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Insecticidal bacterial proteins identify the midgut epithelium as a source of novel target sites for insect control.

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Archives of Insect Biochemistry and Physiology 22:357-371 (1 993)
Insecticidal Bacterial Proteins Identify the
Midgut Epithelium as a Source of Novel
Target Sites for Insect Control
Brian A. Federici
Department of Entomology and Interdepartmental Graduate Program in Genetics, University of
California, Riverside, California
The spore-form ing bacterium, Bacillus thuringiensis (Bt), produces a crystal Iine
parasporal body during sporulation, which i n many subspecies contains one
or more proteins selectively toxic to insect midgut epithelia. Most of these
proteins are protoxins with a molecular mass of about 133,000. When ingested
by a susceptible insect, the inclusions dissolve in the midgut juices and are
activated by proteolytic cleavage, which releases a toxic peptide of about
65,000. In susceptible insects, this peptide binds to sites on the microvillar
membrane, causing cytolysis, apparently by forming transmembrane pores.
The cytolysis of midgut cells results i n paralysis and subsequent death of the
insect. Though less common, naturally truncated protein toxins with masses
of about 70,000 also occur. Three major pathotypes of Bt proteins are known:
Cry1 and Cryll proteins toxic primarily to lepidopterous insects, Crylll proteins
toxic to coleopterous insects, and CrylV proteins toxic to dipterous insects.
The genes encoding more than 50 Bt crystal proteins have been cloned and
sequenced and several of these have already been used to construct recornbinant microbial insecticides and transgenic organisms including viruses,
algae, and insect-resistant plants. Analysis of Bt genes indicate that the protein
toxins they encode consist of at least two functional domains: a seriesoffive
blocks of conserved amino acids that comprise the structural core of the
molecule including the putative toxic moiety, and a hypervariable region
thought to define the spectrum of activity. Determination of the mode of action
at the molecular level and the genetic basis for insect specificity should enable
recombinant DNA technology to be used to expand the insect host range of
Bt as well as increase its toxicity against insects. Moreover, the high toxicity
and specificity of the insecticidal proteins produced by Bt and their action on
midgut microvilli suggest that other types of peptides or simple organic
molecules could be designed as selective insecticides to attack other midgut
functions such as ion channels, peptide and amino acid transport systems,
and midgut proteases. o 1993 WiIey-Liss, Inc.
Key words: Bacillus thuringiensis, insecticidal proteins, midgut epithelium
Received December 16, 1991; accepted March 26, 1992.
Address reprint requests to Dr. Brian A. Federici, Department of Entomology, University of
California, Riverside, CA 92521.
0 1993 Wiley-Liss, Inc.
358
Federici
INTRODUCTION
The critical need for safe and effective alternatives to chemical insecticides
has stimulated considerable interest in using pathogens as biological control
agents for insects of agricultural and medical importance. Although interest
is now greater than ever, the actual use of pathogens in control programs is
low, and accounts for less than 1%of the insect control agents used annually
around the world. The pathogen that has been the most successful and holds
considerable potential for further development is the insecticidal bacterium
Bacillus thuringiensis.
The species Bacillus thuringiensis (Bt) Berliner is actually a complex of
bacterial subspecies, all of which are characterized by the production of a
parasporal body during sporulation. This parasporal body contains one or
more proteins, typically in a crystalline form, and many of these are highly
toxic to certain species of insects. The crystal proteins of insecticidal isolates
are known as endotoxins and typically occur in the parasporal body as
protoxins, which after ingestion are activated by proteolysis in the midgut
lumen. The activated toxins destroy midgut epithelial cells, paralyzing the
insect and eventually causing its death.
Over the past decade, research has intensified on Bt as a result of several
factors, the most important of which are continued pressure to reduce or
eliminate synthetic chemical insecticides from pest control programs, the
safety of the bacterium and its protein toxins to vertebrates and the environment, its unique mode of action, its chemical properties as a protein insecticide, and the development of recombinant DNA technology. The latter two
are particularly important because our knowledge of Bt protein molecular
biology is advancing rapidly, and this knowledge will facilitate the use of
recombinant DNA techniques to develop a much wider range of insect control
agents and strategies. These will likely include recombinant insecticides as
well as transgenic plants protected against insects such as grasshoppers,
aphids, and others for which we currently have no effective Bts or other
control methods. As a result of the enormous potential that recombinant DNA
technology provides for advances in plant protection and other future insect
control strategies based on Bt, almost every major chemical company, and
many emerging biotechnology companies worldwide, are investing heavily
in this area of research.
Aside from this specific focus on Bt, the novel mode of action of Bt proteins,
i.e., the use of midgut microvillar proteins as targets to attack and cause insect
death, suggests that alternative midgut functions or proteins might prove just
as useful as nove1 target sites. In particular, these would include the numerous
enzymatic and transport proteins that occur in the midgut microvillar membrane and are so important to insect growth and development.
In the context of this meeting, which deals primarily with the nervous
system as a source of new target sites for insect control, the purpose of this
paper is to show that what we have learned about the insecticidal proteins
of B . thuringiensis over the past ten years identifies the midgut epithelium
as a potential source of new target sites. This will be done by briefly
reviewing our knowledge of the basic biology of Bt and the insecticidal
Insecticidal Bacterial Proteins
359
proteins it produces, and then suggesting examples of midgut functions that
might serve as target sites.
SYSTEMATICS AND GENERAL BIOLOGY
From the standpoint of bacterial systematics, B. thuringiensis is a very close
relative of B. cereus, and in fact the only reliable character that differentiates
the two is the occurrence of the parasporal body in the former. As a result,
the validity of maintaining the two as separate species has legitimately been
brought into question [l].It also now appears that in almost all known isolates
of B. thuringiensis, the proteins that make up the parasporal body are encoded
on plasmids borne by the bacteria [2]. These plasmids can be lost during
growth and reproduction, and when this happens, the B. thuringiensis strain
“becomes” B. cereus, using accepted methods of bacterial identification and
classification. Nevertheless, because the name is so entrenched in the literature, and the bacterium is becoming so important in pest control, the name
B. thuringiensis is likely to stand, and with the subspecific epithets, will be
used here.
As of 1991, at least several thousand isolates of Bt have been obtained from
a variety of sources such as living and dead insects, soil, grass, grain dust,
and water. These have been divided into groups, of which there are now
nearly 30 [3], that are differentiated from one another on the basis of the H
antigen, i.e., flagellar serotype, which is indicated by a number (e.g., H 1)or
a number and letter combination (e.g., H 3a3b) as well as a serovar name (e.g.,
H 1 is thuringiensis; H 3a3b is kurstukz).
Despite Bt’s growing importance as a pesticide, little is actually known
about its biology and the role of the insecticidal parasporal body in nature. As
noted above, Bt can be isolated from a wide variety of habitats, but it does not
grow well in many of these because it is not a major or dominant species,
Moreover, it is interesting in that, unlike many viruses, fungi, and protozoa,
Bt has never been reported as the cause of large-scale epizootics. Bt is,
however, quite commonly isolated from grain dust, and the original description by Berliner [4]was based on an isolate from the Mediterranean flour
moth, Anugustu kuehniellu (Zeller). In addition, the cadavers of insects killed
by Bt provide very suitable substrates for Bt’s reproduction and sporulation
[ 5 ] .It would appear, therefore, that bacteria which harbor plasmids encoding
insecticidal bacterial proteins have a selective advantage when the spores and
parasporal bodies occur together, and the number of parasporal bodies
ingested by an insect are sufficient to cause death. Despite Bt’s widespread
occurrence in nature, optimal reproduction probably occurs in insects, and its
biology, or perhaps more appropriately that of the plasmids which bear the
insecticidal proteins, may be more meaningfully understood in this context.
MAJOR PATHOTYPES
The numerous isolates of Bt that have been screened for insecticidal activity
can be divided among three major pathotypes: those which exhibit toxicity to
either (a) lepidopterous, (b) dipterous, or (c) coleopterous insects (Table 1).
360
Federici
TABLE 1. Nomenclature and Properties of Insecticidal Proteins and Their Encoding Genes
From Bacillis thurinD.ensis*
Gene taxon
Protein name
Spectrum of activity
cylA(a)
cylA(b)
cyIA(c)
cylB
cyIC
cyID
cyIE
cyIlA
cylIB
cy1IlA
cy1lIB
cylVA
cylVB
cyrvc
cyIVD
CryIA(a)
CryIA(b)
CryIA(c)
CryIB
CryIC
CryID
CryIE
CryIIA
CryIIB
CryIIIA
CryIIIB
CryIVA
CryIVB
CryIVC
CryIVD
CVtA
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera/Diptera
Lepidoptera
Coleoptera
Coleoptera
Dipterab
Diptera
Diptera
Diptera
Diptera/other
CytA
Amino acidsa Mass (kDa)=
1,176
1,155
1,178
1,207
1.189
1,165
1,171
633
633
644
659
1,180
1,136
675
643
248
133.2
131.0
133.3
138.0
134.8
132.5
132.0
70.9
70.8
73.1
74.2
134.4
127.8
77.8
72.4
27.4
*From Hofte and Whiteley [2].
aThese proteins are protoxins which are activated in vivo by proteolytic cleavage in the insect
midgut after ingestion. The Cry proteins are cleaved to form activated toxins in the range of
60-65 kDa, with most of the protein (ca 600 amino acids) in the 130 kDa size range being cleaved
from the c-terminus. A small amount of cleavage also occurs (25-30 amino acids) from the
n-terminus of all Cry proteins.
bCryIVproteins are only of significant toxicity to dipterous insects of the suborder nematocera,
e.g.,insects such as mosquitoes, blackflies, chironomid midges, psychodid flies, and crane flies.
‘In vitro, the CytA protein has been shown to be cytolyticfor a wide range of cell types including
those from invertebrates, as well as vertebrates. Proteolysis yields a broadly cytolytic protein of
25 kDa.
The first successful commercial product for insect control was registered
well over 20 years ago and had as its active ingredient the HD 1isolate of B.
thuringiensis subspecies kurstuki (serotype H 3a3b), an isolate highly and
selectively toxic to lepidopterous insects [6]. Early studies of this isolate
showed that it produced a bipyramidal parasporal body that consisted of a
protein protoxin with a molecular mass of approximately 133,000. When
ingested by a caterpillar, this protein was solubilized in the alkaline midgut
and activated through cleavage by midgut proteases, releasing a toxic core
peptide of about 65,000. Later it was shown that HD 1also produced a protein
with a mass of 65,000 toxic to mosquito larvae and caterpillars, and that this
crystallized separately into a smaller cuboidal inclusion associated with the
bipyramidal parasporal body [7].
Until the mid-l970s, B. thuringiensis was thought only to produce proteins
that were insecticidal to lepidopterous insects. Then in 1976, Goldberg and
Margalit [8] isolated in the Negev desert of Israel what turned out to be a new
subspecies, i.e., isruelensis (H 14), that proved highly toxic to the larvae of
mosquitoes and blackflies. Studies of the parasporal body of this isolate
showed it to be substantially different from the classic bipyramidal crystal. It
contained at least four major proteins with molecular masses of 27,000,72,000,
Insecticidal Bacterial Proteins
361
128,000, and 135,000, respectively, and these were packaged into different
inclusion types and assembled into a spherical parasporal body held together
by a fibrous, lamellar envelope 191-Studies of these proteins showed that those
with a mass of 72,000, 128,000, and 134,000 were similar to the proteins of
lepidoptern-activeisolates, but that the 27,000 protein was markedly different,
being highly cytolytic to a wide range of vertebrate and invertebrate cells in
vitro [lo]. In addition, several studies have provided evidence that the high
toxicity of the B. thuringiensis subspecies isruelensis parasporal body is due to
synergistic interactions among two or more of its proteins [9, 11, 121.
The discovery of B. thuringiensis subspecies isruelensis was followed in the
mid-1980s by the discovery of yet another pathotype of Bt, B. thuringiensis
subspecies tenebrionis (H 8a8b), which was shown to be toxic to certain
coleopterous insects [13]. This new pathotype contains a single toxin gene,
which expresses at different phases of growth two proteins with molecular
masses of about 70,000, which apparently differ from each other by only slight
differences in size.
PROTEIN AND GENE NOMENCLATURE
The first gene encoding an insecticidal Bt protein (133,000) was cloned from
the HD 1isolate of B . thuringiensis subspecies kurstaki [14]. Subsequent studies
of HD 1 employing cloned toxin genes showed that the plasmids it harbored
encoded at least five insecticidal proteins, four of which occurred in its
parasporal bodies, i.e., three slightly different protoxins with molecular
masses of about 133,000 that cocrystallized in the bipyramidal crystal and a
fourth protein of 65,000 that crystallized to form the associated cuboidal
inclusion. Interestingly, when these cloned genes were used as probes, it was
found that the plasmid complements of other isolates of subspecies kurstuki
(H 3a3b) varied, as did the number of toxin encoding genes they harbored,
with some isolates such as HD 73 containing plasmids encoding and expressing only a single protein toxin. These studies were followed by many others
over the past decade, showing that in all subspecies of Bt, the genes encoding
insecticidal proteins are located primarily on large transmissible plasmids (see
[2] for a recent review).
The existence of three different pathotypes, along with the finding that
different proteins expressed by the same pathotype could differ significantly
in toxicity to the same insect species, led several investigators to begin
computer-assisted analyses of the gene sequences to compare the relatedness
of the various Bt genes as well as to attempt to identify important functional
domains of Bt proteins. These studies revealed that with the exception of the
27,000 protein of B. thuringiensis subspecies isruelensis, all Bt protein toxins
have a significant level of nucleotide and amino acid sequence identity,
despite the various names and numbers by which they had been referred to,
which were typically based upon the subspecies from which they were cloned.
To standardize the terminology and provide a nomenclature that contained
informationbased on the gene and protein rather that the bacterial host, Hofte
and Whiteley [2]proposed a system in which the standard types of Bt proteins
are referred to as “Cry” proteins, and the genes “cry” genes (Table 1).This is
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Federici
followed by a roman numeral which indicates pathotype (I and I1 for toxicity
to lepidopterans, I11 for coleopterans, and lV for dipterans), followed by an
upper case letter indicating the chronological order in which genes with
significant differences were described. The I and II for lepidopteran-toxic
proteins also indicate size differences, with the I referring to proteins with
molecular masses in the 135,000 range, and the II to those in the 70,000 range.
Some epithets also include a lower case letter in parentheses, which indicates
minor differences in the nucleotide sequence within a gene type. Thus, CryIA
refers to a 130,000 protein toxic to lepidopterous insects for which the first
gene (cryZA)was sequenced, whereas CryIVD refers to a 72,000 protein with
mosquitocidal activity for which the encoding gene was the fourth from this
pathotype sequenced. Though perhaps not perfect, this system is preferred
to the chaos that existed before it was proposed.
The 27,000 CytA protein first isolated from B. thuringiensis subspecies
isruelensis differs from other Bt proteins not only in its smaller size, but also
in that it is highly cytolytic to a wide range of cell types in vitro, including
those of vertebrates (see [15] for a review). In addition, it shares no apparent
relatedness with Cry proteins. Owing to these differences and its broad
cytolytic activity, Hofte and Whiteley 121 referred to this as the CytA protein
encoded by the CytA gene.
PARASPORAL BODY SHAPE AND COMPOSITION
In general, the shape of the parasporal body is a good but not absolute
indication of an isolate’s pathotype. For example, most isolates of Bt produce a
large bipyramidal parasporal crystal (0.5 x 1 pm) that is almost always only toxic
to lepidopterous insects. In isolates of some subspecies, such as B. thuringiensis
subspecies kurstuki, the bipyramidal crystal may be accompanied by a smaller
cuboidal crystal toxic to lepidopterans and mosquitoes. Others such as B.
thuringiensis subspecies isruelensis (H 14) and the PG-14 isolate of B. thuringiensis
subspecies mowisoni (H 8a8b) produce spherical parasporal bodies (0.7-1 Fm)
that are toxic primarily to nematocerous dipterans (e.g., mosquito and blackfly
larvae), whereas the ”tenebrionis” strain of B. fhuringiensis subspecies mowisoni
(H 8a8b) produces a thin square crystal that is toxic only to certain species of
coleopterans. The degree of protein complexity within these parasporal bodies
can vary considerably. Single crystals can be composed of a single type of protein
molecule or a mixture of as many as three. In addition, a single parasporal body
may be composed of from one (e.g.,certain isolates of subspecies kurstuki) to four
(e.g., the mosquitocidal PG-14 isolate of subspecies mowisoni) crystals [2,15]. An
example of a simple crystal is that of the HD-73 isolate of B. thuringimsis
subspecies kurstuki. This isolate contains one cry gene and encodes only the
CryM(c) protein which forms a typical bipyramidal crystal during sporulation.
The related HD 1 isolate of the same subspecies, however, carries five cry genes
[CryIA(u), (b), (c); cryZIA; q U B ] and produces at least four of these during
sporulation. The three CryA proteins cocrystallizein a single bipyramidal crystal,
whereas the smaller CryIIA protein forms the associated cuboidal inclusion. The
most complexparasporalbody occursin B. thuringiensis subspecies morrisoni (Fig.
1)where 3 CryIV proteins, one Cry1 protein, and the CytA protein crystallize
Insecticidal Bacterial Proteins
363
,144
-135
‘128
- 65
- 27
Fig. 1 . Illustration of some of the key properties of the rnosquitocidal parasporal body of 6acillus
thuringiensis subspecies morrisoni (PC-14). a: Scanning electron micrograph of the basically
spherical parasporal body and a dislodged bipyrarnidal crystal. x 30,000. b: Transmission electron
micrograph illustrating 4 different inclusions of the parasporal body bound together by a fibrous
envelope. x 80,000. c: Comparison of the protein composition of the parasporal bodies from 6.
thuringiensis subspecies israelensis (1) and 6. thuringiensis subspecies morrisoni (2). The numbers
to the right indicate approximate masses of the proteins in kilodaltons.
into four different inclusion types that are bound together in a fibrous
envelope [151.
GENETIC AND STRUCTURALANALYSES OF CRY PROTEINS
Since the cloning and sequencing of the first Bt insecticidal protein gene
[14], more than fifty other genes have been cloned and sequenced. Analysis
364
Federici
of the numerous nucleotide and deduced amino acid sequences available now
for cry genes/Cry proteins has identified two key features common to the
activated portions of most Bt proteins regardless of pathotype [Z].The first is
that most proteins share in common five different blocks of amino acids, in
each of which the sequence from one toxin to another is highly conserved.
These blocks are distributed over the molecule from amino acid position 153
to about 680, and are thought to constitute segments of the molecule critical
to maintaining its tertiary structure as well as forming the key components
that account for the toxicity of Cry proteins. With respect to the latter, the first
two conserved blocks are located in the n-terminus of the activated toxin, and
are likely hydrophobic alpha helices involved in inserting the toxin into the
microvillar membrane and possibly spanning it to form a pore [16].The second
feature of the molecule is that a highly variable block exists in the C-terminal
portion of the activated toxin, spanning the region from about amino acid 280
through 460. The sequence of amino acids in the variable block is thought to
define the spectrum of activity or host range, probably by attaching the toxin
to specific protein binding sites, possibly glycoproteins, on the microvillar
membrane, and contributing to the insertion of the toxin into the membrane.
Evidence in support of the putative functions of these regions has been
provided recently by site-directed mutagenesis of Bt proteins and resolution
of the crystal structure of the CryIIIA protein. In the site-directed mutagenesis
studies, Ge et al. [17] exchanged the highly variable block of amino acids
between the CryIA(a)and CryIA(c)proteins. CryIA(a)is normally highly toxic
for larvae of the silkworm, Bombyx mori, whereas CryIA(c) is virtually nontoxic. Interchanging the variable amino acid block between these two Cry
toxins reversed their toxicity for B. mori. By deletion analysis, these investigators were able to identify the region from amino acid 332 to 450 as the
CryIA(a) segment that determined specificity (and likely binding) of this
endotoxin to B. mori microvilli. Thus, each molecule had the capacity to be
highly toxic for B. mori, provided it had an appropriate variable region, in this
case amino acids 332 to 450 of the CryIA(a)toxin. In the studies of the CryIIIA
crystal structure, Li et al. [18] have shown that the molecule is wedge-shaped
and consists of three domains. Domain I makes up almost half of the molecule,
and stretches from the n-terminus up to and including amino acid 290. This
domain, which contains all of the first conserved block and a major portion
of the second conserved block noted above, is composed of a seven-helix
amphipathic bundle, with six helices surrounding a central helix, a structure
suitable for spanning the lipid portion of the microvillar membrane. Domain
I1 consists of three antiparallel beta sheets around a hydrophobic core, and
stretches from amino acid 291 to 500, and thus includes the hypervariable
block of amino acids that apparently define the spectrum of activity. Within
this domain are portions of conserved blocks three and four. Domain 111 is
comprised of amino acids 501 to 644 and consists of two antiparallel beta
sheets, within which are found the remainder of conserved block number four
along with blocks four and five. The three-dimensional structure resolved by
Li et al. [18] also shows that the five conserved blocks comprise an important
part of the interfacing structure among the three domains, thereby forming
the structural core of the molecule.
Insecticidal Bacterial Proteins
365
The structural and experimental genetic analyses in combination with
analyses of the gene sequences are providing important insights into the
molecular biology and toxicology of Cry proteins. Based on our current
understanding, Cry proteins consist of three domains, with Domain I, and to
a lesser extent Domain 111, containing the structural components that enable
the toxin to insert into and possibly span the microvillar membrane, forming
lethal pores, whereas Domain I1 contains amino acid sequences that initially
bind the toxin to the microvillar membrane, and may also be involved in its
insertion.
MODE OF ACTION
Numerous studies have now shown that though the spore can play a role
in the pathogenicity of B. thuringiensis, it is the parasporal body that causes
the rapid paralysis which ultimately leads to death of the insect (for reviews
see [2]). In the typical Bt, B. fhuringiensis subspecies kurstaski, for example,
the parasporal body dissolves after ingestion upon encountering the alkaline
(pH 8-10) juices of the midgut. As noted above, many of the known Bt toxins
are actually protoxins of about 133,000 daltons (e.g., CryI, CryIVA, and
CryIVB) from which active toxins in the range of 60,000-70,000 daltons are
cleaved by gut proteases. These activated Cry toxin molecules pass through
the peritrophic membrane and bind to specific receptors [19-211, apparently
glycoproteins [20], on the microvilli of midgut epithelial cells. Binding is an
essential step in the intoxication process, and in susceptible insects the toxicity
of a particular Bt protein is often correlated with the number of specificbinding
sites on the microvillar membrane and the affinity of the Bt molecules for these
sites [17,22,23]. However, binding by itself does not, in all cases, lead to
toxicity, indicating that insertion and likely some sort of processing in the
microvillar membrane is required to obtain toxicity [21,24]. Within minutes of
binding to a cell, the microvilli lose their characteristic structure. The microvillar membrane slackens and the microtubules degenerate. Subsequently,
the cells and organelles such as mitochondria become vacuolated and begin
to swell [25,26]. This swelling continues until the cells lyse and slough from
the basement membrane of the midgut epithelium. Both columnar and goblet
cells are destroyed as intoxication advances. As more and more cells slough,
the alkaline gut juices begin to leak into the hemocoel where as a result, the
hemolymph pH rises by a half unit or more. This causes the paralysis and
eventual death of the insect [27].
Though this general picture of the mode of action has been known for some
time, the actual process of intoxication at the molecular level is not well
understood, particularly the series of events that occur after the toxin binds
to the microvillar membrane. There are now a considerable number of studies
on the physiological events that accompany intoxication (see [28] for other
recent key references), but it remains to be determined more accurately which
are causes as opposed to effects. At this point there is general agreement that
binding and insertion of an activated Cry toxin into the microvillar membrane
leads quickly (minutes if not seconds) to an increase in cell membrane cation
permeability and an influx of cations, especially potassium. To balance the
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Federici
cationic influx, the cell takes in water, which leads to hypertrophy and
eventual lysis. Two major hypotheses have been proposed to explain the
cationic influx. The first is that Cry toxins act directly on the microvillar
K+-ATPase pump [29], either intefering with the export of potassium from
the cell or causing a passive leakage inward from the midgut lumen. The
second major hypothesis is that Cry toxins insert in the membrane, forming
nonspecific cation pores [16,30], with each pore consisting of several Cry
molecules, e.g., a hexameric membrane-spanning tubular cluster of molecules
with the channel in the center [31]. The latter is arrived at by a reconfiguration
and aggregation of the Cry molecules after insertion in the membrane in such
a way that two of the alpha helices from each Cry molecule associate to form
the actual channel [31]. In both of these hypotheses, the toxic action takes
place at the microvillar membrane; i.e., no internalization of the toxin occurs
nor is there involvement of a second messenger system leading to inhibition
of metabolic processes inside the cell.
Although the pore-forming hypothesis has gained considerable acceptance
over the past few years, it remains to be clearly validated experimentally. In
this regard, recent evidence that Cry toxins may act inside the cell suggest
that the mode of action may be more complex than currently thought. In
patch-clamp experiments with lepidopteran cells cultured in vitro, Schwartz
et al. [28] have shown that the most immediate effects of Cry intoxication
observed are on the activation of calcium and then chloride ion channels. They
have suggested that binding of the toxin to the cell membrane results in an
activation of calcium channels and an influx of calcium into the cell. This is
followed by internalization of the receptor-toxin complex or endocytosis of
the toxin. In either case, once within the cell, the toxin either unmasks anionic
channels or inserts into the plasma membrane, forming chloride channels.
Subsequently, these events lead to an influx of water and cations, followed
by cell hypertrophy and lysis. These recent observations and discrepancies in
interpretations of the data from toxicological and physiological experiments
at the cellular level demonstrate the need for more research on the molecular
mode of action of Cry toxins.
With respect to the broadly cytolytic CytA toxin, this is also thought to form
pores in the microvillar membrane, but it differs from the Cry toxins in that
it apparently does not require a receptor protein for insertion into the membrane. Instead, it has a high affinity for unsaturated fatty acids in the lipid
bilayer of the cell plasma membranes [32].
GENETIC ENGINEERING OF BACILLUS THUMNGZENSIS
In addition to the development of Bt isolates as conventional insecticides,
transconjugate and genetically engineered strains with novel properties are
now corning to market. For example, Ecogen, Inc. of Langhorne, Pennsylvania, has combined three different plasmids, two encoding proteins toxic to
lepidopterous insects (CryI), the other a protein toxic to coleopterans (CryIII),
into a single strain of B. thuringiensis subspecies kurstaki. This transconjugate strain has an expanded host range that includes both lepidopterous
and coleopterous insects. And recently Mycogen Corporation of San Diego,
Insecticidal Bacterial Proteins
367
California, has received registration for the first genetically engineered bacterial insecticide, MVP, a killed strain of Pseudomonas fluorescens that contains a
Bt toxin active against lepidopterous insects [33]. The apparent advantage of
this strain over conventional Bts is greater residual activity in the field, which
is obtained by fixing the Pseudomonas cell wall around the Bt protein.
Because Bt genes can be manipulated quite easily using recombinant
DNA technology, it is likely that a variety of different approaches to
controlling insects based on Bt’s insecticidal proteins will be developed
over the next few years. For example, fusion proteins with an expanded
host range have already been made by fusing two Bt genes together [34].
Another exciting yet controversial development already at hand is the
successful engineering of Bt genes into plants such as cotton, tomato, and
tobacco to produce insect-resistant transgenic plants [35-381. In all of these
cases, only a single Bt gene was transformed into the plant species, and it
is already known that insects are prone to develop resistance quickly to
engineered bacteria expressing a single gene [39]. This work is controversial primarily from the standpoint of the high risk that the release of such
plants would pose for the development of resistance to both conventional
and engineered Bts used as periodically applied microbial insecticides. In
addition, although there is no evidence whatsoever that transgenic plants
are unsafe to eat, the question of their safety has been raised recently [40].
The concern over the potential for the development resistance for conventional and engineered uses of Bt is valid, and the recent organization of an
industry-based resistant management group hopefully means the threat of
resistance is being taken seriously.
Despite potential problems with the use of Bt, it should be noted that
though Bt has come a very long way in the last five years, it is still at an early
stage of development. In the future, the use of recombinant DNA technology
and an expanded knowledge of Bt’s molecular biology, particularly its mode
of action at the molecular level, should lead to a greater range of novel and
safe insecticides including some for insects for which we currently have few
cost-effectivecontrol methods. For example, if the hypotheses concerning the
specificity and toxin domains are correct, then it should be possible to extend
Bt‘s host range by altering the highly variable region so that the proteins bind
to the midguts of a wider range of insect pests. Another strategy being
investigated is to fuse the Bt toxin domain to proteins capable of binding to
insect midgut cells. Examples of such proteins include the binding domains
of antibodies made against surface proteins on insect gut cells, viral surface
proteins responsible for attaching insect viruses to midgut cells, and other Bt
gut-binding proteins. If successful, it is possible that at least some of these
recombinant proteins may be useful in transgenic plants, providing an opportunity for controlling even sucking insects.
POTENTIAL TARGETS FOR NOVEL INSECTICIDES IDENTIFIED
BY Bt RESEARCH
Perhaps even more importantly, especially with respect to the topic of
this meeting, is that Bt identifies the midgut epithelium as a new source of
368
Federici
target sites for engineered proteins. Since World War 11, the focus of most
insecticide research has been on the development of synthetic organic chemicals that targeted receptors in the nervous, hormonal, or cuticular systems of
insects. The midgut has not been viewed as an important source of target sites
since the arsenicals. The advent of recombinant DNA technology, however, provides an opportunity to reexamine the midgut as a source of target
sites for which inhibitory insecticidal proteins, small peptides, and even
organic molecules can be designed that are highly selective for insects. This
may turn out to be the most important lesson we learn from Bt research.
Needless to say, the midgut in all insect species is an extremely important
and highly active tissue metabolically. The rnicrovillar membrane itself contains a wide range of surface and internal proteins involved in transport
functions such as maintaining ion gradients, absorption of amino acids and
other nutrients, and excretion of enzymes [4143]. All of these represent
potential target sites to be blocked by peptides that can be delivered either in
the form of microbial insecticides, as protein insecticides produced by fermentation, or through transgenic plants. In addition, the developing field of
mirnetics [44]indicates that it may be possible to synthesize simple insecticidal
organic molecules that specifically bind to and inhibit these target proteins.
The rationale behind such strategies has already been developed. For example, monoclonal antibodies can be made that bind to and disrupt the function
of important microvillar surface proteins. Short inhibitory peptides can be
synthesized that mimic the hypervariable region responsible for binding the
antibody to a specific protein. These in turn can be used to synthesize, with
the aid of computer modeling, simple organic molecules, if necessary, as
insecticides. Though no precedent exists for developing insecticides this way,
such a strategy has been used recently to develop peptides and simpler
organic molecules that bound to a cellular viral receptor and inhibited its
ability to induce cellular proliferation [44].
Just ten years ago, virtually nothing was known about the genetics and
molecular biology of insecticidal Bt proteins. The advances over the past
decade have been remarkable, and include the cloning and analysis of over
50 cry genes, the development of transgenic insecticidal plants and bacterial
insecticides, and more recently, resolution of the crystal structure of the first
Cry protein. These developments were made possible largely by the advent
of recombinant DNA technology. If the rapid progress over the recent past is
any indication, further use of this technology in combination with developing
knowledge of insect molecular biology and new chemical technologies such
as mimetics should lead to a range of new types of selective and environmentally safe pesticides in the future. And the midgut provides an excellent
potential source of targets for these.
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