Use of 65kDa mannoprotein gene primers in PCR methods for the identification of five medically important Candida species.

We have developed PCR and Multiplex PCR assays for the detection of medically important Candida spp. using different species and genus-specific PCR primers selected within the MP65 gene, a recently cloned gene encoding a mannoprotein adhesin. The genus-specific PCR primers were able to amplify Candida species DNA (100% positivity) whereas DNA from all other isolates tested, belonging to other fungal genera, was not amplified. The species-specific PCR primers allowed differentiation of each of five Candida species by the amplicon length produced. No amplicons were detected using species- or genus-specific primers in several bacterial or human DNA templates. The methods described in this study are reproducible, simple and specific. The total time required for each PCR method was less than 4 h from the extraction to the visualized amplicons after PCR. In conclusion, we developed PCR methods to differentiate the five most medically important Candida species using primers directed to the MP65 gene.

Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans.

A method for detection of Candida albicans in biological samples (blood, serum, urine) was developed by the use of polymerase chain reaction (PCR) amplification of a DNA fragment of a gene coding for a 65 kDa mannoprotein of C. albicans (CaMP65). The PCR amplifies a 220 bp fragments whose specificity for C. albicans was demonstrated by Southern blot with a non-radioactive probe, leading to the differentiation from all other yeast species or human and bacterial DNA. The sensitivity of this assay was 5-10 C. albicans cells per milliliter of biological sample.

Gene expression of 70 kDa heat shock protein of Candida albicans: transcriptional activation and response to heat shock.

CaHSP70 (70 kDa heat shock protein) is a highly immunogenic protein of Candida albicans. We have studied heat shock-induced expression of the CaHSP70 gene under germ tube-inductive and non-inductive conditions. The CaHSP70 upstream regulatory region was cloned and sequenced. It contains at least three heat shock elements (HSEs), specific DNA sequences that are bound by the heat shock transcription factor (HSF), and one stress response element (STRE), which is an upstream activator sequence (UAS) that causes transcription activation under stress. The binding of HSF to HSE in the CaHSP70 promoter region is constitutive, although the mobility of protein/DNA complexes is altered after heat shock. The CaHSP70 promoter was cloned into a lacZ reporter plasmid, and was able to respond to heat shock in C. albicans as well as in Saccharomyces cerevisiae.

Production of a mouse antiserum to Bacteroides fragilis enterotoxin using a recombinant enterotoxin precursor.

The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.

Generation of a recombinant 65-kilodalton mannoprotein, a major antigen target of cell-mediated immune response to Candida albicans.

A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogen Candida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designated CaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His(6)-tagged protein (rCaMp65) was expressed in Escherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4(+) T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

Generation of a highly immunogenic recombinant enolase of the human opportunistic pathogen Candida albicans.

Enolase, a 46 kDa glycolytic enzyme, is an immunodominant antigen of Candida albicans, an important human opportunistic pathogen. The full-length coding sequence of C. albicans enolase gene was subcloned into the prokaryotic expression vector pDS56/RBSII,His6/E- under the control of an inducible promoter to produce a His6-tagged enolase. The recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography. It was recognized by a monoclonal antibody specific for C. albicans enolase, as well as by anti-enolase antibodies present in human sera (IgG). The recombinant protein promptly elicited antibody in mice and detected immune responses in normal human subjects that were comparable to those generated by the native C. albicans enolase. Thus this new recombinant enolase constitutes a valuable reagent for studying the possible role of this protein in anti-Candida immune response.

A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis.

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-gamma), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-gamma was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-gamma upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

Construction and use of PCR primers from a 70 kDa heat shock protein gene for identification of Candida albicans.

Methods for detection of Candida albicans in culture or biological samples were developed by the use of polymerase chain reaction (PCR) with oligonucleotide primers from C. albicans 70 kDa heat shock protein gene (Cahsp70). The PCR amplifies a 335-base pair fragment which is then hybridized with a non-radioactive probe, leading to the specific identification of C. albicans and its differentiation from all other human pathogenic Candida and/or yeast species. Candida albicans could be rapidly detected in human urine and blood, with a sensitivity of 10 and 50 fungal cells per sample, respectively.