Study Of Novel Killer Toxin Produced By Kluyveromyces Siamensis Hn12-1Isolated From Mangrove Ecosystem

The yeast Kluyveromyces siamensis HN12-1isolated from mangrove ecosystem wasfound to be able to produce killer toxin against the pathogenic yeast (Metschnikowiabicuspidata WCY) in crab. When the killer yeast was grown in medium with pH4.0,0.5%NaCl and at25Co, it could produce the highest amount of killer toxin against thepathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxinagainst the pathogenic yeast M. bicuspidata WCY was the highest when it wasincubated at25C in the assay medium without NaCl and pH4.0.Killer toxin was purified from the supernatant prepared from the cell culture byconcentration, precipitation, gel filtration chromatography, and DEAE Sepharose FastFlow anion-exchange chromatography. Results obtained from SDS-PAGE revealedone single protein band from the final concentrated elute and the relative molecularmass of the purified killer toxin was estimated to be a monomer of66.4kDa.Purified killer toxin was identified by Mass spectrophotometer (MALDITOF/TOF)and the amino acid sequences of these peptide fragments were found to be matchedagainst the lipase (accession number: ZP-00944832) from the bacterium Ralstoniasolanacearum and the score was89.Killing spectra demonstrated that the killer toxin produced by the yeast K. siamensisHN12-1could only kill M. bicuspidata WCY, the pathogenic yeast in crab among tentested yeast and bacterial pathogenic strains.Killer toxin induces its toxic effect in a two-step receptor mediated process in whichthe toxin interacts with cell wall receptor probably chitin and then damages to theplasma membrane. Our results showed that chitin successfully rescued the sensitive cells in the competition assay as compared to other polysaccharides tested. After thesensitive cells were treated by the killer toxin, the data presented by scan electronmicroscopy showed the damaged cell surfaces accompanied by cracks, and dischargeof cell material as compared to control which showed smooth surface indicating thedestruction of the cell wall at first step while results from DAPI and PI staining of thetoxin treated cells revealed that yeast cells were rapidly killed by the toxin, indicatingthe damage of the cytoplasmic membrane function. Lethal activity of killer toxinagainst sensitive cells showed direct relationship between toxin concentrations andthe percentage of cell reduction (dead cells). Killer toxin started its lethal effect within30mints and the minimum concentration of killer toxin and time required to achieve100%dead cells was40μg ml-1and36hours respectively.Effects of density of the sensitive yeast cells (M. bicuspidata WCY) and nutrient oncell growth and killer toxin production by the killer yeast K. siamensis HN12-1showed that when the density of the sensitive yeast cells in the co-culture was lessthan1.010~3cells ml-1, the killer yeast grew well, the considerable amount of killertoxin was produced, percentage of the dead cells in the co-cultures was increased andthe protein concentration in the media was also increased, leading to poor growth ofsensitive yeast. However, when the density of sensitive cells in co-culture was higherthan1.010~3cells ml-1, the killer yeast grew very poorly and the small amount ofkiller toxin was yielded, percentage of the dead cells in co-cultures was decreased andthe protein concentration in the media was also decreased, causing good growth of thesensitive yeast. It was also found that more cell growth and killer toxin productionoccurred in the nutrient rich and deficient media than in the distilled water.This is the first time to report the killer toxin production by the yeast K. siamensisHN12-1isolated from the mangrove ecosystem which is active against pathogenic yeast M. bicuspidata WCY. The presented findings suggested that this novel killertoxin has potential applications.