Ebook Current topics in medical mycology (Vol.4): Part 2

5-Killer System Interactions
L. POLONELLI, G. MORACE, S. CONTI, M. GERLONI,

W.

MAGLIANI, AND C. CHEZZI

Viruses and Fungi
Over the last few years, our concept of yeasts has changed vastly. Once
thought of as "E. coli with a nucleus,"l these organisms currently represent
cells of universal use by modern molecular biologists. Retroviral elements,
ubiquitin, calmodulin, actin, and tubulin are only some of the many biological elements that are being investigated in yeast cells. Ras-related genes,
strongly implicated in the transformation of normal mammalian to cancer
cells, 2 have also been discovered in yeasts, opening the way for exciting new
cytological, biochemical, and experimental genetic strategies that were impossible to carry out in animal cells.
Cytoplasmic viruslike particles (VLPs) were first observed in diseased
mushrooms. 3 Since then, viruses have been detected in more than 100 different fungal species. In most of these species, the persistent viral infection
produces no discernible effect on the fungal host's phenotype.
However, the finding that the antiviral and interferon-inducing activities
of extracts (statolon, hellenin) from Penicillium species were due to the presence of double-stranded RNA (dsRNA) has sparked an explosion of interest
in what has now become a new area for research in mycology.4 The hypovirulence of certain forms of Endothia parasitica, which may cause chestnut
blight disease, has also been found to be related to the presence oflipid-rich
cytoplasmic vesicles containing dsRNA. Reduction of cytochrome oxidase
and respiratory deficiency resulting in abnormal growth and morphology
have been linked to specific dsRNA segments in Ophiostoma ulmi (which
causes Dutch elm disease).
The biological properties of the dsRNA genomes that are in capsid form in
noninfectious VLPs within the fungal cytoplasm have proved to be unique.
These mycoviruses are apparently incapable of extracellular transmission
through lysis of the host cell. They are normally maintained at a relatively
stable copy number and transmitted predominantly by the intracellular

route. In hyphomycetes, for example, transmission occurs through the How
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of protoplasm toward the growing hyphal tip, whereas cytoplasmic mixing
that occurs during budding, mating, or other means of cell fusion is responsible for the spread of these viruses in yeasts. Another unusual characteristic
of the dsRNA VLPs in fungi is that they generally produce no adverse effect
on their hosts and, in some cases, may even be beneficial. In this respect
they may be compared with bacterial plasm ids, in that they act as nonessential extrachromosomal elements which can, however, be quite useful to their
hosts.
In addition to conjugation behavior, enterotoxin production, and antibiotic resistance, bacterial plasmids encode the production of bacteriocins by
certain strains that exert lethal effects on closely related strains. The killer
toxins produced by certain yeasts are extremely similar to the bacteriocins.
The killer characteristics of these yeasts are often compared with those of
bacterial species associated with C factors. The resemblance is even more
striking between killer yeasts and Paramecium species carrying K particles.
In the latter case, a dominant nuclear gene has been found to be responsible
for the organism's ability to maintain these particles. 5 The occurrence of
similar phenomena in organisms with such widely divergent evolutionary
histories is fascinating.

The Killer Phenomenon in Yeast
The yeast killer phenomenon was first observed among Saccharomyces cerevisiae strains. 6 These strains secrete glycoproteins that have toxic effects on
other, sensitive strains of this species as well as closely related ones. The
yeast's ability to produce a killer toxin, its immunity to the effects of that
protein, and its resistance to those produced by other species are all encoded

by satellite dsRNAs.
In S. cerevisiae there are two main recognized killer systems (Kl and K2)
which do not elicit cross-immunity. Kl is found in laboratory strains and is
the most deeply investigated yeast killer system, whereas K2 is exclusively
related to wine yeasts. When no distinction is made, reference to the Kl
killer system is generally intended. In the type I system, cells may have one
offour possible phenotypes: K+ R+ (killer), K- R- (sensitive), K- R+ (neutral), and K+ R- (suicidal).
Two types of dsRNA have been (ound inside icosahedral VLPs of approximately 40 nm in diameter within wild killer strains of S. cerevisiae: L
dsRNA (4.9 kb) is present in approximately 1,000 copies per haploid cell,
while the M dsRNA appears in about 100 copies. Deletion of certain sequences of the latter results in suppressive S dsRNA.7 Both L and M dsRNA
interact with mak (maintenance killer) genes, which are widely scattered
over the chromosomal map of the yeast, and these genes are necessary for
maintaining autoreplication of M dsRNA VLPs. In fact, mutants that have


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lost certain mak genes also lose M dsRNA, though L dsRNA, which is also
present in nonkiller yeast cells, is maintained. Recessive mutations of other
chromosomal genes, such as ski (superkiller), result in increased toxin production by the yeast, presumably because of the roles they play in the replication of M dsRNA.8
Other chromosomal genes are essential for the secretion (sec) and killer
expression (kex) of the toxin as well as for the virus-mediated host immunity
to the toxin (vpl [vacuolar protein localization], end [endocytosis], and rex
[resistance expression] genes) although not necessarily for replication of the
cytoplasmic killer genome. The kex 2 gene product is, however, also required for mating functions and meiotic sporulation.
The relationship between the L and M dsRNA genomes would be analogous to that between a helper and a defective virus rather than that of components of an interdependent segmented mycovirus genome. 9 In the S.
cerevisiae Kl and K2 killer systems, L dsRNA encodes the major capsid
polypeptides of the VLPs. The M and L types of dsRNA probably do not

exist within the same capsid, and although the polypeptide composition of
the VLPs has not been completely described yet, particles containing L
dsRNA reportedly include a large polypeptide of 75,000 Da and two smaller
ones of 55,000 and 37,000 Da. Whether or not the M dsRNA-containing
particles are structurally identical to the L particles cannot be confirmed at
this time. However, in light of the cross-antigenicity observed between the
two VLPs, it is likely that they share at least one polypeptide.
The M dsRNA is related to toxin activity, by encoding the killer as well as
the immune phenotype. Killer toxin-coding cDNA copies of the MdsRNA from
S. cerevisiae have been cloned, and a toxin precursor gene sequence has
been identified. 10.11 This sequence encodes a 35-kDa protein (preprotoxin),
which contains a signal sequence-encoding region. Either this molecule or
a processed product (43-kDa protoxin) of glycosylation in the endoplasmic
reticulum is responsible for immunity of the toxin-producing strain. After
digestion by specific proteolytic enzymes, the protoxin is processed in the
Golgi apparatus or in secretory vesicles into the mature killer toxin, which in
the S. cerevisiae Kl killer system is composed of ~vo disulfide-linked a (9.5kDa) and {3 (9.0-kDa) polypeptide components. 12.13
Mutation of various nuclear genes may drastically affect the yeast's ability
to maintain killer dsRNA VLPs and its killer phenotype. The study ofkillerresistant (kre) mutants has shed light on the mechanism by which killer
toxins destroy sensitive yeast cells. Strains of S. cerevisiae that had been
rendered resistant to S. cerevisiae Kl killer toxin by mutation of the nuclear genes krel and kre2 were found to bind 35S-labeled killer toxin more
weakly than wild, sensitive strains. 14 Although killing activity is not yet fully
understood, .ATe do know that rapid, energy-independent binding of the
toxin to a (1,6)-{3-n-glucan-linked component of the cell wall occurs during
the initial phase and that the products of either the krel or the kre2 nuclear
gene are necessary to this process. Mutations in the nuclear loci krel and


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kre2 result in a reduction and modification of (1,6)-J3-n-glucan content of the
cell wall.
The initial binding to the wall, presumptively by the J3 subunit, might
make the a subunit of the toxin somehow accessible by an energydependent process at a plasma membrane site where the toxic effect is manifested by ion leakage and cell death. 15 When constant concentrations of
sensitive cells were treated with sub saturating concentrations of killer toxin,
linear rates of killing were observed, thus suggesting a single-hit process. 16
Since krel and kre2 mutant spheroplasts are sensitive to the toxin, while
those with mutation of the kre3 nuclear gene are resistant, even though
normal cell wall binding occurs, it may be that the latter gene encodes for or
is in some other way involved with the cytoplasmic membrane receptor site.
Two different hypotheses have been proposed for killer toxin resistance in
the immune cell. The immunity determinant (22-kDa) might alter or mask
the plasma membrane receptor site, rendering its interaction with the a
domains derived from exogenous killer toxin impossible. Alternatively, the
immunity determinant might mediate the relocation or removal of the receptor from the cytoplasmic membrane, a process in which vp2 and end
genes might be involved,17 though protease production by immune strains
for cleaving killer toxin should not be excluded.
Studies using artificial phospholipid bilayer membranes have revealed
that the purified toxin from Pichia kluyveri, like the K1 and K2 S. cerevisiae
toxins, causes ion-permeable channels to form in the bilayer. 18 The formation of pores and proton pumping is not part of the killing effect of a P. mrakii
toxin. This basic polypeptide, composed of 88 amino acid residues, is devoid
of mannosides and has an isoelectric point of 9.1 and a molecular size of
10,721 Da. 19 It selectively inhibits the synthesis of J3-glucan in the cell wall
of sensitive S. cerevisiae cells. 20 Cell wall synthesis of proteins, mannan,
chitin, and the alkali-insoluble, acid-soluble polysaccharides is not affected
by the toxin.
Like S. cerevisiae, the corn smut pathogen Ustilago maydis secretes glycoprotein toxins. 21 There are three different, though closely related, killer
systems (PI, P4, and P6) which are associated with cytoplasmic dsRNAs in

VLPS.22-24 The strains in one system kill those of the other two systems,
though they are immune to their own toxins.
All three toxins consist of a 12.5-kDa and a 1O-kDa peptide chain linked by
disulfide bonds only, and both polypeptides are essential for toxic activity.
Temperature-sensitive, nonkiller mutants secrete an inactive toxin which
lacks the 1O-kDa polypeptide; when the missing peptide is added, the killer
effects of the toxin are restored. The two polypeptides appear to interact
sequentially: the 1O-kDa component initiates the toxic effect by acting as a
recognition element. It interacts with a cell wall receptor to render the cell
accessible to the catalytic effects of the 12.5-kDa polypeptide, which
apparently induces endonucleolytic cleavage of nucleic acids. The U. maydis
killer strains are lethal only for members of their own and very closely re-


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141

lated species and have no effect on yeast isolates that are sensitive to other
killer yeasts.
The plasmids of both prokaryotic and eukaryotic microorganisms are
usually covalently closed circular DNA molecules, though linear forms do
exist.25 The killer system of Kluyveromyces lactis, for example, is mediated
by two linear dsDNA plasmids: pGKI-l (8.9 kb) and pGKI-2 (13.4 kb). There
are approximately 100 copies of each in each haploid cell, and they are cytoplasmically inherited in a non-Mendelian fashion. 26 The pGKI-l plasmid
confers upon the host cell either the killer (gene located in the central part)
or the immunity (gene located in the terminal part of the plasmid) phenotype. Replication and maintenance of the pGKI-l plasmid are probably controlled by the pGKI-2 plasmid.
The K. lactis killer toxin consists of three subunits: a glycosylated
polypeptide with an approximate molecular size of 100,000 Da and two
smaller nonglycosylated components of 30,000 and 27,500 Da. These three

proteins are produced by two distinct RNAs, each of which includes a signal sequence. 27 The first two subunits are derived from a larger precursor.
The toxin inhibits adenylate cyclase in sensitive strains, causing them to
arrest in the G 1 phase of growth. This arrest can be reversed by the addition
of cyclic AMP, which is recognized as necessary for the initiation of a new
mitotic cycle in normal cells.
While many aspects of the killer phenomenon in K. lactis (such as pH and
temperature range for toxin activity, spectrum of activity, and even mode of
action) are different from those of S. cerevisiae, killer cells of both species can
be deprived of their toxic properties by physical and chemical curing processes (growth in cycloheximide or ethidium bromide or at elevated temperatures). Both dsRNA and dsDNA plasmids may be transferred, by
protoplast fusion and transformation techniques, to (heterologous) nonkiller
(sensitive) strains to confer the killer (and resistant) phenotype to the recipient cells. K. lactis dsDNA plasm ids can replicate autonomously and stably
in the S. cerevisiae new host and can coexist with the resident dsRNA,
although they are incompatible with mitochondrial DNA.28 The resultant
killer strains may produce larger amounts of killer toxin than the K. lactis
did. Extracellular transmission of the dsRNA VLPs of S. cerevisiae has also
been demonstrated. 29
The toxin of Pichia anomala (Fig. 5-1a) has proven to be a potent killer of
many different genera of yeasts, including many pathogenic species. 3O The
genetic basis and the mechanism of action have not yet been identified.
Studies of the saturation kinetics of this toxin in Candida albicans cells suggest the presence of a toxin receptor, probably located on the cell wa1l3l
(Fig. 5-1b). Killing activity against C. albicans is greatest during the early
exponential growth phase of P. anomala, while the highest activity against
Saccharomycodes ludwigii occurs during the late phase. This would suggest
that P. anomala produces more than one active component.
Like the toxin of S. cerevisiae, the P. anomala toxin acts by binding to a


Polonelli et aI.

142

KILLER SYSTEM INTERACTIONS
INDIRECT
IMMUNOFLUORESCE 'CE

OCCUI\IIE CE or
PHE OME ON

IOIOTYPIC VACCI ATION

~II.I.ER

PfJyriIuU

~r.lih

rt!mVery

1..1on
YEAST KII.I.ER TOXI

THERAPEUTIC EFFECT

FIG. 5-1. Killer system interactions. For detailed descriptions, see text.

receptor site, but its range of activity is much broader, including microorganisms of various genera. Unlike the toxin of K. lactis, this toxin is not
counteracted by cyclic AMP. These phenomena suggest that the P. anomala
killer toxin has a unique mode of action. 32
Hat-spored species that were formerly classified in the genus Hansenula
have now been included in the genus Pichia, since identity of the two genera
was demonstrated by DNA comparisons.33 Segregation of the recombinant

killer phenotype from the meiotic tetrads of crosses between killer and nonkiller strains of P. anomala have shown that one or more nuclear genes must
be involved in expression of the killer character.34 The search for RNA or
DNA plasmids in many other Pichia (former Hansenula) species have also
failed to yield positive results (N . Gunge, personal communication).
Killer toxin-producing yeast isolates belonging to the pathogenic genera
Cryptococcus and Torulopsis have been isolated from natural habitats,
although the genetic determinants for their toxinogenesis and toxin bioaction
modalities are still unknown.35
The field has been extensively reviewed in whole or in part by several
authors.36- 41 These reviews have been very useful to the authors, and readers will find in them further references and sometimes alternative viewpoints.
The different susceptibilities of potential sensitive yeasts to the activity of


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143

potential killer yeasts must be attributed to different mechanisms of immunity. In the S. cerevisiae Kl system, binding of the killer toxin to the cell wall
receptor is substantially reduced in krel and kre2 mutants,42 which are considered resistant. Binding would thus seem to be a prerequisite for the sensitive phenotype. 43 The Kl killer toxin is lethal for spheroplasts of Candida,
Kluyveromyces, and Schwanniomyces isolates, though intact cells of the
same microorganisms are toxin resistant. Killer spheroplasts themselves also
remain immune to their own toxin. The Kl toxin is poorly retained by the
cell wall of K. iacUs, but the C. albicans wall binds the toxin to more or less
the same extent as do that of sensitive S. cerevisiae species. Thus, yeast
killer toxin specificity is defined by cell wall receptors which are necessary
for binding but not sufficient for toxin action at the plasma membrane of the
intact cells. 44 It is possible that there are additional unidentified cell wall
components for killer toxin activity that are missing in the wall of C. albicans. Alternatively, there could be some structural differences in the wall of
this species which prevent the glucan receptor-bound toxin from reaching
the plasma membrane. Such observations emphasize the need for further

study of the structure and protein transport systems of the yeast cell wall.
Saccharomyces cerevisiae strains carrying a 1.8-kb M dsRNA, which codes
for polypeptide toxin and a resistance function, are resistant to S. cerevisiae
Kl killer toxin, though they do not present a significant reduction in the
number of (1,6)-J3-D-glucan cell wall receptors. Remarkable amounts of killer
toxin are retained by binding to the killer cell's own cell wall. The chromosomal rexl gene is also involved in this form of resistance. 45
Whether this phenomenon represents the mechanism of resistance or not,
all resistant strains bind a certain amount of toxin to the cell walls, indicating
that they contain receptors similar to those that exist in wild-type sensitive
strains. 42 Killer cells themselves present cell wall receptors for their own
toxin, which act as a barrier to the toxin after it passes through the plasma
membrane on its outward path. Binding of the toxin by isolates that contain
sterically compatible cell wall receptors might also reduce toxin activity
against sensitive cells (unpublished data). In krel mutants, which lack such
receptors, a superkiller phenotype results when M dsRNA is introduced.
Phenylmethylsulfonyl fluoride-sensitive protease in the cell wall may also
degrade the toxin before it can be secreted. In fact, mutation of the superkiller (skiS) gene, which controls this enzyme, results in the production of an
active killer toxin more concentrated than that produced by the nonmutant
strain. 46
The various mechanisms of immunity invariably result in differential susceptibility of the yeasts to the toxins of defined killer strains. In a reciprocal
killer assay, yeast isolates were alternatively used as killer or sensitive
strains. Many strains exhibited both killer and susceptible behavior, depending on the strain they were matched against and the conditions under
which the assay was performed. 47
The production of inhibitory factors (killer toxins) can be assumed to play


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an important role in the modification of the ecosystems of natural habitats 34
and infected organisms (amensalism). Studies in animals have clearly shown
that the killer yeast P. anomala is able to secrete toxin in vivo in both immunosuppressed and normal mice after experimental infection. 48
The use of selected killer yeasts during brewing processes has been considered to prevent the growth of contaminating strains. It has been speculated, moreover, that the potential capacity of U. maydis killer proteins to
specifically inhibit U. maydis sensitive strains could be used in the biological
control of cereal smuts, if informational molecules for the production of toxin, introduced into the plant cell cytoplasm, could replicate and express
their killing function. 49

The Killer System as an Epidemiological Marker
The impact of hospital-acquired yeast infections has been dramatically
demonstrated over the last decade, and the need for a simple, reliable, and
sensitive method for differentiating fungal strains beyond the species level
has become increasingly pressing. Several different methods have been used
for this purpose: serotyping, morphologic differentiation, study of mating
behavior, enzyme profiles, chemical analyses, and chemical assimilation or
resistance patterns. 50 The ultimate method for biotyping fungal isolates
would be complete DNA base sequencing, but even if this approach was
technologically feasible, it would probably result in the complete differentiation of all isolates tested, thus invalidating its use as an epidemiological
marker.
First reported among S. cerevisiae strains, 6 the killer yeast phenomenon
has subsequently been observed among many other yeast genera. 51-53 Killer
yeasts have been grouped according to their specificity for killing sensitive
yeasts,54 and conversely, sensitive isolates of the same species have been
differentiated on the basis of their differential susceptibility to the activity of
various killer yeasts. The killer typing system, using zone assays similar to
those used in phage typing of Salmonella species, was first used to differentiate isolates of the pathogenic yeast species C. albicans55 and subsequently
applied to the epidemiological study of nosocomial infections caused by this
yeast species. 56 The adoption of simple test conditions allowed investigators
to evaluate the susceptibility of these isolates to the activity of selected killer
yeasts, primarily those of Pichia spp. Under the same test conditions it was

possible to extend the typing to other opportunistic species of yeasts (C ryptococcus neoformans, C. glabrata, C. parapsilosis, C. pseudotropicalis, and
C. tropicalis). 30 On the basis of their susceptibilities to the panel of selected
killer strains, these opportunistic yeasts could be grouped into reproducible
categories that contained strains that were serotypically heterogeneous.
The ability of the killer yeasts to exert their toxic effects was naturally


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affected by temperature, pH, and composition of the growth medium, conditions that varied according to the requirements of the target species being
studied. To avoid some of these restrictions, isolated and partially purified
toxins were used instead of streaked whole yeasts. 57
The use of killer toxins in place of killer yeasts, although more laborious,
improved the standardization of the system. Killer toxins proved to be stable
when partially purified, concentrated, and stored at 4°C, thus ensuring a
high. degree of test reproducibility. A computer program automatically
divided the C. albicans isolates studied into groups according to their susceptibility to the killer toxins. The program was designed to allow a maximum
error of 5% in strain differentiation. The computer-aided program permitted
the biotyping of the investigated yeast isolates in terms of the group percentage of probable affinity. Computer interpretation of the results eliminated
subjective interpretation. The storage of the data in the computer allowed a
rapid comparison of any new result with the results of the groups coded
previously, thus simplifying its application in epidemiological studies.
The original test conditions were also used to evaluate the occurrence of
sensitivity of hyphomycetes, bacteria, and achlorophyllous microorganisms
to the activity of recognized killer yeasts. 58 Killer toxins appeared to be inhibitory to a wide variety of prokaryotic and eukaryotic microorganisms
other than yeasts (Fig. 5-1c). As expected, the highest activity was displayed
by the isolates of Pichia spp. tested. All of the bacteria and fungi that were
able to grow under the experimental conditions proved to be sensitive to at

least one killer yeast tested. The killing effect was expressed differently
against the various species of bacteria and hyphomycetes and strains within
the same species. The inhibitory effect observed might not necessarily be
caused by the killer toxins themselves but rather by other metabolic products. If the killer phenomenon among yeasts, bacteria, aerobic actinomycetes, hyphomycetes, and achlorophyllous microorganisms could be confirmed with purified killer toxins, this would imply common cell wall binding
receptors and bioaction modalities.
The differential susceptibility of bacteria and hyphomycetes to the killer
strains could be used for epidemiological differentiation of strains within the
same species in much the same way that bacteriocin typing of gram-negative
and gram-positive bacteria is used. 59,60
Gross morphology and microscopic features are inconsistent criteria for
distinguishing intraspecific mycelial cultures. Pleomorphism and lack of
sporulation pose major difficulties to mycologists attempting the biotyping of
mycelial fungi. Unfortunately, with the possible exception of C. albicans61
and C. neoformans, 62 serologic differentiation of fungi has proven to be less
discriminatory than serotyping of pathogenic bacteria.
The exoantigen technique has revealed two serotypes in Blastomyces
dermatitidis,63 and antigenic variability among isolates of Sporothrix schenckii has been detected by indirect immunofluorescence. 64 Monoclonal antibodies have proven to be very useful for serotyping yeastlike and mycelial


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fungi by using the Western blot technique. 65 •66 This immunological
approach, however, requires reagents and technology that are not often
available to small laboratories.
The toxic effect of numerous selected killer yeasts has been studied on
Penicillium camemberti, S. schenckii, Aspergillus niger, Pseudoallescheria
boydii,67 and A. fumigatus and related taxa. 68 The killer system proved to be
a reliable tool for the biotyping of these mycelial cultures.

Although different procedures have been developed for differentiating isolates of gram-positive and gram-negative bacterial species, fewer possibilities have been reported for distinguishing strains of slowly growing bacteria
such as aerobic actinomycetes and mycobacteria.
The killer system, preViously standardized for yeasts and hyphomycetes,
has been adapted to the specific growth conditions of the bacterial isolates.
The (modified) killer system proved to be a convenient and flexible biotyping
method for strain differentiation of Nocardia asteroides, N. brasiliensis, N.
otitidis caviarum, and Actinomadura madurae 69 as well as Acinetobacter
calcoaceticus, Escherichia coli, Pseudomonas aeruginosa, Haemophilus
injluenzae, Neisseria meningitidis (beyond the conventional serotype
level), staphylococcus aureus, group A /3-hemolytic streptococci, Mycobacterium tubercolosis, M. fortuitum, and M. smegmatis. 70
The specific growth conditions of each bacterial species under which killer
yeasts could still exert their potential killer activity were identified, and
there were remarkable variations in pH, temperature, and oxygen concentration. It is likely, however, that more than one killer toxin is produced by
the same yeast, each of which is active under different conditions.
Several typing systems, in addition to serotyping and antibiotic susceptibility, have, of course, been used for such bacterial species: phage
susceptibility,71-73 enzyme production,74,75 bacteriocin production or
susceptibility, 76, 77 DNA hybridization,78 and R plasmid analysis. 79 Most of
these methods, however, are too laborious to be reproduced with a large
number of isolates in small microbiological laboratories. The killer system,
properly adapted to the growth requirements of the sensitive species, may
be a convenient biotyping method for a large number of prokaryotic and
eukaryotic microorganisms. It requires no specific technical expertise and
can be carried out using commercially available media and a set of suitable
killer yeasts.

Antibiotic Potential of Yeast Killer Toxins
Many of the etiologic agents of systemic mycoses have a parasitic form in
infected tissues that is structurally and morphologically different from the
one they present in the cultural phase. The in vitro susceptibility to selected
killer toxins of both the mycelial and yeast forms of S. schenckii isolates were

therefore evaluated comparatively80 (Fig. 5-1d).


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Temperature, pH, aeration, and carbon source are recognized to play important and specific roles in the development of the mycelial or yeast form of
this species, making it impossible to establish the same cultural conditions
for exclusive development of specific morphological types of growth. For this
reason, the original killer system conditions had to be modified to test pure
cultures of mycelial- or yeast-form isolates.
The pure conidial and yeast suspensions were challenged with the same
amounts of killer toxins, and the numbers of colony-forming units (CFU)
were compared with those obtained with control challenges using Sabouraud
broth. Both the yeast and mycelial forms of S. schenckii showed significant
susceptibility to the activity of yeast killer toxins, and this finding, regardless
of the specific mechanism of action, suggests that these toxins or their derivatives might be used in the treatment of systemic mycoses.
In E. coli KB TOOl, the outer membrane receptors for colicins E and
phage BF 23 are also involved in vitamin B12 transport. 81 If the cell wall
receptors for killer toxins were found to have other functions as well, agents
that inhibit the latter might be developed for use as antibiotics. 82 The primary target of the P. mrakii killer toxin on sensitive yeast and mold cells is the
synthesis of J3-glucan. 2o,83 Certain antifungal antibiotics, such as aculeacin A,
echinocandin B, and papulocandin B, have been found to specifically inhibit
the cell wall J3-glucan synthesis in many ascomycetous and deuteromycetous
yeasts. These compounds selectively inhibit J3-glucan synthesis in growing
yeast cells and also the in vitro activity of J3-(1,3)-glucan synthetase obtained
from the yeasts. Thus, the P. mrakii toxin appears to act against sensitive
yeast cells in much the same way that these cell wall-active antibiotic compounds act against their target cells. J3-glucan, especially J3-(1,3)-glucan,
which is recognized as the most important structural component of the yeast

cell wall, might be rendered osmotically fragile and defective by killer toxin
inhibition of J3-glucan synthesis, thus resulting in lytic cell death.
Because of the killer toxins' lability during purification processes and the
instability of the purified molecules, there is little information on the effective antimicrobial spectrum of these toxins. 84 We therefore attempted to
investigate the therapeutic potential of the toxin produced by a P. anomala
isolate (UCSC 25F). This isolate was selected as a reference candidate because it had exhibited the largest sppdrum of activity against prokaryotic and
eukaryotic microorganisms and had proven to be relatively stable at 37°C
and in unbuffered conditions.
Pityriasis versicolor-like lesions were experimentally induced in guinea
pigs and rabbits with a strain of Malassezia furfur, which had previously
been recognized to be sensitive in vitro to the activity of the reference toxin.
The animals recovered well after topical application of the crude toxic extract
(Fig. 5-le). The same behavior was obtained in dogs with otitis media experimentally induced with M. pachydermatis. 85 It was not possible to ascertain the potential therapeutic effect of parenterally administered toxin in
experimental systemic mycoses because toxic effects occurred that were
probably related to the impurity of the substance.


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Monoclonal Antibodies That Neutralize Yeast Killer
Toxin
Since conventional chemical procedures (gel exclusion chromatography,
chromatofocusing, anion-exchange chromatography) were not efficient
enough for the production of large amounts of purified killer toxin, monoclonal antibodies (MAbs) were produced for the one-step immunochemical
purification and characterization of yeast killer toxin by affinity chromatography19 (Fig. 5-1£). Monoclonal antibodies might also be used for analyzing
biologically active epitopes of the toxin in order to chemically synthesize
small peptides with killer activity and poor immunogenicity.
Monoclonal antibodies were obtained after fusion of mouse myeloma cells

with spleen cells isolated from mice primed with a crude extract of yeast
killer toxin produced by the P. anomala UCSC 25F strain. 86 A comparative
enzyme-linked immunosorbent assay, used for selecting the antibodyproducing hybrids, was adapted to determine the reactivity of the antibodies
in the culture fluid. Hybrids that also reacted with the growth medium of the
killer yeast were discarded. The hybrids that produced antibodies which
reacted only with the crude toxic extract were expanded and cloned. Monoclonal antibodies of the IgG class were produced. When the crude P. anomala
killer toxin reference antigen was electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips (Western blot technique), only slight diversity among the MAbs to the killer toxin proteins was
detected.
One of these MAbs (designated KTl) showed a greater avidity than
another (designated KT4), although both reacted with the antigenic determinants that had molecular sizes of 92 and 115 kDa. Antibody-rich fluids produced from the two expanded clones reacted differently to the reference
antigen in immunodiffusion tests (Fig. 5-1g). Monoclonal antibody KT4 produced a clear precipitin band, whereas KTI failed to precipitate with the
antigen. No reactions were observed in immunodiffusion with the growth
medium. When graduated amounts of KT4 (ammonium sulfate-purified
ascitic fluid) were added to the P. anomala UCSC 25F crude killer toxin,
killer activity against a recognized sensitive yeast (C. albicans UCSC 10) was
gradually neutralized (Fig. 5-1h). No neutralization was observed when
equal amounts of a nonspecific MAb (ascitic fluid) were used as a control.
Monoclonal antibody KT4 was also observed to react with toxins produced
by other Pichia species, expecially that of P. mrakii UCSC 255. Cross-reactivity between the killer toxins of these species implies the presence of
common antigenic determinants. However, several yeast killer toxins from
P. mrakii with different chemical and biological properties have been
defined. 87,88 The toxin that has been most fully characterized (as having high
heat and pH stability and a molecular weight of 10,700) is probably not the
one reacting with MAb KT4.
Various killer toxins produced by Saccharomyces, Kluyveromyces, Pichia,


5-Killer System Interactions

149

and Candida species were therefore investigated for reactivity with MAb
KT4. The genetic determinants of some of these toxins have yet to be identified. Double immunodiffusion using the killer toxins as antigens (Fig. 5-1g)
and indirect immunofluorescence on whole killer cells (Fig. 5-li) revealed
that MAb KT4 reacted only with the killer toxins and the whole cells of
yeasts belonging the genus Pichia. 89 This finding suggests that the toxins
coded by different genetic systems are antigenically heterogeneous.
High magnification of yeast killer cells of P. anomala UCSC 25F under
indirect immunofluorescence revealed differential staining depending on the
growth phase of the cells. Apparently, mature blastospores fluoresced more
than younger daughter cells, suggesting that killer toxin secretion is associated with the later phases of growth.
The genus Pichia was formerly limited to a heterogeneous group of species
characterized by hat-shaped, spheroidal, smooth or rough ascospores and
certain common coenzyme properties. 90 On the basis of DNA base sequencing studies,33, many species of the genus Hansenula have recently been
transferred to 'the genus Pichia, eliminating the earlier distinction based on
nitrate assimilation. Species and genus differentiation cannot be based on
the killer toxin phenotype, which is quite variable. In fact, we have observed
a great deal of intrageneric variation in the reactions of 25 Pichia isolates to
MAb KT4. Some of these strains, whose killer activity had been confirmed
previously when tested against sensitive strains, showed no reactivity whatsoever to the MAb. 91 For this reason, the significance of killer interactions
must be interpreted with caution.
Observation of KT4 reactivity with at least one killer toxin produced by
most of the Pichia spp. tested prompted us to attempt the one-step purification of correspondent killer toxin with affinity chromatography. We found
that the resulting toxin was still somewhat toxic and immunogenic to mice,
as might be expected of a large foreign protein (unpublished data). This
finding, together with the toxin's lability at neutral pH and at elevated
temperatures, makes it unlikely that this protein can be used therapeutically
in systemic mycoses. Perhaps the solution to these problems lies in the use
of toxin derivatives as antifungal agents.

Yeast Killer Toxin Mimicking Anti-Idiotypic
Antibodies
The steric interaction that occurs between a yeast killer toxin and the cell
wall receptor might be similar to that which takes place between antigens
and antibodies. The variable regions of antibodies are known to be immunogenic, and the antigenic determinants of these regions are termed
idiotypes. Antibodies produced against these immunogenic regions of the
antibody are called anti-idiotypes or anti-idiotypic antibodies (antilds). Antiidotypic antibodies have been described in a variety of clinical conditions


150

Polonelli et aI.

associated with autoimmunity and other forms of immune pathology, and
their practical application has been proposed as anti-idiotypic vaccines and
in the treatment of B-cell cancers.92 In 1974, Jerne pointed out that some of
the antilds might express the "internal image" of the original antigen (network theory).93 There is substantial evidence suggesting that these types of
antilds may mimick the action of the original antigen at the cell receptor site
and thus reproduce the same biological effect. 94-99
In light of these findings, we attempted to clarify the nature of killer toxin
cell wall receptors in sensitive cells, using antilds raised in New Zealand
rabbits. These animals had been immunized with MAb KT4-secreting hybridoma cells on the assumption that lymphocytes bearing the immunoglobulin
idiotype as receptors might act as a more efficient immunogen lOO (Fig. 5-1j).
The specificity of the antilds raised in the rabbit antiserum was evaluated by
a double-immunodiffusion procedure. The reference system consisted of
ammonium sulfate (50% of saturation)-purified MAb KT4 and the P. anomala
UCSC 25F killer toxin used as an antigen.
The rabbit anti-idiotypic antiserum reacted in immunodiffusion with MAb
KT4, producing a single precipitin band that was homologous to the one of
the reference system (Fig. 5-1g). No reaction was observed when the rabbit

anti-idiotypic antiserum was tested with another MAb produced against an
exoantigen of C. albicans. lOl The results suggested that the anti-idiotypic
antiserum was highly specific for the variable region (idiotype) of MAb KT4.
The precipitin band, moreover, disappeared after adsorption of MAb KT4
with the yeast killer toxin. This observation implies that the antigen blocked
the combining site of MAb KT4. The rabbit antiserum thus appeared to
contain subpopulations of antilds that shared structural similarities with the
portion of the killer toxin that binds with MAb KT4, while the combining
site of MAb KT4 can be considered as the cell wall receptor-like idiotype.

Detection of Yeast Killer Toxin Microbial Receptors
Ideally, MAb KT4 could be used to visualize yeast killer toxin bound to
sensitive cells and to ascertain the presence of common cell wall receptors on
the surfaces of taxonomically unrelated microorganisms. The cell wall of C.
albicans has been recognized to contain a (1,6)-f3-D-glucan, 102 which is the
likely site of binding for the toxin. However, previous studies using a conventional sandwich constituted of sensitive cell, yeast killer toxin, yeast
killer toxin MAb, anti-mouse fluorescein-conjugated immunoglobulins (or
anti-mouse biotinylated antibodies and fluorescein-streptavidin) have failed
to detect killer toxin receptors in sensitive C. albicans cells (unpublished
data). This negative finding may have been caused by the dissociation of the
bound toxin during washings, to the masking of specific killer toxin epitopes
for the MAb by cell wall receptors themselves, or other reasons.
In contrast, the use of antilds that mimicked the activity of yeast killer


5-Killer System Interactions

151

toxin made it possible to visualize directly the interaction with the cell wall

receptors on sensitive C. albicans yeast cells by indirect immunofluorescence (Fig. 5-1k). Reactivity was clearly detectable on the outer cell wall of
the yeast cells. The degree of fluorescence varied according to the growth
phase of each cell. Immunofluorescence was mainly detectable in budding
cells and germ tubes and never inside yeast cell. No fluorescence was
observed when antilds, previously adsorbed with MAb KT4, were used
which attests to the specificity of the reaction 103 (Fig. 5-11). Interestingly,
cell wall receptors were also revealed by the antilds in killer cells of P. anomala, which were obviously resistant to their own toxin, and in K. lactis
cells, which proved to be sensitive to the P. anomala killer toxin (Fig. 5-1m).
Preliminary results have suggested that taxonomically unrelated eukaryotic and prokaryotic isolates, such as S. schenckii (yeast and mold form of
growth) and gram-positive (S. aureus) and gram-negative (E. coli, P. aeruginosa) bacteria, which had previously proven to be susceptible to killer
yeasts, might show the presence of compatible yeast killer toxin cell wall
receptors under some circumstances (Fig. 5-1n). The therapeutic implications of such a finding become even more evident when we consider that no
fluorescence was detectable on animal cells (HeLa, Vero, HEp2) in vitro
(unpublished data) (Fig. 5-10). Theoretically, it should be possible to develop new antibiotics that react with a specific physiological target (the yeast
killer toxin cell wall receptor). It is reasonable to hope that these antilds
antibiotics will create fewer adverse side effects than killer toxins themselves
or other more conventional drugs adopted as antifungal agents in clinical
use.

Antibiotic Activity of Anti-Idiotypic Antibodies
Anti-idiotypic antibodies purified by affinity chromatography against immobilized MAb KT4 were used to investigate their in vitro killing activity on
sensitive C. albicans (CDC B385) cells (Fig. 5-1p). After overnight adsorption, CFUs were compared in inocula incubated with antilds and with
phosphate-buffered saline used as a control. The former repeatedly showed
statistically significant reduction of CFU when compared with the control.
It was also interesting to observe that in the same experimental design,
the antilds were able to kill P. anomala cells secreting UCSC 25F killer toxin
that had previously shown resistance to the action of their own toxin (Fig.
5-1q). The cell's normal mechanisms of immunity to its own toxins (proteases, reduction in cell wall receptors) are apparently ineffective against the
toxin-mimicking antilds.
The neutralization of the killer activity of antilds after adsorption with the

complementary MAb KT4 should attest to the specificity of their action 104
(Fig. 5-1r). The activity of antilds against toxin-resistant killer cells such as P.
anomala UCSC 25F implies that antilds may have wider ranges of activities


152

Polonelli et aI.

than the toxins themselves. It may be, however, that binding of the antiIds
to a cell wall receptor alone, as visualized in prokaryotic microorganisms, is
not sufficient for killing activity.

Idiotypic Vaccination
An important aspect of the therapeutic potential of antilds is the fact that
they can be elicited in vivo by immunization with the complementary MAb
(idiotypic vaccination). Administration of MAb KT4 to syngeneic mice
according to standardized schedules of immunization elicited high titers of
antilds which were associated with a significant degree of protection in vivo
against lethal infections with C. albicans cells (Fig 5-1s).
Increased survival rates were seen with lower infecting doses and higher
MAb immunization doses. The specificity of this immunoprotection was
demonstrated by high antild levels detected during the entire course of
immunization by neutralization enzyme-linked immunosorbent immunometric assay exploiting the competition of antilds with yeast killer toxin
for the binding site of MAb KT4 as well as by the in vitro killer activity of
mouse antilds purified by affinity chromatography against MAb KT4 tested
in the CFU assay on the yeast cells used for infection (manuscript in publication) (Fig. 5-lt).
Specific immunity may be evoked in animals against an infectious agent by
the whole antigen (or its fractions), as occurs in conventional vaccination, by
its derivatives, as occurs in recombinant vaccination, or through the mediation of internal images of the antigen, as demonstrated in anti-idiotypic vaccination.

Our approach, in which the immunizing agent was represented by MAb
KT4, constitutes a unique type of vaccination in that the protective immunoglobulins that it elicits (antilds) act more like antibiotics ("antibiobodies") than like antibodies against the target organism (C. albicans).

Perspectives
New techniques that are capable of generating human or chimeric MAbs
could provide autologous antibodies for the induction of antilds in humans.
Alternatively, the hypervariable regions of murine MAbs could be sequenced to allow synthesis of artificial haptens that could be carried on human proteins. These approaches would theoretically eliminate the problem
of toxicity associated with the therapeutic use of either killer toxins themselves or heterologous immunoglobulin derivatives while maintaining the
high levels of antimicrobial activity. The network theory implies that properly engineered antiIds could be used not only prohylactically but also
therapeutically in animals and humans already affected by systemic mycoses.


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153

Acknowledgments
This work was supported by the National Research Council (C.N.R) Target
Projects on Biotechnology and Bioinstrumentation, Prevention and Control
of Diseases Factors (FAT-MA) and by the Istituto Superiore di Sanita'
(I.S.S.) Third Project on AIDS Research (1990), contract 5204008.

References
1. Herskowitz I. Yeast as the universal cell. Nature (London) 1985; 316: 678-679.
2. Nurse P. Yeast aids cancer research. Nature (London) 1985; 313:631-632.
3. Hollings M. Viruses associated with a die-back disease of cultivated mushroom.
Nature (London) 1962; 196: 962-965.
4. Kleinschmidt WJ, Cline JC, Murphy EB. Interferon production induced by
statolon. Proc Natl Acad Sci USA 1964; 52:741-744.
5. Beale GH. The genetics of Paramecium aurelia. University Press, Cambridge.

1954.
6. Bevan EA, Makower M. The physiological basis of the killer character in yeast,
in Geerts SJ (ed), Genetics Today, XIth Int Congr Genet (Vol 1). Pergamon
Press, Oxford. 1963; pp 202-203.
7. Fried HM, Fink GR. Electron microscopic heteroduplex analysis of "killer"
double-stranded RNA species from yeast. Proc Natl Acad Sci USA 1978;
75: 4224-4228.
8. Wickner RB, Leibowitz MJ. mak mutants of yeast: mapping and characterization. ] Bacteriol1979; 140: 154-160.
9. Bostian KA, Sturgeon JA, Tipper DJ. Encapsidation of yeast killer doublestranded ribonucleic acids: dependence of M on L. ] Bacteriol1980; 143 :463470.
10. Bostian KA, Bussey H, Tipper DJ. Toxin precursor-gene sequence in MIdsRNA. Abstr Cold Spring Harbor Lab Meet Mol Bioi Yeast.l983; p 289.
11. Skipper N. Synthesis of a double-stranded cDNA transcript of the killer toxincoding region of the yeast M1 double-stranded RNA. Biochem Biophys Res
Commun 1983; 114:518-525.
12. Bussey H, Boone C, Dmochowska A, et al. Secretion and action of yeast K1
killer toxin, in Koltin Y, Leibowitz MJ (eds), Viruses of Fungi and Simple
Eukaryotes. Marcel Dekker Inc, New York. 1988; pp 161-178.
13. Sturley SL, EI-Sherbeini M, Kho S-H, et al. Acquisition and expression of the
killer character in yeast, in Koltin Y, Leibowitz MJ (eds). Viruses of Fungi and
Simple Eukaryotes. Marcel Dekker Inc, New York. 1988; pp 179-208.
14. Bussey H, Saville D, Hutchins K, et al. Binding of yeast killer toxin to a cell
wall receptor on sensitive Saccharomyces cerevisiae. ] Bacteriol 1979;
140: 888-892.
15. de la Pena P, Barros F, Gasc6n S, et al. Effect of yeast killer toxin on sensitive
cells of Saccharomyces cerevisiae. ] Bioi Chem 1981; 256: 10420-10425.
16. Bussey H. Effects of yeast killer factor on sensitive cells. Nature (London) New
Bioi 1972; 235: 73-75.
17. Douglas CM, Sturley SL, Bostian KA. Role of protein processing, intracellular
trafficking and endocytosis in production of and immunity to yeast killer toxin.
EurJ Epidemiol1988; 4: 400-408.
18. Kagan BL. Mode of action of yeast killer toxins: channel formation in lipid
bilayer membranes. Nature (London) 1983; 302: 709-710.

154

Polonelli et al.

19. Yamamoto T, Imai M, Tachibana K et al. Application of monoclonal antibodies
to the isolation and characterization of a killer toxin secreted by Hansenula
mrakii. FEBS Lett 1986; 195:253-257.
20. Yamamoto T, Hiratani T, Hirata H, et al. Killer toxin from Hansenula mrakii
selectively inhibits cell wall synthesis in a sensitive yeast. FEBS Lett 1986;
197:50-54.
21. Koltin Y, Day PRo Inheritance of killer phenotypes and double-stranded RNA
in Ustilago maydis. Proc Natl Acad Sci USA 1976; 73: 594-598.
22. Koltin Y. Virus like particles in Ustilago maydis: mutants with partial genomes.
Genetics 1977; 86:527-534.
23. Koltin Y, Kandel JS. Killer phenomenon in Ustilago maydis: the organization of
the viral genome. Genetics 1978; 88:267-276.
24. Koltin Y. The killer system of Ustilago maydis: secreted polypeptides encoded
by viruses, in Koltin Y, Leibowitz MJ (eds), Viruses of Fungi and Simple
Eukaryotes. Marcel Dekker Inc, New York. 1988; pp 209-242.
25. Gunge N. Some structural aspects oflinear DNA plasmids in yeast, in Wickner
RB, Hinnebusch A, Lainbowitz AM, et al. (eds), Extrachromosomal Elements
in Lower Eukaryotes. Plenum Press, New York. 1986; pp 449-461.
26. Gunge N, Tamaru A, Ozawa F, et al. Isolation and characterization of linear
deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid
associated killer character. J Bacteriol1981; 145:382-390.
27. Stark MJ, Boyd A. The killer toxin of Kluyveromyces lactis: characterization of
the toxin subunits and identification of the genes which encode them. EMBO J
1988; 5: 1995-2002.

28. Gunge N, Yamane C. Incompatibility of linear DNA killer plasmids pGKIl and
pGK12 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae. J Bacteriol1984; 159: 533-539.
29. El-Sherbeini M, Bostian KA. Viruses in fungi: infection of yeast with Kl and K2
killer viruses. Proc Natl Acad Sci USA 1987; 84:4923-4927.
30. Morace G, Archibusacci C, Sestito M, et al. Strain differentiation of pathogenic
yeasts by the killer system. Mycopathologia 1984; 84: 81-85.
31. Sawant AD, Abdelal AT, Ahearn DG. Anti-Candida albicans activity of Pichia
anomala as determined by a growth rate reduction assay. Appl Environ Microbioll988; 54:1099-1103.
32. Sawant AD, Abdelal AT, Ahearn DG. Purification and characterization of the
anti-Candida toxin of Pichia anomala WC 65. Antimicrob Agents Chemother
1989;33:48-52
33. Kurtzman CPo Synonymy of the yeast genera Hansenula and Pichia demonstrated through comparison of deoxyribonucleic acid relatedness. Antonie van
LeeuwenhoekJ Microbiol SeroI1984; 50:209-217.
34. Starmer WT, Ganter PF, Aberdeen V, et al. The ecological role of killer yeasts
in natural communities of yeasts. Can J Microbiol1987; 33: 783-796.
35. Middelbeek EJ, Peters GJWH, Stumm C, et al. Properties of a Cryptococcus
laurentii killer toxin and conditional killing effect of the toxin on Cryptococcus
albidus. FEMS Microbiol Lett 1980; 9:81-84.
36. Pietras DF, Bruenn JD. The molecular biology of yeast killer factor. Int J
Biochem 1976; 7: 173-179.
37. Bruenn JD. Virus-like particles of yeast. Annu Rev Microbiol1980; 34:49-68.
38. Bussey H. Physiology of killer factor in yeast. Adv Microb Physiol. 1981;
22:93-112.
39. Tipper DJ, Bostian KA. Double-stranded ribonucleic acid killer system in
yeasts. Microbiol Rev 1984; 48: 125-156.
40. Wickner RB. Killer yeasts, in McGinnis MR (ed), Current Topics in Medical
Mycology. Springer-Verlag, New York. 1985; pp 286-312.


5-Killer System Interactions

155

41. Fujimura T, Esteban R, Wiclmer RB. Double-stranded DNA replication in
Saccharomyces cerevisiae: genetic control and in vitro replication, in Koltin Y,
Leibowitz MJ (eds), Viruses of Fungi and Simple Eukaryotes. Marcel Dekker
Inc, New York. 1988; pp 9l-115.
42. Hutchins K, Bussey H. Cell wall receptor for yeast killer toxin: involvement of
(1,6)-J3-n-glucan. ] Bacteriol1983; 154: 161-169.
43. Al-Aidroos K, Bussey H. Chromosomal mutants of Saccharomyces cerevisiae
affecting the cell wall binding site for killer factor. Can] Microbiol 1978;
24:228-237.
44. Zhu H, Bussey H. The K1 toxin of Saccharomyces cerevisiae kills spheroplasts
of many yeast species. Appl Environ Microbiol1989; 55: 2105-2107.
45. Wiclmer RB. Chromosomal and non chromosomal mutations affecting the killer
character of Saccharomyces cerevisiae. Genetics 1974; 76:423-432.
46. Bussey H, Steinmetz 0, Saville D. Protein secretion in yeast: two chromosomal
mutants that oversecrete killer toxin in Saccharomyces cerevisiae. Curr Genet
1984: 7: 449-456.
47. Rogers D, Bevan EA. Group classification of killer yeasts based on cross reactions between strains of dilferent species and origin. ] Gen Microbiol 1978;
105: 199-202.
48. Polonelli L, Conti S, Gerloni M, et al. Production of yeast killer toxin in experimentally infected animals. Mycopathologia, 1990; 110: 169-175.
49. Koltin Y, Day PRo Specificity of Ustilago maydis killer proteins. Appl Microbiol
1975; 30: 694-696.
50. Odds FC. Biotyping of medically important fungi, in McGinnis M (ed), Current
Topics in Medical Mycology. Springer-Verlag, New York. 1985; pp 155-171.
51. Philliskirk G, YoungTW. The occurrence of killer character in yeasts of various
genera. Antonie van Leeuwenhoek] Microbiol Serol1975; 41: 147-151.
52. Stumm C, Hermans JMH, Middelbeek EJ, et al. Killer-sensitive relationships
in yeasts from natural habitats. Antonie van Leeuwenhoek] Microbiol Serol

1977; 43: 125-128.
53. Kandel JS, Stern TA. Killer phenomenon in pathogenic yeast. Antimicrob
Agents Chemother 1979; 15:568-571.
54. Young TW, Yagiu M. A comparison of the killer character in different yeasts and
its classification. Antonie van Leeuwenhoek] Microbiol Serol1978; 44: 59-77.
55. Polonelli L, Archibusacci C, Sestito M, et al. Killer system: a simple method for
dilferentiating Candida albicans strains. ] Clin Microbiol1983; 17: 774-780.
56. Polonelli L, Morace C. Yeast nosocomial infections. Ig Mod 1983; 79:315-334.
57. Polonelli L, Castagnola M, Rossetti DV, et al. Use of killer toxins for computeraided dilferentiation of Candida albicans strains. Mycopathologia 1985;
91:175-179.
58. Polonelli L, Morace C. Reevaluation of the yeast killer phenomenon. ] Clin
Microbiol1986; 24: 866-869.
59. Holland lB. Physiology of colicin action. Adv Microb Physiol1975; 12:56-139.
60. Tagg JR, Dajani AS, Mannamaker LW. Bacteriocins of gram-positive bacteria.
Bacteriol Rev. 1976; 40: 722-756.
61. Hasenclever HR, Mitchell WOo Antigenic studies of Candida. I. Observation
of two antigenic groups in Candida albicans. ] Bacteriol1961; 82: 570-573.
62. Wilson DE, Bennett JE, Bailey JW. Serologic grouping of Cryptococcus
neoformans. Proc Soc Exp Bioi Med 1968; 127: 820-823.
63. Kaufman L, Standard PC, Weeks RJ, et al. Detection of two Blastomyces dermatitidis serotypes by exoantigen analysis. ] Clin Microbiol1983; 18: 110-114.
64. Nishikawa T, Harada T, Harada S, et al. Serologic dilferences in strains of Sporothrix schenckii. Sabouraudia 1975; 13:285-290.
65. Polonelli L, Morace C. Specific and common antigenic determinants of Candi-


156

66.
67.
68.

69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.

Polonelli et aI.

da albicans isolates detected by monoclonal antibody. ] Clin Microbiol 1986;
23: 366-368.
Polonelli L, Castagnola M, Morace G. Identification and serotyping of Microsporum canis isolates by monoclonal antibodies.] Clin Microbiol1986; 23: 609615.
Polonelli L, Dettori G, Cattel C, et al. Biotyping of mycelial fungus cultures by
the killer system. Eur] Epidemiol1987; 3:237-242.
PoloneIIi L, Conti S, Campani L, et al. Biotyping of Aspergillus fumigatus and
related taxa by the yeast killer system, in Samson RA, Pitt JI (eds), Modern

Concepts in Penicillium and Aspergillus Classification. Plenum Press, New
York, 1990; pp. 225-233.
Morace G, Dettori G, Sanguinetti M, et al. Biotyping of aerobic actinomycetes
by modified killer system. Eur] Epidemiol1988; 4:99-103.
Morace G, Manzara S, Dettori G, et al. Biotyping of bacterial isolates using the
yeast killer system. Eur] Epidemiol1989; 5:303-310.
Marsik FJ, Paris is JT. Bacteriophage types and 0 antigen groups of Escherichia
coli from urine. Appl MicrobioI1971; 22: 26-31.
Smith PB. Bacteriophage typing of Staphylococcus aureus, in Cohen JO (ed),
The Staphylococci. Wiley Interscience, New York. 1972; pp 431-441.
Lindberg RB, Latta RI. Phage typing of Pseudomonas aeruginosa: clinical and
epidemiologic considerations. ] Infect Dis. 1974; 130(Suppl): S33-S42.
Aber RC, Mackel DC. Epidemiologic typing of nosocomial microorganisms.
Am] Med 1981; 70:899-905.
Janda JM, Bot,tone EJ. Pseudomonas aeruginosa enzyme profiling: predictor of
potential invasiveness and use as an epidemiological tool.] Clin Microbiol1981;
14:55-60.
Bauerfeind A, PetermuIIer C, Schneider R. Bacteriocins as tools in analysis of
nosocomial Klebsiella pneumoniae infections.] Clin Microbiol1981; 14: 15-19.
Fyfe JAM, Harris G, Govan JRW. Revised piocyn typing method for Pseudomonas aeruginosa.] Clin MicrobioI1984; 20:47-50.
Moseley SL, Hug I, Alim ARMA, et aI. Detection of enterotoxinogenic Escherichia coli by DNA colony hybridization.] Infect Dis 1980; 142:892-898.
Tompkins LS, Plorde JJ, Falkow S. Molecular analysis of R-factors from multiresistant nosocomial isolates. ] Infect Dis 1980; 141: 625-636.
Polonelli L, Conti S, Campani L, et aI. Yeast killer toxins and dimorphism. ]
Clin Microbiol1989; 27: 1423-1425.
Di Masi DR, Klute JC, Schnaitman CA, et aI. Transport of vitamin Bl2 in
Escherichia coli: common receptor sites for vitamin Bl2 and the E colicins on
the outer membrane of the cell envelope.] Bacteriol1973; 115:506-513.
Bussey H, Skipper N. Killing of Torulopsis glabrata by Saccharomyces cerevisiae killer factor. Antimicrob Agents Chemother 1976; 9:352-354.
Yamamoto T, Uchida K, Hiratani T. In vitro activity of the killer toxin from
yeast Hansenula mrakii against yeasts and molds.] Antibiot 1988; 41 :398-403.

Otha Y, Tsukada Y, Sugimori T. Production, purification and characterization of
HMI, an anti-yeast substance produced by Hansenula saturnus. Agric BioI
Chem 1984; 48:903-908.
Polonelli L, Lorenzini R, De Bernardis F, et al. Potential therapeutic effect of
yeast killer toxin. Mycopathologia 1986; 96: 103-107.
Polonelli L, Morace G. Production and characterization of yeast killer toxin
monoclonal antibodies.] Clin Microbiol1987; 25:460-462.
O'Leary E. Secretion of multiple killer toxins by Williopsis mrakii. Abstr Annu
Meet Am Soc Microbiol1988; p 394.
Ashida S, Shimazaki T, Kitano K, et aI. New killer toxin of Hansenula mrakii.
Agric BioI Chem 1983; 47: 2953-2955.


5-Killer System Interactions

157

89. Polonelli L, Manzara S, Conti S, et al. Serological study of yeast killer toxins by
monoclonal antibodies. Mycopathologia 1989; 108:211-215.
90. Yamada Y, Okada T, Ueshima 0, et al. Coenzyme Q system in the classification
of the ascosporogenous yeast genera Hansenula and Pichia. ] Gen Appl Microbioi 1973; 19: 189-208.
91. Polonelli L, Conti S, Campani L, Gerloni M, Morace G, Chezzi C. Differential
toxinogenesis in the genus Pichia detected by an anti-yeast killer toxin monoclonal antibody. Antonie van Leeuwenhoek 1991; 59: 139-145.
92. Burdette S, Schwartz RS. Idiotypes and idiotypic networks. N EnglJ Med 1987;
317:219-224.
93. Jerne NK. Towards a network theory of the immune system. Ann Immunol
(Inst Pasteur) 1974; 125C: 373-389.
94. Williams WV, Weiner DB, Cohen JC, et al. Development and use of receptor
binding peptides derived from antireceptor antibodies. Bio/Technology 1989;
7:471-475.

95. Marasco MA, Becker EL. Anti-idiotype as antibody against the formyl peptide
chemotaxis receptor of the neutrophil. ] Immunol1982; 128: 963-968.
96. Nepom JT, Weiner HL, Dichter MA, et al. Identification of a hemagglutininspecific idiotype associated with reovirus recognition shared by lymphoid and
neural cells. ] Exp Med 1982; 155: 155-167.
97. Sacks DL, Esser KM, Sher A. Immunization of mice against African trypanosomiasis using anti-idiotypic antibodies. ] Exp Med 1982; 155: 1108-1119.
98. Sacks DL, Sher A. Evidence that anti-idiotypic induced immunity to experimental African trypanosomiasis is genetically restricted and requires recognition of combining sites-related idiotopes. ] Immunol1983; 131: 1511-1515.
99. Sege K, Peterson PA. Use of anti-idiotypic antibodies as cell-surface receptor
probes. Proc Natl Acad Sci USA 1978; 75:2443-2447.
100. Polonelli L, Morace G. Yeast killer toxin-like anti-idiotypic antibodies. ]
Clin Microbiol1988; 26: 602-604.
101. Polonelli L, Morace G. Specific and common antigenic determinants of Candida albicans isolates detected by monoclonal antibodies. ] Clin Microbiol1986;
23: 366-368.
102. Gopal PK, Sheperd MG, Sullivan PA. Analysis of wall glucans from yeast,
hyphal and germ-tube forming cells of Candida albicans. ] Gen MicrobioI1984;
130: 3295-3301.
103. Polonelli L, Fanti F, Conti S, Campani L, Gerloni M, Castagnola M, Morace
G, Chezzi C. Detection by immunofluorescent anti-idiotypic antibodies of
yeast killer toxin cell wall receptors of Candida albicans. ] Immunol Methods
1990; 132:205-209.
104. Polonelli L, Conti S, Gerloni M, Campani L, Castagnola M, Morace G, Chezzi
C. "Antibiobodies": antibiotic-like anti-idiotypic antibodies. ] Med Vet Mycology, in press.


6-Allylamine Antifungal Drugs
NEIL

S.

RYDER AND HUBERT MIETH

The allylamines constitute a recently developed class of synthetic antimycotics characterized functionally by their action as squalene epoxidase
inhibitors. 1 Figure 6-1 shows the structures of three representative allylamines. Naftifine, the first of these compounds to be discovered, was first
synthesized in 1974,2 and its antifungal properties were identified during
routine screening. The potent antifungal activity of naftifine in vitr03 and in
viv0 4 led to its clinical development, and this drug has been marketed since
1985 as a topical antimycotic. Naftifine provided the basis for an extensive
program of chemical derivatization 5 - 8 aimed at improving the antimycotic
efficacy, especially with regard to oral administration. This goal was achieved
in the form of terbinafine (SF 86-327),1,6-13 the efficacy of which has now
been confirmed in numerous clinical studies involving both topical and oral
application. Parallel to this development, detailed investigations were carried out concerning the mechanism of action of the allylamines, 14- 26 including much basic research on the biochemistry of ergosterol biosynthesis in
pathogenic fungi.
A considerable body of literature on the experimental and clinical properties of the· allylamines has now arisen in the decade since the first
presentation27 of naftifine. In this chapter, we aim to present a comprehensive overview of the available data on this new class of antimycotics, with
emphasis on the underlying biological and biochemical aspects of relevance
to their clinical application.

Experimental Antimycotic Activity
Spectrum of Antifungal Action In Vitro
In in vitro tests, both naftifine and terbinafine are active against a broad
spectrum of pathogenic fungi. 3 ,9,10,12,13,28 Activity is extremely high against
dermatophytes, with minimum inhibitory concentrations (MICs) in the
158


6-Allylamine Antifungal Drugs

159

FIG. 6-1. Structures of the allylamine antimycotics naftifine (1), terbinafine (2Land

SDZ 87-469 (3).

nanogram/milliliter range in the case of terbinafine and generally very good
against the majority of filamentous and dimorphic fungi. The 3-chloro-7benzo[b]thienyl derivative SDZ 87-469 is even more active than terbinafine
against most fungi tested. 29.30 Terbinafine has also been reported to have
activity against a wide range of unusual fungal pathogens. 31 - 34 Table 6-1
summarizes the published data on the spectrum of action of these three
allylamines against filamentous and dimorphic fungi, and Table 6-2 shows
data for yeasts. In addition to its effects on human and animal pathogens,
terbinafine has also been reported to inhibit several fungi of agricultural
importance as plant pathogens. 42-45
Activity of the allylamines against yeasts is quite variable between different species and strains. For example, Candida parapsilosis is very susceptible whereas C. glabrata shows little response (Table 6-2). Susceptibility of
C. albicans varies considerably among strains. SDZ 87-469 is markedly more
active than terbinafine against pathogenic yeasts in generaJ.29 Of particular
interest is the much higher susceptibility to allylamines of the filamentous
form ofC. albicans,22.46.47 which plays an important role in pathogenicity of
this organism. This is probably a significant factor in the clinical efficacy of
allylamines against Candida infections.
It should be noted that the data in Tables 6-1 and 6-2 are derived from
several workers using a variety of methods and are intended only as a guide
to the spectrum of activity of the allylamines. In vitro antifungal data are
notoriously variable and can be strongly influenced by factors such as
medium composition, pH, inoculation size, and endpoint interpretation. 48


Dermatophytes
Epidennophyton Jloccosum
Microsporum species
Trichophyton species
Filamentous fungi

Aspergillus fumigatus
A.Jlavus
A. niger
A. terreus
Aspergillus species
Pseudallescheria boydii
Scopulariopsis brevicaulis
Zygomycetes b
Acremonium species

Fungus

0.02-5.0
0.01-0.5
0.005-0.5
0.05-5.0
0.005-5.0
32->64
0.8-8:0
64->128
1.0-4.0

2.0-12.5
0.25

64- >128

0.8-12.5
16-64

0.001-0.006
0.002-0.01
0.001-0.01

Terbinafine

<0.06-0.5
<0.06-2.0
0.05-1.0

Naftifine

SDZ 87-469<'

0.05

0.02

0.01-0.05

0.0004-0.02
(all dermatophytes)

Range of MIC values (lLg/ml)

TABLE 6-1. Spectrum of action in vitro of naftifine, terbinafine, and SDZ 87-469 against filamentous and dimorphic fungi

9,10,12,31,36-38
9,31,36-38
36-38

36,38
3,31,36,38
9,31
10,31,36
9
31

3,9,10,35,36

Reference(s)

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~0.05-2.0

~0.05-0A

sO.05-0.2

sO.05-0A
sO.06-8.0

0.25-0.5
0.05->64
1.0-4.0
0.25-0.50
1.0-4.0

8-64
0.1
1.0-4.0
<0.06-2.0
<0.06-0.5
0.01-1.0
0.01-0.03
0.001-0.025

~0.05-0A

0.25-1.0
0.5-4.0

data for SDZ 87-469 are from references 29 and 30.
Rhizopus species.
C Fonseceae, Phialophora, and Cladosporium species.
d Exophiala jeanselmei, C. bantianum, and W. dermatitidis.

b Mucor and

a All

Fusarium species
Hendersonula toruloidea
Lasiodiploidia theobromae
Madurella species
Paecilomyces species
Phialophora parasitica
Scytalidium hyalinum

Chromoblastomycosis agentsc
Pheohyphomycosis agents d
Cladosporium bantianum
Dactylaria constricta
Wangiella dermatitidis
Dimorphic fungi
Blastomyces dermatitidis
Histoplasma capsulatum
Sporothrix schenckii

C urvularia fallax

0.02-0.1

9
9
3,9,10,31,36

31
31,33,34
31
31
31
31
32
31
9,31
9
56
56

39,56

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