Killer yeasts
Encyclopedia
A killer yeast is a yeast
, such as Saccharomyces cerevisiae
, which is able to secrete one of a number of toxic proteins which are lethal to receptive cells. The phenomenon was first observed by Louis Pasteur
These yeast cells are immune to the toxic effects of the protein due to an intrinsic immunity. Killer yeast strains can be a problem in commercial processing because they kill desirable strains. The killer yeast system was first described in 1963. Study of killer toxins helped to better understand the secretion pathway of yeast, which is similar to those of higher eukaryotes. It also can be used in treatment of some diseases, mainly that caused by fungi.
(Saccharomyces cerevisiae
), which was found to spoil brewing
of beer. In S. cerevisiae are toxins encoded by a double-stranded RNA virus, transcribed as a precursor, cleaved and secreted outside of the cells, where they may affect susceptible yeast.
There are other killer systems in S. cerevisiae, such as KHR and KHS genes encoded on chromosome.
, L-A, is an icosahedral virus of S. cerevisiae comprising a 4.6 kb genomic segment and several satellite double-stranded RNA
sequences, called M dsRNAs. The genomic segment encodes for the viral coat protein and a protein which replicates the viral genomes. The M dsRNAs encode the toxin, of which there are at least three variants in S. cerevisiae, and many more variants across all species.
L-A virus uses yeast Ski complex
(super killer) and MAK (maintenance of killer) chromosomal genes for its preservation in the cell. The virus is not released into the environment. It spreads between cells during yeast mating.
The initial protein product from translation of the M dsRNA is called the preprotoxin, which is targeted to the yeast secretory pathway
. The preprotoxin is processed and cleaved to produce an α/β dimer
, which is the active form of the toxin, and is released into the environment.
The two most studied variant toxins in S. cerevisiae are K1 and K28.
K1 binds to the β-1,6-D-glucan
receptor
on the target cell wall, moves inside, and then binds to the plasma membrane receptor Kre1p. It forms a cation-selective ion channel
in the membrane, which is lethal to the cell.
K28 uses the α-1,6-mannoprotein receptor to enter the cell, and utilizes the secretory pathway in reverse by displaying the endoplasmic reticulum
HDEL signal. From the ER, K28 moves into the cytoplasm and shuts down DNA synthesis
in the nucleus, triggering apoptosis
.
before secretion, and although the toxin reenters through the cell wall it is unable to reactivate TOK1. However Breinig, Tipper and Schmitt (2002) showed that the TOK1 channel was not the primary receptor for K1, and that TOK1 inhibition does not confer immunity. Vališ, Mašek, Novotná, Pospíšek and Janderová (2006) experimented with mutants which produce K1 but do not have immunity to it, and suggested that cell membrane receptors were being degraded in the secretion pathway of immune cells, apparently due to the actions of unprocessed α chains.
Breinig, Sendzik, Eisfeld and Schmitt (2006) showed that K28 toxin is neutralized in toxin-expressing cells by the α chain in the cytosol, which has not yet been fully processed and still contains part of a γ chain attached to the C terminus. The uncleaved α chain neutralizes the K28 toxin by forming a complex with it.
are associated with linear DNA plasmids
, which have on their 5'end
associated proteins, which enable them to replicate themselves, in way similar to adenoviruses. It's an example of protein priming
in DNA replication
. MAK genes are not known. The toxin consists of three subunits, which are matured in golgi complex by signal peptidase
and glycosylated.
The mechanism of action is poorly understood. Affected cells are arrested in G1 phase
and lose viability.
and 223 other Candida
strains.
Others experimented with using killer yeasts to control undesirable yeasts. Palpacelli, Ciani and Rosini (1991) found that Kluyveromyces phaffii was effective against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii – all of which cause problems in the food industry. Polonelli et al. (1994) used a killer yeast to vaccinate against C. albicans in rats. Lowes et al. (2000) created a synthetic gene for the toxin HMK normally produced by Williopsis mrakii, which they inserted into Aspergillus niger
and showed that the engineered strain could control aerobic spoilage in maize silage and yoghurt. Ciani and Fatichenti (2001) used a toxin-producing strain of Kluyveromyces phaffii to control apiculate yeasts in wine-making. Da Silvaa, Caladoa, Lucasa and Aguiar (2007) found a toxin produced by Candida nodaensis was effective at preventing spoilage of highly salted food by yeasts.
Several experiments suggest that antibodies that mimic the biological activity of killer toxins have application as antifungal agents.
The greatest potential for control of killer yeasts appears to be the addition of the L-A virus and M dsRNA, or an equivalent gene, into the industrially-desirable variants of yeast, so they achieve immunity to the toxin, and also kill competing strains.
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, such as Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
, which is able to secrete one of a number of toxic proteins which are lethal to receptive cells. The phenomenon was first observed by Louis Pasteur
Louis Pasteur
Louis Pasteur was a French chemist and microbiologist born in Dole. He is remembered for his remarkable breakthroughs in the causes and preventions of diseases. His discoveries reduced mortality from puerperal fever, and he created the first vaccine for rabies and anthrax. His experiments...
These yeast cells are immune to the toxic effects of the protein due to an intrinsic immunity. Killer yeast strains can be a problem in commercial processing because they kill desirable strains. The killer yeast system was first described in 1963. Study of killer toxins helped to better understand the secretion pathway of yeast, which is similar to those of higher eukaryotes. It also can be used in treatment of some diseases, mainly that caused by fungi.
Saccharomyces cerevisiae
The best characterized toxin system is from yeastYeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
(Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
), which was found to spoil brewing
Brewing
Brewing is the production of beer through steeping a starch source in water and then fermenting with yeast. Brewing has taken place since around the 6th millennium BCE, and archeological evidence suggests that this technique was used in ancient Egypt...
of beer. In S. cerevisiae are toxins encoded by a double-stranded RNA virus, transcribed as a precursor, cleaved and secreted outside of the cells, where they may affect susceptible yeast.
There are other killer systems in S. cerevisiae, such as KHR and KHS genes encoded on chromosome.
RNA virus
The virusVirus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
, L-A, is an icosahedral virus of S. cerevisiae comprising a 4.6 kb genomic segment and several satellite double-stranded RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
sequences, called M dsRNAs. The genomic segment encodes for the viral coat protein and a protein which replicates the viral genomes. The M dsRNAs encode the toxin, of which there are at least three variants in S. cerevisiae, and many more variants across all species.
L-A virus uses yeast Ski complex
Ski complex
The Ski complex is a multi-protein complex involved in the 3' end degradation of messenger RNAs. The complex consists of three main proteins, the RNA helicase Ski2 and the proteins Ski3 and Ski8. In yeast, the complex guides RNA molecules to the exosome complex for degradation via a fourth protein,...
(super killer) and MAK (maintenance of killer) chromosomal genes for its preservation in the cell. The virus is not released into the environment. It spreads between cells during yeast mating.
Toxins
Secretory pathway
The secretory pathway is a series of steps a cell uses to move proteins out of the cell; a process known as secretion. The path of a protein destined for secretion has its origins in the rough endoplasmic reticulum, a membrane-bound compartment in the cell...
. The preprotoxin is processed and cleaved to produce an α/β dimer
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
, which is the active form of the toxin, and is released into the environment.
The two most studied variant toxins in S. cerevisiae are K1 and K28.
K1 binds to the β-1,6-D-glucan
Beta-glucan
β-Glucans are polysaccharides of D-glucose monomers linked by β-glycosidic bonds. β-glucans are a diverse group of molecules that can vary with respect to molecular mass, solubility, viscosity, and three-dimensional configuration...
receptor
Receptor (biochemistry)
In biochemistry, a receptor is a molecule found on the surface of a cell, which receives specific chemical signals from neighbouring cells or the wider environment within an organism...
on the target cell wall, moves inside, and then binds to the plasma membrane receptor Kre1p. It forms a cation-selective ion channel
Ion channel
Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane of cells by allowing the flow of ions down their electrochemical gradient. They are present in the membranes that surround all biological cells...
in the membrane, which is lethal to the cell.
K28 uses the α-1,6-mannoprotein receptor to enter the cell, and utilizes the secretory pathway in reverse by displaying the endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
HDEL signal. From the ER, K28 moves into the cytoplasm and shuts down DNA synthesis
DNA synthesis
DNA synthesis commonly refers to:*DNA replication - DNA biosynthesis *Polymerase chain reaction - enzymatic DNA synthesis *Oligonucleotide synthesis - chemical synthesis of nucleic acids...
in the nucleus, triggering apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
.
Immunity
Sestia, Shiha, Nikolaevaa and Goldstein (2001) claimed that K1 inhibits the TOK1 membrane potassium channelPotassium channel
In the field of cell biology, potassium channels are the most widely distributed type of ion channel and are found in virtually all living organisms. They form potassium-selective pores that span cell membranes...
before secretion, and although the toxin reenters through the cell wall it is unable to reactivate TOK1. However Breinig, Tipper and Schmitt (2002) showed that the TOK1 channel was not the primary receptor for K1, and that TOK1 inhibition does not confer immunity. Vališ, Mašek, Novotná, Pospíšek and Janderová (2006) experimented with mutants which produce K1 but do not have immunity to it, and suggested that cell membrane receptors were being degraded in the secretion pathway of immune cells, apparently due to the actions of unprocessed α chains.
Kluyveromyces lactis
Killer properties of Kluyveromyces lactisKluyveromyces lactis
Kluyveromyces lactis is a Kluyveromyces yeast commonly used for genetic studies and industrial applications. Its name comes from the ability to assimilate lactose and convert it into lactic acid.- Use :...
are associated with linear DNA plasmids
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
, which have on their 5'end
Directionality (molecular biology)
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5′-end and a 3′-end...
associated proteins, which enable them to replicate themselves, in way similar to adenoviruses. It's an example of protein priming
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
in DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
. MAK genes are not known. The toxin consists of three subunits, which are matured in golgi complex by signal peptidase
Signal peptidase
Signal peptidases are enzymes that convert secretory and some membrane proteins to their mature form by cleaving off their N-terminal targeting signals....
and glycosylated.
The mechanism of action is poorly understood. Affected cells are arrested in G1 phase
G1 phase
The G1 phase is a period in the cell cycle during interphase, before the S phase. For many cells, this phase is the major period of cell growth during its lifespan. During this stage new organelles are being synthesized, so the cell requires both structural proteins and enzymes, resulting in great...
and lose viability.
Other Yeast
Other toxin systems are found in other yeasts:- PichiaPichiaPichia is a genus of yeasts in the family Saccharomycetaceae with spherical, elliptical or oblong acuminate cells. Pichia is a teleomorph, and forms during sexual reproduction hat-shaped, hemispherical or round ascospores. The anamorphs of some Pichia species are Candida species...
and Williopsis - Hanseniaspora uvarum
- Zygosaccharomyces bailiiZygosaccharomyces bailiiZygosaccharomyces bailii is the type species for the genus Zygosaccharomyces. It was initially described as Saccharomyces bailii by Lindner in 1895, but in 1983 was reclassified as Zygosaccharomyces bailii in the work by Barnett et al....
- Ustilago maydis
Use of toxins
The susceptibility to toxins varies greatly between yeast species and strains. Several experiments have made use of this to reliably identify strains. Morace, Archibusacci, Sestito and Polonelli (1984) used the toxins produced by 25 species of yeasts to differentiate between 112 pathogenic strains, based on their sensitivity to each toxin. This was extended by Morace et al. (1989) to use toxins to differentiate between 58 bacterial cultures. Vaughan-Martini, Cardinali and Martini (1996) used 24 strains of killer yeast from 13 species to find a resistance signature for each of 13 strains of S. cerevisiae used as starters in wine-making. Buzzini and Martini (2001) showed that sensitivity to toxins could be used to discriminate between 91 strains of Candida albicansCandida albicans
Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans. Systemic fungal infections including those by C...
and 223 other Candida
Candida (genus)
Candida is a genus of yeasts. Many species are harmless commensals or endosymbionts of animal hosts including humans, but other species, or harmless species in the wrong location, can cause disease. Candida albicans can cause infections in humans and other animals, especially in immunocompromised...
strains.
Others experimented with using killer yeasts to control undesirable yeasts. Palpacelli, Ciani and Rosini (1991) found that Kluyveromyces phaffii was effective against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii – all of which cause problems in the food industry. Polonelli et al. (1994) used a killer yeast to vaccinate against C. albicans in rats. Lowes et al. (2000) created a synthetic gene for the toxin HMK normally produced by Williopsis mrakii, which they inserted into Aspergillus niger
Aspergillus niger
Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food...
and showed that the engineered strain could control aerobic spoilage in maize silage and yoghurt. Ciani and Fatichenti (2001) used a toxin-producing strain of Kluyveromyces phaffii to control apiculate yeasts in wine-making. Da Silvaa, Caladoa, Lucasa and Aguiar (2007) found a toxin produced by Candida nodaensis was effective at preventing spoilage of highly salted food by yeasts.
Several experiments suggest that antibodies that mimic the biological activity of killer toxins have application as antifungal agents.
Control methods
Young and Yagiu (1978) experimented with methods of curing killer yeasts. They found that using a cycloheximine solution at 0.05 ppm was effective in eliminating killer activity in one strain of S. cerevisiae. Incubating the yeast at 37°C eliminated activity in another strain. The methods were not effective at reducing toxin production in other yeast species. Many toxins are sensitive to pH levels; for example K1 is permanently inactivated at pH levels over 6.5.The greatest potential for control of killer yeasts appears to be the addition of the L-A virus and M dsRNA, or an equivalent gene, into the industrially-desirable variants of yeast, so they achieve immunity to the toxin, and also kill competing strains.