accomplishments are not described but work is continuing.
Impacts
(N/A)
Publications
- No publications reported this period
|
Progress 10/01/99 to 09/30/00
Outputs
Progress continues to be made on in vitro and in vivo analysis of the genes and their protein products.
Impacts
(N/A)
Publications
- Kapteyn, J.C., L.L.Hoyer, J.E. Hecht, W.H. Muller, A. Andel, A.J. Verkleij, M. Markarow, H. Van Den Ende, and F.M. Klis. 2000. The cell wall architecture of Candida albicans wild-type cells and cell-wall-defective mutants. Molecular Microbiology 35:601-611.
- Hoyer, L.L. and J.E. Hecht. 2000. The ALS6 and ALS7 genes of Candida albicans. Yeast 16:847-855.
|
Progress 10/01/98 to 09/30/99
Outputs
The goal of this project is to characterize ALS genes from the pathogenic fungus Candida albicans, and to define the function of Als proteins. During the past year, more ALS genes were cloned and characterized. The ALS family now includes nine genes. Als proteins were used to verify that the structure of the C. albicans cell wall largely conforms to the accepted model of the S. cerevisiae cell wall. Als proteins extracted from the cell wall with beta-1,6-glucanase migrate at nearly triple their predicted size due mainly to the presence of abundant N- and O-linked carbohydrate on the protein. During the past year, we created a set of C. albicans strains that overexpress Als proteins from a high-level constitutive promoter. These strains were used to confirm other data which suggest that there is a link between Als proteins and cell-surface hydrophobicity in C. albicans.
Impacts
(N/A)
Publications
- Hoyer LL, Clevenger J, Hecht JE, Ehrhart EJ, Poulet FM. 1999. Detection of Als proteins on the cell wall of Candida albicans in murine tissues. Infect. Immun. 67:4251-4255.
- Kapteyn JC, Hoyer LL, Hecht JE, Muller WH, Andel A, Verkleij AJ, Makarow M, Van Den Ende H, Klis FM. 2000. The cell wall architecture of Candida albicans wild-type cells and cell-wall-defective mutants. Mol. Microbiol. (In press).
|
Progress 10/01/97 to 09/30/98
Outputs
The goal of this project is to characterize ALS genes from the pathogenic fungus Candida albicans, and to define the function of ALS proteins. During the past year, several more ALS genes were cloned and characterized. These new genes were found to conform to the basic three-domain structure of ALS genes which has been described previously. Southern blotting evidence demonstrated that additional, uncharacterized ALS genes are present in the C. albicans genome. Biochemical characterization of the mature ALS proteins demonstrated the presence of N- and O-linked glycosylation, a feature that was predicted from analysis of the ALS protein sequence. ALS protein products were localized to the C. albicans cell surface using indirect immunofluorescence techniques. Additional studies, which investigated ALS protein production in a murine model of disseminated candidiasis, demonstrated that ALS proteins are found in the C. albicans cell wall. Immunohistochemical staining of
tissues from mice with disseminated candidiasis showed that ALS proteins are produced in a diversity of body sites during infection. Expression of ALS proteins in the non-adherent yeast Saccharomyces cerevisiae conferred adhesion to cultured human cells supporting our hypothesis that ALS proteins function in adhesion of C. albicans to host tissue.
Impacts
(N/A)
Publications
- Hoyer, L.L., Payne, T.L., Bell, M., Myers, A.M., Scherer, S. 1998. Candida albicans ALS3 and insights into the nature of the ALS gene family. CURR. GENET. 33:451-459.
- Hoyer, L.L., Payne, T.L., Hecht, J.E. 1998. Identification of Candida albicans ALS2 and ALS4 and localization of ALS proteins to the fungal cell surface. J. BACTERIOL. 180:5334-5343.
|
Progress 01/01/97 to 12/31/97
Outputs
The goal of this project was to define the ALS gene family from the pathogenic fungus Candida albicans, which encodes cell-surface glycoproteins that are potentially involved in adhesion to host tissue. DNA fragments originally identified as encoding genes in the ALS family were characterized from Candida albicans strain 1161. These fragments encoded four distinct genes (ALS1, ALS2, ALS3 and ALS4) which all share a common central domain consisting of varying numbers of copies of a 108-bp tandemly repeated motif. Sequences 5' of this repeat domain were approximately 85% identical in each gene while sequences 3' of the repeat domain were unique. Each gene predicted an amino acid sequence with hallmarks of a cell-surface glycoprotein. The four ALS genes were found in all laboratory and clinical isolates of C. albicans examined. Unique probes were developed for each ALS gene; these were used to demonstrate that the genes were dispersed in the genome on chromosomes 6 and
R. Unique probes were also used in Northern blot analysis to demonstrate that ALS genes were differentially regulated by physiologically relevant mechanisms including cell morphology and growth stage. Probing genomic Southern blots with common sequences from 5' of the tandem repeat domain revealed additional hybridizing fragments that suggest the ALS family is larger than originally estimated.
Impacts
(N/A)
Publications
- No publications reported this period
|
|