Therapeutic potential of yeast killer toxin-like antibodies and mimotopes

This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy. KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms. They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats. KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.     

From <Emphasis Type="Italic">Pichia anomala</Emphasis> killer toxin through killer antibodies to killer peptides for a comprehensive anti-infective strategy

“Antibiobodies”, antibodies (Abs) with antibiotic activity, internal image of a Pichia anomala killer toxin (PaKT) characterized by microbicidal activity against microorganisms expressing β-glucans cell-wall receptors (PaKTRs), were produced by idiotypic vaccination with a PaKT-neutralizing monoclonal Ab (PaKT-like Abs) or induced by a protein-conjugated β-glucan. Human natural PaKT-like Abs (PaKTAbs) were found in the vaginal fluid of women infected with KT-sensitive microorganisms. Monoclonal and recombinant PaKT-like Abs, and PaKTAbs proved to be protective against experimental candidiasis, cryptococcosis and aspergillosis. A killer decapeptide (KP), synthesized from the sequence of a recombinant PaKT-like Ab or produced in transgenic plants, showed a microbicidal activity in vitro, neutralized by β-glucans, a therapeutic effect in vivo, against experimental mucosal and systemic mycoses, and a prophylactic role in planta, against phytopathogenic microorganisms, respectively. KP showed fungicidal properties against all the defective mutants of a Saccharomyces cerevisiae library, inclusive of strains recognized to be resistant to conventional antifungal drugs. KP inhibited in vitro, ex vivo and/or in vivo HIV-1 and Influenza A virus replication, owing to down-regulation of CCR5 co-receptors, physical block of the gp120-receptor interaction and reduction in the synthesis of glycoproteins, HA and M1 in particular. KP modulated the expression of costimulatory and MHC molecules on murine dendritic cells, improving their capacity to induce lymphocyte proliferation. KP, proven to be devoid of cytotoxicity on human cells, showed self-assembly-releasing hydrogel-like properties, catalyzed by β 1,3 glucan. PaKT’s biotechnological derivatives may represent the prototypes of novel antifungal vaccines and anti-infective drugs characterized by different mechanisms of action.     

Protective antifungal yeast killer toxin-like antibodies

After several years of controversy, antibodies (Abs) are now believed to play an important role in the protection against fungal infections. Among them, recent data are strongly supporting the relevance of protective yeast killer toxin-like Abs ("antibiobodies", KT-Abs), which are able to exert a direct microbicidal activity by mimicking a killer toxin (PaKT) and its interaction with cell wall receptors on susceptible cells essentially constituted by beta-glucans. This review will focus on the implications of the yeast killer phenomenon, and, particularly, the occurrence and antimicrobial activity of protective antifungal KT-Abs, such as those produced during the course of experimental and natural infections caused by PaKT-sensitive microorganisms or produced by idiotypic vaccination with a PaKT-neutralizing mAb. The strong therapeutic activity exerted against different experimental mucosal and systemic mycoses by monoclonal and recombinant microbicidal KT-Abs (either in their soluble forms or expressed on human commensal bacteria) as well as by a synthetic killer peptide (KP, an antibody fragment engineered from the sequence of a recombinant KT-Ab) will be discussed. The surprisingly wide antimicrobial spectrum of activity against eukaryotic and prokaryotic pathogenic agents, such as fungi, bacteria and protozoa, of these Abs and Ab-derived molecules suggests new potential strategies for transdisease anti-infective prevention and therapy.     

New strategies for treatment of Candida vaginal infections

New strategies for treatment of vaginal candidiasis have been recently exploited, due to widespread occurrence of this disease, in particular as recurrent infections, limitations of safe and efficacious antifungals as well as the lack of reliable preventative approaches. In this review new chemotherapeutic and immunotherapeutic strategies, based on the improved understanding of the immunopathogenesis of this prevalent human infection, will be discussed. The role of killer antibodies (or their molecular derivatives), i.e. antibodies that show antibiotic activity bearing the internal image of a yeast killer toxin (KT), characterized by a wide spectrum of microbicidal activity, and of the specific cell wall KT receptor as putative new therapeutic agents and preventative or therapeutic vaccines, respectively, will be particularly outlined.     

Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts

A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii. KT4561 was precipitated by ethanol and purified by ion-exchange chromatography. The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa. Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h. The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C. glabrata, 0.03 and 0.23 microg/ml for I. orientalis and 1.50 and 2.25 microg/ml for P. guilliermondii. Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics. The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

The relationship between the acoustic behaviour and surface activity of killer whales (<Emphasis Type="Italic">Orcinus orca</Emphasis>) that feed on herring (<Emphasis Type="Italic">Clupea harengus</Emphasis>)

We describe the acoustic behaviour of piscivorous killer whales in Norwegian and Icelandic waters. Whales were assigned to one of three activities (feeding, travelling or other), and sound recordings were made in their proximity with a single hydrophone and a digital audiotape (DAT) recorder. A quantitative analysis of the production of pulsed calls, whistles and echolocation clicks in the three activities revealed that there was a significant effect of activity on the production of these sound types. Both killer whales in Icelandic and Norwegian waters produced high rates of clicks and calls during feeding and low rates of click, calls and whistles during travelling. The differences can be used as acoustical markers and provides new possibilities for acoustic monitoring of killer whales in these areas. Based on the similarity between their prey choice, hunting strategies, phenotype and acoustic behaviour, we suggest that the killer whales in Icelandic and Norwegian waters belong to the same ecotype: Scandinavian herring-eating killer whales.     

Biology of killer yeasts

Killer yeasts secrete proteinaceous killer toxins lethal to susceptible yeast strains. These toxins have no activity against microorganisms other than yeasts, and the killer strains are insensitive to their own toxins. Killer toxins differ between species or strains, showing diverse characteristics in terms of structural genes, molecular size, mature structure and immunity. The mechanisms of recognizing and killing sensitive cells differ for each toxin. Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. They are also suitable to study the mechanisms of protein processing and secretion, and toxin interaction with sensitive cells. This review focuses on the biological diversity of the killer toxins described up to now and their potential biotechnological applications. Electronic Publication     

嗜杀酵母的生物学功能及其应用

嗜杀酵母能够分泌毒素蛋白,杀死敏感酵母。嗜杀酵母对自身分泌的嗜杀毒素具有免疫力。嗜杀酵母的嗜杀特性与两种双链线状RNA(dsRNA)有关,即编码产生毒素蛋白的M-dsRNA和编码自身和M-dsRNA外壳蛋白的L-dsRNA。嗜杀毒素破坏细胞跨膜化学质子梯度,造成ATP和钾离子泄漏,导致细胞死亡。应用嗜杀酵母可避免野生型酵母污染,净化发酵体系,改善发酵产物品质;嗜杀毒素也可作为抵制病原酵母和类酵母微生物的抗真菌剂。     

Yeast killer toxins: from ecological significance to application

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.     

Zygocin,a secreted antifungal toxin of the yeast Zygosaccharomyces bailii,and its effect on sensitive fungal cells

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.     

Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study.

A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a 'punctuated' labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra-thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra-thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts

Two yeast killer toxins active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces are here described for the first time. The two toxins produced by Pichia anomala (DBVPG 3003) and Kluyveromyces wickerhamii (DBVPG 6077), and named Pikt and Kwkt, respectively, differ for molecular weight and biochemical properties. Interestingly, the fungicidal effect exerted by Pikt and Kwkt against Dekkera bruxellensis is stable for at least 10 days in wine. Thus, a potential application for the two toxins as antimicrobial agents active on Dekkera/Brettanomyces during wine ageing and storage can be hypothesised.     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

Dimorphism in Itersonilia perplexans: Yeast and hyphal phases differ in their sensitivity to mycocins produced by tremellaceous yeasts

The monokaryotic yeast phase of the heterobasidiomycete Itersonilia perplexans, unlike the hyphal phase, was found to be sensitive to mycocins produced by killer strains of Cryptococcus humicola, Cr. laurentii, Cystofilobasidium bisporidii and Rhodotorula fujisanense. Both the yeast and hyphal phases wer resistant to mycocins of Cr. podzolicus, Filobasidium capsuligenum, Rhodotorula glutinis, Rh. mucilaginosa, Rh. pallida, Sporidiobolus johnsonii, Sb. pararoseus and Sporobolomyces alborubescens. The different sensitivity patterns of yeast and hyphal phases are probably caused by biochemical differences in the cell walls.     

酵母菌杀菌系统的杀菌活性

探讨酵母菌杀菌系统（即３０株杀菌酵母菌）对３８株真菌或细菌的杀菌活性。结果显示杀菌酵母菌不仅对念珠菌、新型隐球菌和烟曲霉菌等真菌有杀菌活性,而且对其不相关的葡萄球菌、肠杆菌科、绿脓杆菌、不动杆菌和副溶血性弧菌也有杀菌活性,提示该系统具有广泛的杀菌活性,特别是Ｋ３６。     

In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci

BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling of new antibiotics with broad antibacterial spectra to tackle the emergence of microbial resistance.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.     

Production of yeast killer toxin in experimentally infected animals

The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.