Sec20p-interacting proteins (Tip20p, Ufe1p) in the retrograde secretory pathway of the fungal pathogen Candida albicans.

Sec20p is an essential Type-II membrane protein of the human fungal pathogen Candida albicans, which is thought to be involved in mediating retrograde vesicle traffic from the Golgi to the endoplasmic reticulum (ER). Using an epitope-tagged Sec20p we obtained evidence for its localization in ER membranes, which is consistent with its proposed role in an ER-tSNARE complex. Two genes encoding potential interaction partners for Sec20p, Tip20p and Ufe1p, were identified in genomic sequences of C. albicans; these show 18% and 27% identity, respectively, to homologues in Saccharomyces cerevisiae. An interaction between the cytoplasmic domain of Sec20p and Tip20p was demonstrated by two-hybrid analysis; in addition, Tip20p was found to form homodimers. Interaction between Sec20p and Tip20p in vivo was verified by co-immunoprecipation experiments. CaUFE1, which encodes a potential ER-tSNARE, was able to complement a thermosensitive ufe1 mutation in S. cerevisiae, suggesting functional conservation between the two fungal proteins. Thus, although the sequences of some components of the ER-tSNARE complex have diverged considerably during evolution, it appears that they have retained similar functions in C. albicans and S. cerevisiae.

Th1/Th2 cytokine profiles in saliva of HIV-positive smokers with oropharyngeal candidiasis.

Oropharyngeal candidiasis (OPC) is a common opportunistic infection among HIV-positive individuals and often correlates with a CD4 cell number < 200 cells/microl. This study further examined the association of smoking and OPC in HIV-positive persons. A strong association between smoking and OPC was seen in HIV-positive individuals with > or =200 CD4 cells/microl. In HIV-positive persons with > or =200 CD4 cells/microl, OPC+ smokers had lower gamma-interferon (IFN-gamma) concentrations and a trend toward higher interleukin (IL)-4 concentrations in whole saliva compared to OPC- persons with > or =200 CD4 cells/microl, a cytokine profile consistent with that observed in HIV+OPC+ persons with < 200 CD4 cells/microl. These results suggest that premature OPC in HIV-positive smokers is associated with altered oral host defence mechanisms that cannot be overcome by levels of systemic CD4 cells that are otherwise sufficient to protect against OPC.

Potential role for a carbohydrate moiety in anti-Candida activity of human oral epithelial cells.

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety.

Divergence of eukaryotic secretory components: the Candida albicans homolog of the Saccharomyces cerevisiae ++Sec20 protein is N terminally truncated, and its levels determine antifungal drug resistance and growth.

Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20 alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3p and PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20 strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.

Candida-specific systemic cell-mediated immune reactivities in human immunodeficiency virus-positive persons with mucosal candidiasis.

Oropharyngeal candidiasis (OPC), as opposed to vulvovaginal candidiasis (VVC), is a common opportunistic infection in human immunodeficiency virus (HIV)-positive persons that correlates with reduced CD4 T cell counts. Although cell-mediated immunity (CMI) by CD4 Th1-type cells is considered to be the predominant host defense against mucosal candidiasis, the immune factors associated with susceptibility to OPC in HIV-positive persons are not well understood. This study investigated Candida-specific systemic CMI in HIV-positive persons with OPC and/or VVC. Reductions in delayed skin test reactivity to Candida antigen were observed in HIV-positive persons with CD4 cell counts <200 cells/microL, irrespective of the presence of mucosal infection. Likewise, despite the correlate of OPC with reduced CD4 cell counts in HIV-positive persons, differences in Candida-specific peripheral blood mononuclear cell proliferation and Th1/Th2 cytokine production between HIV-positive and HIV-negative persons were not consistent in a manner to suggest that deficiencies in Candida-specific systemic CMI account solely for the susceptibility to OPC.

Isolation of the melanoma-associated antigen p23 using antibody phage display.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.

Growth inhibition of Candida by human oral epithelial cells.

Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons. Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C. albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C. albicans in vitro. In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species. Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity. Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC. Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus.

Mucosal candidiasis is common in human immunodeficiency virus (HIV) infection. Susceptibility to such infections may be attributed to reduced host defense mechanisms and/or virulence of the organism. In the present study, we compared the virulence of mucosal Candida albicans isolates from HIV-infected people, with and without fluconazole-refractory infection, in established murine models of systemic and vaginal candidiasis. Compared with the mortality rate ( approximately 70%) after intravenous challenge with 2 virulent reference isolates, challenge with most clinical isolates (66%-77%) resulted in prolonged survival. In contrast, fungal burden induced by intravaginal challenge of nearly all (97%) isolates was similar to that of the virulent controls. There were no differences in in vitro growth rates for any of the isolates, and there was no association between reduced mortality and clinical failure to fluconazole, in vitro antifungal susceptibility, site of infection, or other host factors. These results suggest that virulence of C. albicans is tissue specific and is not a factor in the development of fluconazole-refractory infections in advanced HIV disease.

Sequence analysis of SLA2 of the dimorphic yeasts Candida albicans and Yarrowia lipolytica.

We report the complete nucleotide sequence of SLA2 of the dimorphic yeasts Candida albicans and Yarrowia lipolytica. In Saccharomyces cerevisiae, SLA2 codes for an actin binding protein. The deduced amino acid (aa) sequences of C. albicans CaSla2p and Y. lipolytica YlSla2p consist of 1063 and 1054 aa, respectively. The alignment of the deduced proteins of Saccharomyces cerevisiae, Y. lipolytica and C. albicans shows regions of identity in the N-terminal part of the proteins, which are essential for growth at 37 degrees C, endocytosis and actin organization in S. cerevisiae. The Sla2p proteins have also several conserved regions in the C-terminal moiety, the I/LWEQ boxes, displaying homology to the talin protein of mouse, Dictyostelium discoideum, Caenorhabditis elegans and to human huntingtin interacting protein (Hip 1p). The sequence data of C. albicans SLA2 are registered in the EMBL database (AJ009556), and for the Y. lipolytica gene in GenBank (U65409).

Combined Fmoc-Alloc strategy for a general SPPS of phosphoserine peptides; preparation of phosphorylation-dependent tau antisera.

A block method for the solid phase synthesis (SPPS) of serine phosphopeptides has been developed using a combination of Fmoc and Alloc strategies. Alloc-Ser[PO(OCH2CH CH2)2] OH2, prepared in a one pot procedure from Alloc-Ser-OH, was introduced at the N-terminus of a sequence prepared by standard Fmoc-SPPS. Global cleavage of the allyl ester based protecting groups, followed by coupling of a tripeptide fragment, led to the tau phosphopeptide, 1. Using tau phosphopeptides a series of phosphorylation state-dependent antisera to human tau protein have been raised. These antisera are valuable tools for studying the tau protein which is found in an abnormal, hyperphosphorylated form in Alzheimer's disease brain.

The carboxyl termini of beta-amyloid peptides 1-40 and 1-42 are generated by distinct gamma-secretase activities.

We have studied the effects of peptide aldehyde protease inhibitors on the secretion of beta-amyloid peptide 1-40 (Abeta(1-40)) and Abeta(1-42) by HEK 293 and COS-1 cells expressing beta-amyloid precursor protein with the Swedish double mutation. A multiphasic SDS-polyacrylamide gel electrophoresis system was used for the discrimination of Abeta(1-40) and Abeta(1-42). Calpain inhibitor I, carbobenzoxyl-Leu-Leu-leucinal, and calpeptin were found to reduce the amount of Abeta(1-40) released into the medium in a dose-dependent manner. The reduction of Abeta(1-40) after treatment with 50 microM calpain inhibitor I or 5 microM carbobenzoxyl-Leu-Leu-leucinal was accompanied by a slight increase of Abeta(1-42) released into the medium. These observations suggest that the cleavages at residues 40 and 42 are accomplished by different enzyme activities.

Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase.

The Candida albicans ADH1 gene encodes an alcohol dehydrogenase which is immunogenic during infections in humans. The ADH1 gene was isolated and sequenced, and the 5'- and 3'-ends of its mRNA were mapped. The gene encodes a 350 amino acid polypeptide with strong homology (70.5-85.2% identity) to alcohol dehydrogenases from Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. The cloned C. albicans ADH1 gene was shown to be functional through complementation of adh mutations and efficient production of active alcohol dehydrogenase in S. cerevisiae. Northern analysis of C. albicans RNA revealed that ADH1 mRNA levels were regulated in response to carbon source and during batch growth. During growth on glucose, ADH1 mRNA levels rose to maximum levels during late exponential growth phase and declined to low levels in stationary phase. The ADH1 mRNA was relatively abundant during growth on galactose, glycerol, pyruvate, lactate or succinate, and less abundant during growth on glucose or ethanol. Alcohol dehydrogenase levels did not correlate closely with ADH1 mRNA levels under the growth conditions studied, suggesting either that this locus is controlled at both transcriptional and post-transcriptional levels, or that other differentially regulated ADH loci exist in C. albicans.

Conformations of synthetic beta peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells.

The secondary structures of peptides beta 25-35 (the active toxic fragment) and beta 35-25 (reverse sequence and non-toxic fragment), as well as of the amidated beta (25-35)-NH2 peptide were investigated in aqueous solution and in the solid state by means of Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy. The conformations of the beta 25-35 and beta 35-25 in solid state were identical and contained mostly beta-sheet structures. In solid state the amidated beta (25-35)-NH2 peptide also contained mostly beta-sheet structures. Freshly prepared aqueous solutions of the beta 25-32 (0.5 - 3.8 mM) contained a mixture of beta-sheet and random coil structures. Within 30-60 min incubation at 37 degrees C in water or in phosphate-buffered saline solution (PBS), beta 25-35 was almost fully converted to a beta-sheet structure. Decreasing the temperature from 37 degrees C to 20 degrees C decreased the rate of conversion from random coil to beta-sheet structures, 1-2 h being required for complete conversion. In contrast beta 35-25 in water or in PBS buffer had mostly a random coil structure and remained so for 6 days. The amidated beta(25-35)-NH2 peptide in water (2.7 mM) was also mostly random coil. However, when this peptide (2-2.7 mM) was dissolved in PBS (pH 7.4) or in 140 mM NaCl, a gel was formed and its conformation was mostly beta-sheet. Decreasing the concentration of beta (25-35)-NH2 peptide in 140 mM NaCl aqueous solution from 2 mM to 1 mM or below favored the conversion from beta-sheet structures to random coil structures. The beta 25-35 was toxic to PC12 cells while beta 35-25 was not. The amidated peptide beta (25-35)-NH2 was at least 500-fold less toxic than beta 25-35. Structural differences between these beta peptides in aqueous solutions may explain the difference in their respective toxicities.

Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans.

Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the cDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strong homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans delta hsp90/delta hsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans.

Structure and regulation of a Candida albicans RP10 gene which encodes an immunogenic protein homologous to Saccharomyces cerevisiae ribosomal protein 10.

The Candida albicans clone cDNA10 was isolated on the basis that it encodes a protein which is immunogenic during infections in humans (R. K. Swoboda, G. Bertram, H. Hollander, D. Greenspan, J. S. Greenspan, N. A. R. Gow, G. W. Gooday, and A. J. P. Brown, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45 degrees C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.

Regulation of the gene encoding translation elongation factor 3 during growth and morphogenesis in Candida albicans.

The level of the TEF3 mRNA, which encodes the fungal-specific translation elongation factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans. In contrast to a previous report, TEF3 mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with the addition of 10% (v/v) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with an increase in the pH of the medium (from pH 4.5 to 6.5). TEF3 mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast to cultures undergoing morphological changes. TEF3 mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in TEF3 expression that accompany the dimorphic transition in C. albicans appear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se. For this reason TEF3 mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3 mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation factor might be subject to post-transcriptional regulation.

The amyloid precursor protein fragment His 657-Lys 676 inhibits noradrenaline- and enkephaline-induced suppression of voltage sensitive calcium currents in NG108-15 hybrid cells.

We have investigated the effects of the C-terminal amyloid precursor protein fragment His 657-Lys 676 upon calcium currents in NG108-15 neuroblastoma x glioma hybrid cells. The amyloid precursor protein fragment His 657-Lys 676 (1-10 microM) did not affect calcium currents per se, but clearly blocked the calcium current suppression mediated by both adrenergic alpha 2B- and opioid delta receptors in a concentration-dependent manner. The reverse amyloid precursor protein fragment Lys 676-His 657 and the shorter amyloid precursor protein fragment Gly 659-Lys 676 did not affect calcium current suppression by adrenergic alpha 2B- and opioid delta receptors. The similar interaction of C-terminal amyloid precursor protein with adrenergic alpha 2B- and opioid delta receptors suggest that the effect occurs downstream of the receptor, possibly via the GTP binding protein Go.

Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism.

The levels of pyruvate kinase (PYK1), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast-to-hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25 degrees C to 37 degrees C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25 degrees C to 37 degrees C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late-exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.