A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

The C-terminal antibody binding domain of Candida albicans mp58 represents a protective epitope during candidiasis

The 58-kDa surface mannoprotein of Candida albicans (mp58) elicits strong antibody responses during infection. Epitope mapping with sera from patients with candidiasis and control individuals indicated the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. These immunoreactive regions were similar to the ones previously identified using sera from immunized animals. Two of the epitopic regions (including the C-terminal domain) showed increased reactivity with antibodies present in sera from patients with candidiasis as compared to control individuals. Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis. A monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematogenously disseminated candidiasis. Together, these observations indicate the carboxy-terminal antibody binding domain of C. albicans mp58 represents a protective epitope during candidiasis.     

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells.     

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

Immune system function in candidiasis patients

To compare the clinical picture and the immunological characteristics, 58 candidiasis patients differing by the severity and dissemination of the disease were examined. Chronic candidiasis of the skin and mucous membranes, the most severe and disseminated form of the disease, is associated with a decrease in the number of T-lymphocytes and changes in their subpopulations, as well as high titers of Candida albicans antigen and antibodies to it in blood sera. The immune system of patients with visceral candidiasis and chronic vulvovaginal candidiasis was similar to that of healthy persons in the characteristics under study. Immediate and mixed hypersensitivity occurred in candidiasis patients more frequently than in healthy persons. In extremely severe forms of Candida infection immediate hypersensitivity prevailed.     

Expression of the fibrinogen binding mannoprotein and the laminin receptor of Candida albicans in vitro and in infected tissues

Abstract We have previously reported a 37 kDa laminin-binding protein (p37) and a 58 kDa fibrinogen-binding mannoprotein (mp58) on the surface of Candida albicans . A few yeast cells expressed both functional receptors at the surface while germ tubes expressed a functional mp58 fibrinogen but not a functional p37 laminin receptor. These receptors were heterogeneously dispersed at the surface as shown by binding of rabbit antiserum to mp58 (PAb anti-mp58) and antiserum to the human high affinity laminin receptor. In this report we have used a dual fluorescence technique to determine if the two receptors colocalize, perhaps as part of a receptor complex. Fibrinogen was used as a probe for mp58 and polyclonal antiserum generated to the p37 (PAb anti-p37) was used as a probe for the 37 kDa laminin-binding protein. Both receptors were heterogeneously distributed, but the receptors were not colocalized as the areas of concentration of each receptor were different. Immunohistochemical analysis of tissue sections from patients with disseminated and superficial candidiasis with PAb anti-p37 and PAb anti-mp58 revealed that both receptors were also expressed in infected tissues. The patterns of morphological expression were similar to the in vitro patterns detected by immunofluorescence.     

Horizontal transmission of Candida albicans and evidence of a vaccine response in mice colonized with the fungus

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis.     

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2-DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.     

Amphotericin B-metronidazole combination against Candida spp

Oropharyngeal candidiasis (OPC) remains a common opportunistic infection in HIV-infected patients. Candida albicans is the most frequent causative agent of OPC. However, non-albicans spp. are being increasingly isolated. Candidal cell wall proteins and mannoproteins play important roles in the biology and patogenesis of candidiasis. In the present study, we have analyzed the proteinaceous components associated with cell wall extracts from C. albicans, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii and Candida rugosa obtained from HIV-infected patients with recurrent OPC. Cell wall proteinaceous components were extracted with beta-mercaptoethanol and analyzed using electrophoresis, immunoblotting (with antisera generated against C. albicans cell wall components, and with serum samples and oral saline rinses from patients with OPC), and lectin-blotting (concanavalin A) techniques. Numerous molecular species were solubilized from the various isolates. Major qualitative and quantitative differences in the polypeptidic and antigenic profiles associated with the cell wall extracts from the different Candida spp. were discernible. Some of the antibody preparations generated against C. albicans cell wall components were able to recognize homologous materials present in the extracts from non-albicans spp. Information on cell wall antigens of Candida species may be important in the therapy and prevention of HIV-related OPC.     

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.     

Defining criteria for anti-mannan antibodies to protect against candidiasis

Prevention of hematogenously disseminated candidiasis and mucocutaneous disease, including Candida vaginitis, through immunological approaches is appealing for the following reason. A long-acting and safe vaccine that protects against both serotypes of Candida albicans and other important species, such as C. tropicalis and C. glabrata, should significantly reduce the incidence of various forms of candidiasis by these etiologic agents. Through extensive experimentation on protective responses in experimental animals against Candida mannan components, others and we have evidence that antibodies specific for short-chain beta-linked oligomannosides are protective against candidiasis. Although the mechanism of protection against vaginal infection requires further investigation, experimentally the ability of antibody to rapidly deposit high amounts of complement factor C3 onto the yeast cell wall is requisite for enhancing resistance against disseminated candidiasis.     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to theC. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluo-rescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.     

Conversion of α1,2-mannosidase E-I from Candida albicans to α1,2-mannosidase E-II by limited proteolysis

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble α1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, α1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted α1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for α1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.     

Characterization with crossed immunoelectrophoresis of some antigens differentiating a virulent Candida albicans from its derived, avirulent strain

Antigenic differences between a wild-type virulent Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed immunoelectrophoresis. Yeast cell extracts as well as soluble protein and mannoprotein fractions obtained by affinity chromatography on concanavalin A (Con A) were analyzed. Sera from patients with candidiasis and antisera from rabbits infected with live wt cells and boosted with wt extracts or rabbits immunized with purified wt cell wall preparation were used as counter reactants. Qualitative differences in serum precipitins formed by patients with suspected or culture-proven candidiasis to polysaccharide antigens of wt and m-10 origin were observed. In comparison, except for a spike-formed precipitate detected only with the wt extract, the serum from infected rabbits precipitated the wt and m-10 cell wall polysaccharide antigens about equally. The same type of precipitate was also found with the Con A wt mannoprotein fractions but was again lacking with the m-10 mannoproteins. This precipitate, with extremely slow electromobility in the first dimension, may be related to some special immunodeterminant of the wt mannan molecule. No substantial differences in the precipitation patterns of the Con A wt and m-10 proteins were found when analyzed with patients' sera or rabbit anti-cell sera. However, using these protein fractions with anti-cell wall sera revealed a larger number of precipitates for the wt as opposed to the m-10 strain. The observed antigenic differences between the virulent- and the avirulent-derived strains seem to be mainly associated with cell wall determinants (components) and might be related to the greater adherence and infectivity of the wild strain.     

A proteomic-based approach for the identification of Candida albicans protein components present in a subunit vaccine that protects against disseminated candidiasis

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.     

Induction of salivary antibodies to inhibit Candida albicans adherence to human epithelial cells by tonsillar immunization in rabbits

To examine the possibility of a vaccine for Candida albicans infection in the oral cavity, we induced salivary antibodies by immunization of killed-C. albicans ATCC 18804 on the palatine tonsils of rabbits. The enzyme-linked immunosorbent assay reaction of salivary antibodies was high against C. albicans serotype A. The saliva antibodies greatly inhibited C. albicans adherence to cloned epithelial cells from human gingiva. Tonsillar immunizations of C. albicans ATCC 18804 induce salivary antibodies that prevent C. albicans adherence to epithelial cells, and thus should prove useful in the prevention of oral candidiasis caused by C. albicans serotype A.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.     

Effect of antigen used in the detection of anti -Candida albicans antibodies

Antigens of different origins were used in the investigation of anti-Candida albicans antibodies. This can influence the results obtained.We have assayed three different antigenic preparations weekly for 8 weeks in the study of anti-C. albicans antibodies induced by cutaneous, digestive, and systemic inoculations with C. Albicans ATCC 26555 in rabbits free of specific antibodies, and using indirect immunofluorescence (IIF), direct agglutination (DA) and counterimmunoelectrophoresis (CIE) as serological methods.In IIF and DA, two antigens were used (C. albicans ATCC 26555 and C. albicans NCPF 3153). In CIE we also used a third commercial antigen. All three somatic antigens were used at three different concentrations.Using IIF and DA the titres obtained with both antigens were similar in different inoculations. The IIF was somewhat earlier in the detection of antibodies, and the titre reached was higher when the antigen used was obtained from the inoculated strain.The detection of precipitins by CIE was in most cases only positive with the antigen obtained from the homologous strain, the highest level being reached in the systemic inoculation.     

Identification of protein and mannoprotein antigens of Candida albicans of relevance for the serodiagnosis of invasive candidiasis.

Antigens from Candida albicans blastoconidia and germ tubes were identified by two-dimensional electrophoresis and Western blotting and characterized by microsequencing, reactivity with concanavalin A, and a panel of human sera. Antigens identified included a polydispersed area in the acidic high-molecular-mass regions of blastoconidium and germ-tube extracts, and 16 antigens varying in molecular masses and isoelectric points (pIs). The majority of the detected antigens, especially those in the polydispersed region, showed mannosyl groups, as determined by concanavalin A reactivity. Antibodies present in sera from patients with invasive candidiasis showed high reactivity with a number of antigens not detected with sera from blood donors. Eight of the 16 antigens could be identified by reactivity with monoclonal antibodies or by microsequencing. Five antigens showed homology with five enzymes previously described as antigens in C. albicans: enolase, phosphoglycerate kinase, malate dehydrogenase, and two isoforms of the fructose biphosphate aldolase. However, to our knowledge, this is the first report of the immunogenic activity of a kexin precursor, a mitochondrial complex I chaperone, and a diacylglycerol kinase catalytic domain from C. albicans. Antigens described in this study may be of potential interest for the serodiagnosis of invasive candidiasis.