Candida albicans Hgt1p, a multifunctional evasion molecule: complement inhibitor, CR3 analogue, and human immunodeficiency virus-binding molecule.

BACKGROUND: The complement system is tightly controlled by several regulators. Two of these, factor H (FH) and C4b-binding protein (C4BP), can be acquired by pathogens conveying resistance to complement attack. The aim of the study was to characterize the FH binding molecule of Candida albicans, a potentially life-threatening yeast. METHODS: The gene coding for this molecule was identified by probing an expression library and homozygous deletion mutants of the respective gene were constructed. Binding and functional assays were undertaken to compare wild-type and knockout strains. RESULTS: The high-affinity glucose transporter 1 (CaHgt1p) was identified as an FH-binding molecule. Homozygous hgt1Δ/Δ deletion mutants, but not the restored strain in which HGT1 was reintegrated, showed a decreased binding of FH and even of C4BP, demonstrating its function as an FH- and C4BP-binding protein. This led to an enhanced terminal complement complex deposition after incubation with human serum; CaHgt1p thus functions as complement inhibitor. hgt1Δ/Δ mutants failed to form rosettes with complement-coated sheep erythrocytes, and show reduced binding to HIV-gp160, implying that a complement receptor 3 (CR3) moiety, known as fungal HIV binding molecule is lacking. CONCLUSIONS: CaHgt1p is a multifunctional evasion molecule, as complement inhibitor, CR3 analogue and HIV receptor.

HER2 is unlikely to be involved in directly regulating angiogenesis in human breast cancer.

Angiogenesis is a fundamental component of oncogenesis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet derived-endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) are generated from tumor cells to provide tumor growth and are thought to be regulated via the HER2 oncogene, whose amplification is the most common genetic alteration in breast cancer. The present study aimed to evaluate the immunoreactivity of angiogenic factors (VEGF, PD-ECGF/TP) and microvessel density (MVD) via epidermal growth factor receptor (EGFR) and HER2, and to correlate their expression with clinicopathologic features. Two hundred one invasive human breast cancer specimens were tested immunohistochemically for the expression of these proteins. In addition, MVD was examined using computerized image analysis. VEGF could be an additional interesting prognostic variable, as it was significantly associated with tumor grade (P=0.002), stage (P=0.018), and negative estrogen receptor status (P=0.011). EGFR was significantly related to invasive ductal carcinoma (P=0.030), tumor grade (P=0.009), VEGF expression (P=0.013), PD-ECGF/TP expression (P=0.024), and MVD (P=0.050). The finding that VEGF is not correlated to MVD does not rule out a crucial role of VEGF as a key factor in angiogenesis. HER2 could not be correlated to MVD, VEGF expression, or PD-ECGF/TP expression, indicating that this factor is unlikely to be involved in directly regulating angiogenesis, whereas the significant correlations between EGFR and histologic tumor type, tumor grade, the angiogenic factors VEGF and PD-ECGF/TP, and MVD point out that EGF is the major modulating growth factor for angiogenesis in breast cancer.

The vesicle transport protein Vac1p is required for virulence of Candida albicans.

The putative vesicle transport protein Vac1p of the human pathogenic yeast Candida albicans plays an important role in virulence. To determine the cellular functions of Vac1p, a null mutant was generated by sequential disruption of both alleles. The vac1 null mutant strain showed defective endosomal vesicle transport, demonstrating a role of Vac1p in protein transport to the vacuole. Vac1p also contributes to resistance to metal ions, as the null mutant strain was hypersensitive to Cu(2+), Zn(2+) and Ni(2+). In addition, the loss of Vac1p affected several virulence factors of C. albicans. In particular, the vac1 null mutant strain showed defective hyphal growth, even when hyphal formation was induced via different pathways. Furthermore, Vac1p affects chlamydospore formation, adherence to human vaginal epithelial cells, and the secretion of aspartyl proteinases (Saps). Avirulence in a mouse model of systemic infection of the vac1 null mutant strongly suggests that Vac1p of C. albicans is essential for pathogenicity. In summary, the Vac1p protein is required for several cellular pathways, in particular those that control virulence and pathogenicity. Consequently, Vac1p is a novel and interesting target for antifungal drugs.

Phosphatidylinositol 3-kinase VPS34 of Candida albicans is involved in filamentous growth, secretion of aspartic proteases, and intracellular detoxification.

The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans influences vesicular intracellular transport, filamentous growth and virulence. To get a clearer understanding how these phenomena are connected, we analysed hyphal growth in a matrix under microaerophilic conditions at low temperature, the detoxification of metal ions and antifungal drugs, the secretion of aspartic proteinases (Saps), as well as expression of adhesion-associated proteins of the C. albicans vps34 null mutant strain. The hyphal growth in a matrix, which is repressed in the wild-type strain by Efg1p, was derepressed in the mutant. CZF1, which encodes an activator of hyphal growth in a matrix, was up-regulated in the mutant. In addition, CZF1 expression was pH-dependent in the wild-type. Expression of EFG1 was not changed. Examination of Saps secretion showed a reduction in the vps34 null mutant. Determination of sensitivity against metal ions and antimycotic drugs revealed defects in detoxification. Expression studies indicated that the vps34 mutant reacts to the phenotypical defects with an up-regulation of genes involved in these processes, including the aspartyl proteinases SAP2 and SAP9, adhesion proteins ALS1 and HWP1, and the ABC transporters CDR1 and HST6. We also found an increased expression of the PI 4-kinase LSB6 indicating a complex feed-back mechanism for the compensation of the multiple defects arising from the lack of the PI3-kinase VPS34.

Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue.

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (kappa = 0.661) and HPV18 (kappa = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.