Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Expression of the receptor for advanced glycation end products in acquired reactive perforating collagenosis.

BACKGROUND: Acquired reactive perforating collagenosis (ARPC) is a rare skin disorder characterized by transepidermal elimination of dermal collagen. There is little data regarding the pathogenesis of ARPC. The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that plays an important role in inflammatory responses and may be involved in the pathogenesis of ARPC. AIM: To explore the expression of RAGE in ARPC. METHODS: Paraffin-embedded punch biopsy specimens of 41 patients with ARPC and of 11 healthy controls with normal skin were obtained from the Department Of Pathology. Clinical data of all patients were reviewed from the medical files. All specimens were stained immunohistochemically with antibody to RAGE (Anti-RAGE). The intensity of RAGE expression was assessed semi-quantitatively on epidermal cells, microvascular endothelium, dermal fibroblasts and inflammatory cells and graded as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The patients were divided into diabetic and nondiabetic groups for analysis. RESULTS: RAGE expression in microvascular endothelium, inflammatory cells and fibroblasts of patients with ARPC was more intense than normal tissues of healthy participants (P values are 0.005, 0.017 and P > 0.05). LIMITATIONS: Our method of assessment of RAGE expression was semi-quantitative. CONCLUSION: We observed an overexpression of RAGE in lesional samples of patients with ARPC which was independent of the presence of diabetes.

Evidence that the MAPK-docking site in MAPKK Dpbs2p is essential for its function.

In eukaryotes, mitogen-activated protein kinase (MAPK) pathways are very important signal transduction modules that regulate various cellular processes. Although eukaryotic cells possess a number of MAP kinase pathways, normally the MAPKKs selectively activate their cognate MAPK. Recent studies suggest that the MAPK-docking site in MAPKK facilitates this specific recognition and activation. However, the role of the docking site under in vivo conditions has not been demonstrated. In yeast external high osmolarity activates HOG (high osmolarity glycerol) MAPK pathway that consists of MAPKKK (Ste11p or Ssk2p/Ssk22p), MAPKK (Pbs2p), and MAPK (Hog1p). Previously, we have isolated a Pbs2p homologue (Dpbs2p) from osmo-tolerant and salt-tolerant yeast Debaryomyces hansenii that complemented pbs2 mutation in Saccharomyces cerevisiae. Here we show, for the first time, the presence of a MAPK-docking domain in Dpbs2p that is essential for its function in vivo. Mutation in this motif completely abolished its binding to Hog1p in vitro.

Evidence that C-terminal non-kinase domain of Pbs2p has a role in high osmolarity-induced nuclear localization of Hog1p.

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus. In baker's yeast external high osmolarity activates high osmolarity glycerol (HOG) MAPK pathway which consists of two upstream branches (SHO1 and SLN1) and common downstream elements Pbs2p MAPKK and Hog1p MAPK. Activation of this pathway causes rapid nuclear accumulation of Hog1p, essentially leading to the expression of target genes. Previously we have isolated a PBS2 homologue (DPBS2) from osmo-tolerant and salt-tolerant yeast Debaryomyces hansenii that partially complemented pbs2 mutation in Saccharomyces cerevisiae. Here we show that by replacing C-terminal region of Dpbs2p with the homologous region of Pbs2p we could abrogate partial complementation exhibited by Dpbs2p and this was achieved due to increase in nuclear translocation of Hog1p. Thus, our result showed that in HOG pathway, MAPKK has important role in nuclear translocation of Hog1p.

Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1Delta strain of Saccharomyces cerevisiae.

The HOG1 gene encodes a MAP kinase that plays an essential role in maintaining water homeostasis in the yeast Saccharomyces cerevisiae. A gene homologous to S. cerevisiae HOG1 has been isolated from a highly salt-tolerant yeast, Debaryomyces hansenii, by phenotypic complementation. DNA sequencing of the clone revealed the presence of an open reading frame encoding a protein 387 amino acids long. The deduced amino acid sequence showed very high similarity with homologous genes identified from S. cerevisiae, Candida albicans and Zygosaccharomyces rouxii. In addition, it has also TGY motif characteristics of hyperosmolarity-activated MAP kinases. The Genbank Accession No. of this sequence is AF185278.