Rat monoclonal yeast killer toxin (KT)-like immunoglobulin M (IgM) anti-idiotypic antibodies (KT-IdAbs) were produced by idiotypic vaccination with a mouse monoclonal antibody (MAb; MAb KT4) that neutralized a Pichia anomala KT characterized by a wide spectrum of antimicrobial activity. The characteristics of the KT-IdAbs were demonstrated by their capacity to compete with the KT to the idiotype of MAb KT4 and to interact with putative KT cell wall receptors (KTRs) of sensitive Candida albicans cells. The internal-image properties of KT-IdAbs were proven by their killer activity against KT-sensitive yeasts. This lethal effect was abolished by prior adsorption of KT-IdAbs with MAb KT4. These findings stressed the potential importance of antibody-mediated immunoprotection against candidiasis and suggested a feasible experimental approach for producing antimicrobial receptor antibodies without purifying the receptor. KT-IdAbs might represent the basis for producing engineered derivatives with a high potential for effective therapeutic antifungal activity. MATERIALS AND METHODS Strains. P. anomala ATCC 96603 (formerly defined as UP 25F) was used for KT production. P. anomala UM3 and C. albicans UP 10, two yeast isolates known to be sensitive to the activity of the P. anomala KT, were also used in this study. These yeasts were subcultured on Sabouraud dextrose agar plates (Difco Laboratories, Detroit, Mich.) and are maintained in sterile distilled water in our fungus collection. KT production. KT was produced by a previously described procedure (21). Briefly, P. anomala ATCC 96603 was grown for 24 h at 25ЊC in Sabouraud broth (Difco) buffered at pH 4.6 with 0.1 M citric acid and 0.2 M sodium phosphate. The supernatant was filtered and concentrated 50 times with Minicon B15 concentrators (Amicon Division, W. R. Grace & Co., Beverly, Mass.). The concentrated KT was tested for killer activity against a recognized sensitive strain by conventional well assay (33) and was refrigerated (4ЊC) until it was used. Animals. Fischer-344 rats (CPA Rot, Roquemaure, France) were used in this study to produce KT-IdAbs. Immunogen. An immunoglobulin G1 (IgG1) MAb (MAb KT4) which neutralized the activity of P. anomala KT against recognized KT-sensitive strains of C. albicans and which was produced by standard procedures as described previ

The expression of Candida albicans antigenic determinants reacting with secretory IgA from patients with oral and vaginal candidiasis was investigated under different in vitro conditions. Reversible antigenic transitions were inducible in synthetic medium by temperature shifts, as the yeast cells were positive by an indirect immunofluorescence assay after being incubated at 37 degrees C but not at 25 degrees C. In vitro temperature-inducible C. albicans antigenic determinants reactive with secretory IgA were characterized by radioimmune Western blot as mannoproteins with molecular masses of 180-200, 130-150, 90-110, and 60-70 kDa. This is the first report on the expression of mannoproteins regulated by temperature involved in the secretory immune response during mucosal candidiasis.

The distribution of p-1,2-linked oligomannosides among glycoconjugates of various Candida species was investigated by Western blotting, using monoclonal and polyclonal antibodies which react with these epitopes. Expression of b1,2-linked oligomannosidic epitopes on a 14-18 kDa polydisperse antigen nonreactive with concanavalin A (ConA), previously identified as a C. albicans serotype A phospholipomannan (PLM), appeared to be restricted to C. albicans serotypes A and B (including var. C. stellatoidea types I and II) and C. tropicalis. In C. albicans, p1,2-linked oligomannosidic epitopes also appeared to be slightly associated with high molecular mass (> 100 kDa) polydisperse ConA-reactive mannoproteins. For all the other Candida strains investigated, belonging to the species C. parapsilosis, C. kmsei, C. glabrata and C. robusta (4 cemwisiae), &1,2=linked oligomannosidic epitopes were found to be present in association with medium molecular mass (18-1 00 kDa) and high molecular mass ConA-reactive mannoproteins, giving reproducible labelling profiles that varied between species.

A monoclonal antibody specific for /?-1,2-linked oligomannosides was used to study the association of these residues with Candida albicans mannan and phospholipomannan (PLM) in relation to growth conditions and in mannan mutant strains. Double immunof luorescence assays performed on cells grown under standard conditions indicated a highly heterogeneous cell surface expression of these epitopes in comparison with the homogeneous expression of a-linked oligomannosidic epitopes. Growth in the presence of tunicamycin, which inhibits mannan N-glycosylation, resulted in an absence of / l -1, 2-oligomannosidic epitopes on the cell surface, although PLM synthesis still occurred as shown by autoradiography. Similarly, growth in acidic conditions, which inhibits the incorporation of /l-l,2-oligomannosides in mannan, resulted in an absence of ~-1,2-oligomannosidic epitopes a t the cell surface, although they still associated with PLM as shown by Western blotting. Western blots of C. albicans mutant strains with reduced amounts or an absence of phosphorus and acid-labile B-1,2-oligomannosides in their mannan confirmed that the association of /l-1,2-linked oligomannosides with mannan and with PLM involves different mannosylation processes.Keywords : p-1,2-oligomannosidic epitopes, mannan, phospholipomannan, pmannosylation processes INTRODUCTIONHomopolymers of p-1,2-linked oligomannosides were initially described in Candida albicans cell wall phosphopeptidomannan (PPM), also called mannan, where they correspond to the antigenic factor 5 (Shibata e t al., 1992).Such P-1,2-linked oligomannosidic epitopes were further observed on a C. albicans 14-18 kDa phospholipomannan (PLM) (Trinel e t al., 1992), a glycolipid that induced TNFa production by macrophages (jouault e t al., 1994). Mapping of these p-l,2-oligomannosidic epitopes among glycoconjugates of Candida species revealed that the presence of PLM was restricted to the most pathogenic species of the genus Candida, C. albicans and C. tropicalis (Cantelli e t al., 1995) and also demonstrated that in other t Present address: Facolta di Medecina e Chirurgia, lstituto di Microbiologia, Via Gramsci 14, 43100 Parma, Italy.Abbreviations: AERC, alkaline extraction in reducing conditions; ConA, concanavalin A; IFA, immunofluorescence assay; PLM, phospholipomannan; PPM, phosphopeptidomannan; TNF, tumour necrosis factor.Candida species, such as C. parapsilosis, C. krusei and C. glabrata, p-1,2-oligomannosidic epitopes were shared by numerous mannoproteins according to species-specific profiles.From a physiopathological point of view, recent observations increasingly suggest that P-1,2-oligomannosides are important molecules involved in the host-Candida interplay during pathogenic processes. P-1,2-0ligo-mannosides have been shown to bind to host cell membranes (Li & Cutler, 1993), to trigger the cytokine network (Jouault e t al., 1995) and elicit an antibody response different from those supported by a-Man residues (Poulain e t al., 1993) during candidosis. In contrast to these studies,...

The interaction of the killer yeast Pichia anomala UP 25F with the killer toxin-sensitive clinical isolate Candida albicans UCSC 10S and its natural toxin-resistant mutant derivative C. albicans UCSC 10R were studied under various conditions. A differential inhibition was shown to occur in vitro at pH and temperature values, which are not encountered in vivo, only by using preformed killer toxin, since antagonism due to yeast growth proved to be predominant on the killer effect. Under adverse growth conditions, the P. anomala killer yeast proved to be able to produce an anatoxin antigenically related to the active or heat inactivated killer toxin. These findings suggest that killer toxins may not function as potential virulence factors in the competition between the opportunistic killer yeast P. anomala and sensitive microorganisms for colonization in the course of natural human infections.