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.

TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a ß-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding ß-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking.

AIMS: The yeast strain Tetrapisispora phaffii DBVPG 6706 (formerly Kluyveromyces phaffii) secretes a killer toxin (Kpkt) that has antimicrobial activity against apiculate yeasts. The aim of this study was to evaluate the killer activity of Kpkt towards Hanseniaspora uvarum under winemaking conditions. METHODS AND RESULTS: The zymocidial activity of Kpkt on H. uvarum was assayed in microfermentation trials inoculated with free and immobilized T. phaffii cells. The microbial evolution and fermentation profiles of the wines were evaluated to determine the effects of Kpkt on apiculate yeasts, in comparison with SO(2). The results indicate that the fungicidal activity of Kpkt against H. uvarum is stable for at least 14 days in wine, and the zymocin can control the proliferation of apiculate yeasts. The analytical composition of wines with the inoculum of T. phaffii immobilized cells did not differ from the wines with SO(2). In contrast to wines without this control of apiculate yeasts, an increase in ethyl acetate was seen. CONCLUSIONS: Tetrapisispora phaffii is an excellent candidate for the biological control of undesired proliferation of apiculate yeasts during the first steps of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Tetrapisispora phaffii cells in an immobilized form can be used as a biocontrol agent to reduce the need for SO(2) addition.

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin.

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.

Osmophilic linear plasmids from the salt-tolerant yeast Debaryomyces hansenii.

Three novel linear plasmids, pDHL1 (8.4 kb), pDHL2 (9.2 kb) and pDHL3 (15.0 kb), were discovered in the halophilic (salt-tolerant) yeast Debaryomyces hansenii. Exonuclease treatment indicated that all three plasmids were blocked at their 5' ends, presumably, by analogy with most other eukaryotic linear plasmids which involved protein attachment. The Debaryomyces plasmids were entirely cured simply by growing cells in normal culture medium, but were stably maintained in culture medium containing salts, sorbitol or glycerol at suitable concentrations. This suggested that the pDHL plasmids required an osmotic pressure for stable replication and maintenance. The Debaryomyces yeast secreted a killer toxin against various yeasts species. Toxin activity was demonstrated only in the presence of salts such as NaCl or KCl, but this killer phenotype was not associated with the pDHL plasmids. Analysis of the plasmid-curing pattern suggested that pDHL3 may play a key role in the replication of the Debaryomyces plasmids. Southern hybridization showed that an extensive homology exists between specific regions of pDHL1 and pDHL2, whereas pDHL3 is unique.

Killer toxin of Hanseniaspora uvarum.

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000. Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by beta-1, 6-D-glucans.

Effect of Mycobacterium leprae-infected Schwann cells and their supernatant on lymphocyte neuroglia interaction.

Since the resolution of neural lesions and subsequent nerve damage in leprosy must inevitably involve the participation of immune cells sensitized to Mycobacteria, we have used the dissociated Schwann cell culture model to study the relationship between M. leprae-infected Schwann cells and sensitized immune cells. Our earlier study on light and ultrastructural observations showed that on infection with M. leprae, the cytomorphology of Schwann cells remains unaffected, while degenerative changes suggestive of apoptosis are seen in extraneous lymphocytes which are subsequently phagocytosed by the Schwann cells. We now present additional evidence confirming that the phagocytosis of splenic cells by Schwann cells is indeed a two-step process. The first involves M. leprae-dependent cytotoxicity to splenic cells. This is followed by phagocytosis of these cells, which is a secondary and M. leprae-independent phenomenon. This finding has implications particularly on the weak inflammatory response observed in nerve lesions of a majority of lepromatous patients.