Characterization of Candida albicans cell wall antigens with monoclonal antibodies

Abstract

The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI).

Cell Wall and Secreted Proteins ofCandida albicans: Identification, Function, and Expression

“…There is a growing body of experimental evidence indicating that the properties-expression, distribution, and chemical characteristics-of cell wall proteins and glycoproteins observed in vitro and in vivo are dependent on multiple factors. These include growth conditions, organism-related factors (such as growth state, morphology of the cells, strain and serotype, phenotypic switching, cell surface hydrophobic or hydrophilic status), and the nature of the biological specimens (intact cells or isolated wall preparations) that are subjected to analysis (5,7,24,44,57,63,106,159,190,191,195,210,284,297,301,326,416,422,477,513,529). Iron availability, which has been shown to be important for pathogens in establishing infection (46,400,566), affects the cell surface (529).…”

“…On the other hand, a protein that is not detected at the cell surface by indirect immunofluorescence but is present in cell wall extracts is located only in the interior of the wall (8). Using a panel of MAbs to localize proteins, Pontón et al (416) demonstrated various distribution classes of cell wall proteins: (i) expressed only on the germ tube surface, (ii) expressed on the germ tube surface and within the yeast cell wall, (iii) expressed on both yeast cell and germ tube surfaces, and (iv) expressed within the wall of both germ tubes and yeast cells. A fifth category, expressed only on yeast surfaces, is also reported (296).…”

“…Since the cell wall is the structure ultimately responsible for shape, and since the final steps of cell wall assemblage occur externally to the plasma membrane, the research efforts of a number of investigators in different laboratories have concentrated in the study of this morphogenetic event, as well as on the characterization of cell wall components (especially proteins and mannoproteins) that are growth phase specific. These studies have been fueled by four important considerations: (i) the interest in morphogenesis itself as an important biological phenomenon, (ii) the commonly postulated relationship between growth in the mycelial form and pathogenicity, (iii) the coordination between expression of adhesion factors and germ tube formation, and (iv) the idea that characterization of mycelium-specific antigens would provide the basis for the development of improved serodiagnostic test for the detection of invasive candidiasis (50,64,79,96,209,352,416,418,453,480).…”

Recognition ofCandida albicansAls3 by the germ tube-specific monoclonal antibody 3D9.3

“…These antibodies were most frequently directed toward carbohydrates carried by cell wall proteins. Among these antibodies, three MAbs have been demonstrated by indirect immunofluorescence assay (IFA) to only recognize C. albicans germ tubes and hyphae (Ollert & Calderone, 1990; Marot‐Leblond et al , 1993, 1995, 2000; Ponton et al , 1993). Molecular genetic approaches have identified genes such as ALS3, HWP1 and HYR1 that encode hypha‐specific surface proteins that may contribute to differences in cell wall structure and functions (Bailey et al , 1996; Hoyer et al , 1998; Staab & Sundstrom, 1998).…”

Differentiation of Candida strains by lectin‐mediated agglutination kinetics

“…A variety of differentiation methods exist to type yeast strains. These are phenotypical differentiation techniques of yeast strains such as serotyping [4], morphological typing [5, 6], typing by assessment of resistance toward antimycotic agents or chemical substances [7, 8]. Typing by use of killer yeasts also belongs with the phenotypical methods [9, 10].…”