Phenotypic and Cell Wall Proteomic Characterization of a DDR48 Mutant Candida albicans Strain
1Department of Natural Sciences, Lebanese American University,Lebanon, 2Department of Chemistry, Technical University of Munich, Germany
J. Microbiol. Biotechnol. 2019; 29(11): 1806-1816
Published November 28, 2019
Copyright © The Korean Society for Microbiology and Biotechnology.
Infections caused by
Ddr48, a stress-associated protein on the cell wall, is composed of 212 amino acids and is among the major immunodominant antigens expressed during candidiasis . Ddr48 had been previously studied in our lab by generating a haploinsufficient
In this study, we aim to further elucidate the role of Ddr48 by subjecting our mutant strain to additional assays. Since Ddr48 is a cell wall protein we hypothesize that a mutant strain would have an impact on the proteomic constitution, architecture, and rigidity of the cell wall. Characterization was performed through classical phenotypic assays, and through a proteomic/bioinformatics approach; a method previously utilized by our lab to characterize
Materials and Methods
Media Preparation and Culture Conditions
Rich potato dextrose agar (PDA) medium (HiMedia, India) was used to grow strains during routine culturing and for oxidative stress tolerance, cell surface disruption resistance, temperature sensitivity, and adhesion experiments. PDA plates were supplemented with histidine and uracil throughout all phenotypic characterization experiments. For the biofilm assay, yeast nitrogen base (YNB) synthetic medium (Fluka, Switzerland) was supplemented with uracil and histidine. For routine growth to exponential phase for oxidative stress tolerance, cell surface disruption resistance, temperature sensitivity, and adhesion experiments, rich potato dextrose broth (PDB) liquid media (Hi Media, India) was used, and strains were incubated at 30°C under aerobic conditions.
For proteomic analysis, strains were grown in PDB media supplemented with histidine and uracil until exponential phase. For filamentous growth, PDB was supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C under aerobic conditions.
It is important to note that only proteins appearing in half of the extractions or having a minimum of 2% sequence coverage (equating to an average of 12 amino acids identified in the exact order) were considered successful hits . All peptide sequences identified by MASCOT but unassigned to any protein were BLAST searched on
Fig. 1. Methodology flowchart. A combinatory phenotypic and proteomic approach was utilized to achieve characterization of Ddr48.
The wild-type and mutant strains were subjected to a battery of tests at different concentrations. For each test a representative picture is shown in Fig. 2.
Fig. 2. Resistance to stress. (
A) Control, untreated cells. ( B) 10 mM hydrogen peroxide. ( C) 0.5 mM menadione. ( D) 2 mM diamide. ( E) 0.05% SDS. ( F) 10 mg/ml NaCl. ( G) Heat shock at 42°C. ( H) 100 µg/ml Congo red. ( I) 100 µg/ml calcofluor white. Note that the mutant strain is more sensitive to oxidative stress agents, osmotic stress, and SDS than the wild type whereas the revertant strain restored growth under these stresses, and that the mutant and wild-type strains can equally tolerate heat shock, Congo red, and calcofluor white.
Fig. 3. Pathogenicity related attributes. No significant difference between the mutant and wild type strains was shown as far as ability to form cell wall chitin (left column,
p-value = 0.1404). Our mutant however was slightly defective in adhesion with a significant 10% reduction in adhesion to human epithelial cells (right column, p-value = 0.0002). The decrease in adhesion was mirrored by a significant 15% decrease in biofilm production seen as a decrease in crystal violet absorbance at 590 nm (middle column, p-value = 0.03). Mutant values for chitin content are expressed as percentage of the wild-type strain. All experiments were performed in triplicates with error bars representing the average along with the -/+ SEM.
Fig. 4. Virulence test. For each strain, six mice were intravenously injected in the tail with
Candida albicansand survival was monitored for 15 days. Both strains were able to cause disseminated candidiasis and kill mice within 2 weeks of infection, however, the mutant exhibited a slight-but-not-statistically-significant delay in causing death ( p-value = 0.4).
The cell wall proteome profiles of the mutant and the wild-type strains were compared in order to detect differentially expressed CWPs responsible for the previously described phenotypes. It is important to note that in our study, the main focus is on the proteins exclusive to the wild type and lacking in the mutant strain since the latter was found to be more attenuated in our phenotypic studies.
This current study aimed at further elucidating the role of Ddr48 in
The wild type and mutant were also subjected to stresses induced by cell surface perturbing agents. The rationale behind such an experiment is that deletion of cell wall proteins has been previously shown to affect cell surface architecture and rigidity as in many instances it generates a less rigid cell wall, or alternatively the cell overcompensates for the deletion by turning on salvage pathways and increasing the chitin content resulting in an increasingly rigid cell wall leading to increased resistance to cell surface disrupting agents . We found that our mutant strain was slightly less resistant to SDS than the wild type and that the revertant strain showed restoration of SDS resistance. This implies that Ddr48 is involved in protection against cell wall stress. Both strains however were equally sensitive to calcofluor white and Congo red. These agents have distinct modes of action since SDS affects and solubilizes the plasma membrane proteins , whereas Congo red and calcofluor white act on β-glucans and chitin fiber assembly respectively [36, 37]. A recent study showed that
In our study, we also tested both strains for their tolerance to heat shock with no discernable differences observed between both strains implying that Ddr48 is not required for thermal resistance. The ability of both strains to form biofilm was also assessed. We found the mutant strain to be slightly deficient in biofilm formation. Knowing that adhesion is an essential step in biofilm formation , we also performed an adhesion assay on human epithelial cells. As expected, the mutant strain mirrored the biofilm data and showed a significant drop in adhesion as well. This is in line with the observation of Cleary et al.  that a ddr48 deficient strain fails to self-aggregate and flocculate properly. We thus confirm that Ddr48 affects adhesion and biofilm formation in
Moreover, virulence was assayed through a murine model of disseminated candidiasis. All infected mice died within two weeks post intravenous injection, with a slight yet statistically insignificant delay in the mutant strain. This shows that Ddr48 does not play a significant role in virulence. The slight drop in biofilm formation, adhesion, and tolerance to stresses mentioned above was not enough to impart an attenuated virulence trait to the mutant.
We then proceeded to analyze the cell surface proteome of the mutant and wild-type strains to determine possible changes in the cell surface proteome that can explain the observed phenotypic results. It is interesting to note that the number of differentially detected proteins observed was relatively low compared to other studies with similar techniques [23–25]. This makes sense in fact and can be explained by the fact that our mutant phenotypes were not severe. It is noteworthy in this proteomic approach that the lack of detection of a particular protein in a sample does not necessarily mean that the protein is absent or not expressed. The protein could still be present in the sample but at an insufficiently low concentration for it to be successfully detected. We performed eight independent cell wall extractions for each strain.
Three proteins detected exclusively in the wild-type strain have functions in combating oxidative stress: Sod4, Sod6, and Vma2. Sod4 and Sod6 are superoxide dismutases that function in converting the damaging superoxide radicals generated by the host’s macrophages and neutrophils into less harmful hydrogen peroxide that can be later transformed into water by catalases . Vma2, a subunit of the vacuolar ATPase complex involved in the acidification of intracellular organelles, is needed for the resistance of
The extent of cell wall elasticity and integrity reflects the ability of
Biofilm formation is a multi-step mechanism in which various proteins and factors are recruited. Als3 is a cell surface adhesin that has a major role in yeast aggregation and consequently in biofilm formation . Hsp90 is a heat shock protein that is a prime regulator of antifungal tolerance and dispersion of
In addition to the above-mentioned proteins, Eft2 and Tim21 were solely detected in the wild-type strain. However, these proteins are ribosomal and mitochondrial proteins respectively as referenced in
In this study, we further characterized the
We would like to thank Dr. Brigitte Wex Dr. Georges Khazen, and Dr. Rony Khnayzer for their helpful advice and information.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
- Sorgo AG, Heilmann CJ, Dekker HL, Brul S. 2010. Mass spectrometric analysis of the secretome of
Candida albicans. Yeast 27: 661-672.
- Southern P, Horbul J, Maher D, Davis DA. 2008.
C. albicanscolonization of human mucosal surfaces. PLoS One 3: e2067.
- Viudes A, Pemán J, Cantón E, Úbeda P. 2002. Candidemia at a tertiary-care hospital: epidemiology, treatment, clinical outcome and risk factors for death.
Eur. J. Clin. Microbiol. Infect. Dis. 21: 767-774.
- Martins N, Ferreira IC, Barros L, Silva S. 2014. Candidiasis:predisposing factors, prevention, diagnosis and alternative treatment.
Mycopathologia 177: 223-240.
- Barada G, Basma R, Khalaf RA. 2008. Microsatellite DNA genotyping and identification of
Candida albicansfrom Lebanese clinical isolates. Mycopathologia 165: 115-125.
- Yazbek S, Barada G, Basma R, Mahfouz J. 2007. Significant discrepancy between real-time PCR identification and hospital identification of C.
albicans from Lebanese patients. Med. Sci. Monit. 13: MT7-MT12.
- Pfaller MA, Diekema DJ. 2007. Epidemiology of invasive candidiasis: a persistent public health problem.
Clin. Microbiol. Rev. 20: 133-163.
- Cheng S, Clancy CJ, Checkley MA, Handfield M. 2003. Identification of
Candida albicansgenes induced during thrush offers insight into pathogenesis. Mol. Microbiol. 48: 1275-1288.
- Mayer FL, Wilson D, Hube B. 2013.
Candida albicanspathogenicity mechanisms. Virulence 4: 119-28.
- Jacobsen ID, Wilson D, Wächtler B, Brunke S. 2012.
Candida albicansdimorphism as a therapeutic target. Expert. Rev. Anti. Infect. Ther. 10: 85-93.
- Brown AJ, Odds FC, Gow NA. 2007. Infection-related gene expression in
Candida albicans. Curr. Opin. Microbiol. 10: 307313.
- Sudbery PE. 2011. Growth of
Candida albicanshyphae. Nat. Rev. Microbiol. 9: 737-748.
- Gow NA, Latge J, Munro CA. 2017. The fungal cell wall:structure, biosynthesis, and function.
Microbiol. Spectr. 5(3).
- Masuoka J. 2004. Surface glycans of
Candida albicansand other pathogenic fungi: physiological roles, clinical uses, and experimental challenges. Clin. Microbiol. Rev. 17: 281-310.
- Tronchin G, Poulain D, Herbaut J, Biguet J. 1981. Localization of chitin in the cell wall of
Candida albicansby means of wheat germ agglutinin. Fluorescence and ultrastructural studies. Eur. J. Cell. Biol. 26: 121-128.
- Chaffin WL. 2008.
Candida albicanscell wall proteins. Microbiol. Mol. Biol. Rev. 72: 495-544.
- De Groot PW, Hellingwerf KJ, Klis FM. 2003. Genome-wide identification of fungal GPI proteins.
Yeast 20: 781-796.
- Erwig LP, Gow NA. 2016. Interactions of fungal pathogens with phagocytes.
Nat. Rev. Microbiol. 14: 163-176.
- Bitar I, Khalaf RA, Harastani H, Tokajian. 2014. Identification, typing, antifungal resistance profile, and biofilm formation of
Candida albicansisolates from Lebanese hospital patients. Biomed. Res. Int.931372.
- Thomas DP, Viudes A, Monteagudo C, Lazzell AL. 2006. A proteomic-based approach for the identification of
Candida albicansprotein components present in a subunit vaccine that protects against disseminated candidiasis. Proteomics 6: 6033-6041.
- Dib L, Hayek P, Sadek H, Beyrouthy B. 2008. The
Candida albicansDdr48 protein is essential for filamentation, stress response, and confers partial antifungal drug resistance. Med. Sci. Monit. 14: BR113-BR21.
- Cleary IA, M acGregor NB, S aville S P, T homas DP. 2012. Investigating the function of Ddr48p in
Candida albicans. Eukaryot Cell 11: 718-724.
- Awad A, El Khoury P, Wex B, Khalaf RA. 2018. Proteomic analysis of a
Candida albicans pga1null strain. EuPA Open Proteom. 18: 1-6.
- Awad A, El Khoury P, Wex B, Khalaf RA. 2018. Tandem mass spectrometric cell wall proteome profiling of a
Candida albicans hwp2mutant strain. Curr. Mol. Pharmacol. 11: 211225.
- El Khoury P, Awad A, Wex B, Khalaf RA. 2018. Proteomic analysis of a
Candida albicans pir32null strain reveals proteins involved in adhesion, filamentation and virulence. PLoS One 3: e0194403.
- Zohbi R, Wex B, Khalaf RA. 2014. Comparative proteomic analysis of a
Candida albicans DSE1mutant under filamentous and non-filamentous conditions. Yeast 31: 441-448.
- Pedreño Y, González-Párraga P, Martínez-Esparza M, Sentandreu R. 2007. Disruption of the
Candida albicansATC1 gene encoding a cell-linked acid trehalase decreases hypha formation and infectivity without affecting resistance to oxidative stress. Microbiology 153: 1372-1381.
- Bahnan W, Koussa J, Younes S, Abi Rizk M. 2012. Deletion of the
Candida albicans PIR32results in increased virulence, stress response, and upregulation of cell wall chitin deposition. Mycopathologia 174: 107-119.
- Plaine A, Walker L, Da Costa G, Mora-Montes HM. 2008. Functional analysis of
Candida albicansGPI-anchored proteins:roles in cell wall integrity and caspofungin sensitivity. Fungal Genet. Biol. 45: 1404-1414.
- Peeters E, Nelis HJ, Coenye T. 2008. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.
J. Microbiol. Methods 72: 157-65.
- Tsuchimori N, Sharkey LL, Fonzi WA, French SW. 2000. Reduced virulence of
HWP1-deficient mutants of Candida albicansand their interactions with host cells. Infect. Immun. 68: 1997-2002.
- Daher JY, Koussa J, Younes S, Khalaf RA. 2011. The
Candida albicansDse1 protein is essential and plays a role in cell wall rigidity, biofilm formation, and virulence. Interdiscip. Perspect. Infect. Dis.504280.
- Munro CA, Whitton RK, Hughes HB, Rella M. 2003. CHS8-a fourth chitin synthase gene of
Candida albicanscontributes to in vitro chitin synthase activity, but is dispensable for growth. Fungal. Genet. Biol. 40: 146-158.
- Cabezón V, Llama-Palacios A, Nombela C, Monteoliva L. 2009. Analysis of
Candida albicansplasma membrane proteome. Proteomics. 9: 4770-4786.
- Barrett J, Brophy PM, Hamilton JV. 2005. Analysing proteomic data.
Int. J. Parasitol. 35: 543-553.
- Brasch J, Kreiselmaier I, Christophers E. 2003. Inhibition of dermatophytes by optical brighteners.
Mycoses 46: 120-125.
- Nodet P, Capellano A, Fevre M. 1990. Morphogenetic effects of Congo red on hyphal growth and cell wall development of the fungus
Saprolegnia monoica. J. Gen. Microbiol. 136: 303310.
- Ene I, Walker LA, Schiavone M, Lee KK. 2015. Cell wall remodeling enzymes modulate fungal cell wall elasticity and osmotic stress resistance.
MBio 6: e00986-15.
- Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M. 2010. Dispersion as an important step in the
Candida albicansbiofilm developmental cycle. PLoS Pathog. 6: e1000828.
- Frohner IE, Bourgeois C, Yatsyk K, Majer O. 2009.
Candida albicanscell surface superoxide dismutases degrade hostderived reactive oxygen species to escape innate immune surveillance. Mol. Microbiol. 71: 240-252.
- Rane HS, Bernardo SM, Hayek SR, Binder JL. 2014. The contribution of
Candida albicansvacuolar ATPase subunit V 1 B, encoded by VMA2, to stress response, autophagy, and virulence is independent of environmental pH. Eukaryot. Cell 13: 1207-1221.
- Alberti-Sequi C, Morales AJ, Xing H, Kessler MM. 2004. Identification of potential cell-surface proteins in
Candida albicansand investigation of the role of a putative cellsurface glycosidase in adhesion and virulence. Yeast 21: 285302.
- Spreghini E, Davis DA, Subaran R, Kim M. 2003. Roles of
Candida albicansDfg5p and Dcw1p cell surface proteins in growth and hypha formation. Eukaryot. Cell. 2: 746-755.
- Klotz SA, Gaur NK, De Armond R, Sheppard D. 2007.
Candida albicansAls proteins mediate aggregation with bacteria and yeasts. Med. Mycol. 45: 363-370.
- Nobile CJ, Fox EP, Nett JE, Sorrells TR. 2012. A recently evolved transcriptional network controls biofilm development in
Candida albicans. Cell 148: 126-138.
- Nobile CJ, Andes DR, Nett JE, Smith FJ. 2006. Critical role of Bcr1-dependent adhesins in
C. albicansbiofilm formation in vitro and in vivo. PLoS Pathog. 2: e63.
- Sentandreu M, Elorza MV, Sentandreu R, Fonzi WA. 1998. Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of
Candida albicans. J. Bacteriol. 180: 282-289.
- Peltroche-Llacsahuanga H, Goyard S, D'Enfert C, Prill SK. 2006. Protein O-mannosyltransferase isoforms regulate biofilm formation in
Candida albicans. Antimicrob. Agents. Chemother. 50: 3488-3491.
- Cornet M, Richard ML, Gaillardin C. 2009. The homologue of the
Saccharomyces cerevisiaeRIM9 gene is required for ambient pH signalling in Candida albicans. Res. Microbiol. 160: 219-223.
- Bassilana M, Blyth J, Arkowitz RA. 2003. Cdc24, the GDPGTP exchange factor for Cdc42, is required for invasive hyphal growth of
Candida albicans. Eukaryot. Cell 2: 9-18.
- Almeida RS, Brunke S, Albrecht A, Thewes S. 2008. The hyphal-associated adhesin and invasin Als3 of
Candida albicansmediates iron acquisition from host ferritin. PLoS Pathog. 4: e1000217.