Download BACILLUS SPHAERICUS TOXINS: Molecular Biology and Mode of

BACILLUS SPHAERICUS TOXINS: Molecular Biology and Mode of
Action
ZHOU Xian-zhi
PERSPECTIVES AND OVERVIEW
The use of microorganisms as a source of biological compounds for insect pest control started after the
discovery of the highly insecticidal bacteria Bacillus thuringiensis . The discovery of the strain of
B.thuringiensis serovar israelensis (38,44) made possible efficient microbiological control of Diptera
Nematocera vectors of diseases, such as mosquitoes (Culicidae) and black flies (Simuliidae).
The first reported Bacillus sphaericus strain active against mosquito larvae was isolated from moribund
mosquito larvae (49). The larvicidal activity of this isolate was so low that their use in mosquito control
would never have been considered. The identification of the strain SSⅡ-1 in India (70) renewed the search
for more active strains. But only after the isolation (in Indonesia from dead mosquito larvae) of strain 1593which exhibited a much higher level of mosquitocidal activity (71) was the potential of B.sphaericus as a
biological control agent for mosquitoes taken seriously (72). More than 380 B. sphaericus strains which are
toxic to the larvae of mosquitoes in at least three genera (Culex, Aedes, and Anopheles) have been identified
(7, 36). These strains have been subdivided into the low-toxicity strains (50% lethal concentration [LC50],
~105 cells per ml) and the high-toxicity strains (LC50, 102 to 103 cells per ml) (7, 63). Until recently, only
two types of mosquitocidal toxin have been found in B.sphaericus a binary toxin (comprising 51.4- and
41.9-kDa proteins) produced during sporulation in high-toxicity strains (7) and Mtx toxin (compring 100-,
31.8- and 35.8-kDa proteins) synthesized in both the low- and high toxicity strains (54, 77).
B.sphaericus (56) is an aerobic bacterium that produces terminal spherical spores. It lacks several
biochemical pathways and thus can not use sugars as metabolites. The findings of various genetic and
biochemical studies indicate that this species is heterogeneous. The increasing number of isolated strains
has made differentiating between toxic and atoxic strains. And between the toxic strains themselves,
difficult.
Numerous methods have been used to classify this very heterogenic species. For exemble, relationships
between strains of B.sphaericus have been examined in terms of DNA homology (50). Based on the
percentage of homology, five groups were identified, and group Ⅱ was further subdivided into ⅡA
(related over 79% homology and almost contain all toxic strains) and ⅡB. Classification via two other
systems, bacteriophage typing (82, 83) and serotyping using flagellar antigens (36, 39), yielded a similar
grouping for the group ⅡA mosquito pathogenic strains. Nine serotypes ( list in table 1) are known to
contain active strains. Such as numerical classification based on taxonomy of phenotypic features (2, 45),
cellular fatty acid composition analysis (43) ribotyping (3) and random amplified polymorphic DNA
analysis (80)are recently developed techniques, indicate most toxic strains are recovered in a few groups.
None of these methods allows us to predict the level of toxicity of a given strain. The three
mosquitocidal activity levels do not correspond to the groups defined by any other method classification
(Table 1). The genes encoding various toxins have been cloned, and they might be used in hybridization
experiments to predict activity (4). However, some strains react positively with toxin genes but display only
weak larvicidal activity. Therefore, to identify potentially valuable strains and to further elucidate the nature
of the mosquito-larvicidal activity, we must continue to determine toxicity levels by using mosquito larvae.
Table1 Characterization of some mosquito-larvicidal B.sphaericus*
strain
origin
serotype
Phage
DNA
Toxici-
Crystal
type
group
ty**
genes
Mtx1
Mtx2
Mtx3
Kellen K
USA
H1
1
ⅡA
L
-
+
+
+
Kellen Q
USA
H1
1
ⅡA
L
-
+
+
+
BS-197
India
H1
NA
NA
M
+
NA
NA
NA
SSⅡ-1
India
H2
2
ⅡA
L
-
+
+
+
IAB881
Ghana
H3
-
NA
H
+
-
NA
NA
LP1-G
Singapore
H3
8
ⅡA
H
+
-
NA
NA
2362
Nigeria
H5
3
ⅡA
H
+
+
+
+
2317-3
Thailand
H5
-***
NA
H
+
+
+
NA
C3-41
China
H5
NA
NA
H
+
NA
NA
NA
IAB-59
Ghana
H6
3
NA
H
+
+
+
+
31
Turkey
H9
8
NA
L
-
+
+
+
2297
Sri Lanka
H25
4
ⅡA
H
+
+
+
+
2173
India
H26
-***
ⅡA
M
-
-
-
NA
IAB872
Ghana
H48
3
NA
H
+
NA
NA
NA
14577
USA
H4
-***
Ⅰ
-
-
NA
NA
NA
Data compiled from references23, 40, 52, 54, 66; ** Based on the lethal concentrition 50% after 48h on the fourth-instar larvae of Culex
quinquefasciatus L≥10-3 spore crystal complex dilution; M≈10-4; H≤10-6: -*** inactive to all bacteriophages；+present； -absent；NA : no
information available
B.sphaericus strains are generally highly active against larvae of Anopheles and Culex species and are
poorly or not toxic to larvae of Aedes species. However, susceptibility appears to depend on the species of
mosquito and can thus vary within a genus (13). Toxitity levels vary among the larvicidal serotypes and
even within the same serotype (79). Therefore, the relative potency of each strain is currently evaluated and
described in terms of specific activity titers on the different mosquito species and in terms of activity ratios
derived from such titers (79).
Like B.thuringiensis Lepidoptera. Singer (70) first suggested that B.sphaericus acts by toxemin rather
than septicemia. A few years later, Davidson and Myers (32) reported the presence of parasporal inclusions,
which they suspected participate in the toxic action of B.sphaericus. Indeed, all of the mpst toxic strains
produce parasporal inclusions during sporulation. These inclusions have a crystalline ultrastructure and are
released into the medium along with the spore after the completion of sporulation. The relationship
between sporulation, crystal formation, and mosquitocidal activity was first clearly established for strains
belongings to serotypes H5a5b (16, 56), H25 (36, 48, 81), and H6 (38). Later, the partial purification of
crystals toxic to mosquito larvae (62), and the study of mutants blocked at early stages of sporulation that
fail to make crystals and are not mosquitocidal (21), confirmed the toxic nature and protein composition of
the crystals, more recently, the identification of a group of toxins different from crystal toxins, the Mtx
toxins, renewed interested in B.sphaericus as a mosquitocidal agent (77, 78). Two other reviews discuss
the genetics of B.sphaericus toxins and their mode of action (10, 63). And an overview of each is given
below.
BIOCHEMISTRY AND GENETICS OF B. SPHAERICUS TOXINS
Two kinds of toxins (crystal and Mtx toxins) seem to account for the larvicidal activity of B.sphaericus.
They differ both in their composition and time of synthesis. The crystal toxins are present in all highly
active strains and are produced during sporulation. The Mtx toxins are responsible for the avtivity of most
of the weakly active strains. Mtx proteins synthesized only during the vegetative phase.
Crystal Toxins
The crystal toxin is composed of two proteins that are synthesized in equmolar amounts and assembled in
crystal structures visible at about stage Ⅲ of sporulation (11, 49, 81). The proteins are designated as P51
and P42 on the basis of their predicted molecular masses of 51.4 and 41.9 kDa (5, 8, 9, 12, 14, 46, 47). and
they also named BinA and BinB, respectively. The genes encoding both proteins have been cloned from
sverval high-toxicity strains. They appear to be organized in an operon with a 174- to 176-base pair (bp)
intergenic region. A stem-loop structure (characteristic of transcription terminators) lies downstream from
the P42 gene (figure 2a) (8, 46). No sequence similar to Bacillus subtilis sporulation promoters have been
found upstream from the gene encoding P51. however, both the P51 and P42 genes are expressed only
during sporulation in B.subtilis (6). Moreover, lacZ fusions to the promoter of the crystal protein genes
indicate that, in B.sphaericus, transcription begins immediately before the end of exponential growth and
continues into stationary phase (1). Thus, enough protein for crystal formation accumulates before stage
Ⅲ.
The genes for P51 and P42 are chromosomal, at least in strains 1593, 2362, and 2297, although these
strains contain plasmids (3). Both genes have been reported in diverse strains, and all highly toxic strains
tested contain similar sequences as assessed using DNA hybridization (4). The reverse is not true:
moderately toxic strains such as LPI-G also contain these genes (Table 1). The toxin-coding and flanking
regions of strains 1593, 2362, and 2317-3 are identical over a span of 3479 nucleotides, whereas the
sequences in strains IAB59 and 2297 differ by 7 and 25 nucleotides, respectively (14). The resulting
differences in amino acid sequences of P51 are 5 and 3 amino acids, respectively and those for P42 are 1
and 5. these variations appear to be responsible for the differences in specificity of these strains (see below).
Interestingly, LPI-G is only weakly active , although it produces the crystal toxin (Table 1). The sequence
of crystal-toxin genes from this strain may indicate which regions of the protein are involved in the loss of
toxicity.
The amino acid sequences of P51 and P42 are not similar to those of any other bacterial toxins, including
those produced by B.thuringiensis serovar israelensi (Bti). However , P51 and P42 share four segments of
sequence similarity (8) , the significance of which remains unclear . therefore, P51 and P42 of B.sphaericus
constitute a separate family of insecticidal toxins (8).
The aggregation of both P51 and P42 has been analyzed using crystal-toxin components expressed
separately or together in homologous or heterologous Bacillus hosts. When expressed independently in
B.subtilis and B.sphaericus or in B.thuringiensis crystal-negative hosts, the proteins form amorphous
inclusions (20, 58); in contrast, crystals similar to those produced by naturely occurring, highly toxic
B.sphaericus strains are produced when the two genes are simultaneously expressed in either B.sphaericus
or B.thuringiensis (20, 58). No crystal can be detected in B.thuringiensis contain factors that help stabilize
the proteins and subsequent crystallization of the toxin.
In vivo, P42 is slowly converted into a stable protein of ~39kDa, whereas P51 is rapidly converted into a
stable fragment of ~43kDa (11, 16). In vitro deletion analysis led to the delineation of the minimal active
fragments of both proteins, which indeed correspond to the activated fragments (18, 25, 61, 67 ). At the N
and C terminus of P51, 32 and 53 amino acids, respectively, can be eliminated without loss of toxicity (25);
deletions of 10 and 17 amino acids at the N and C terminus of P42, respectively, result in a protein similar
to the 39-kDa activated fragment (18) (Figure 2a).
Subcloning experiments have shown that P42 alone is toxic for mosquito larvae (Culex pipiens pipiens),
although the activity is weaker than that of crystals containing both proteins (58). In contrast, P51 alone is
not toxic, but its presence enhances the larvicidal activity of P42, suggesting synergy between the
polypeptides (18, 40, 58,61). In vitro assays on mosquito cell lines also revealed that the activated form of
P42 alone is toxic to Culex pipiens quinquefasciatus cells, whereas P51 appears to be inactive; however, no
synergy between the components was observed in vitro (7). Although simultaneous presence of both
proteins appears necessary for full toxicity, the activity strains depends on the origin of P42, as shown by
analysis of toxins mutated in vitro (13). For example, when amino acids were substituted in a region
centered around position 100 of P42, the IAB59 and 2297 P42 proteins became similar to the protein from
strain 2362,: the activity and specificity of the mutant toxins toward Culex and Aedes larvae were
comparable, un like those of the wild-type. This finding indicates that this region is involved in specificity.
Together, these observations suggest that P42 is the most important determinant of specificity and activity.
Based on their larvicidal activity and compose of amino acids, binary toxin divided into four types. TypeⅠ:
high toxic to Culex, medium toxic to Anopheles, nontoxic to Aedes; typeⅡ: high toxic to Culex, medium
toxic to Anopheles, low toxic to Aedes; typeⅢ: high toxic to Culex and Anopheles, nontoxic to Aedes; type
Ⅳ: nontoxic to all mosquito larvae.
Table 2 Comparison of Bin amino acid sequences from various B.sphaericus strains
Polypeptide
Amino acid
typeⅠ
typeⅡ
typeⅢ
typeⅣ
IAB59
1593
2297
LP-G
2362
2317-3
C3-41
41.9kD
51.4
93
Leu
Leu
Leu
Ser
99
Val
Val
Phe
Val
104
Glu
Ala
Ser
Ser
125
His
His
Asn
Asn
135
Tyr
Tyr
Phe
Phe
267
Arg
Arg
Lys
Lys
69
Ala
Ser
Ser
Ser
70
Asn
Asn
Lys
Asn
110
Thr
Thr
Ile
Ile
314
His
Leu
Tyr
His
317
Leu
Phe
Leu
Leu
389
Leu
Leu
Met
Met
Mtx Toxins
Three types of Mtx toxins have been described to date: Mtx1, Mtx2 and Mtx3, with molecular masses of
100, 31.8 and 35.8 kDa, respectively ; genes for these toxins have been cloned from the medium-toxicity
strain SSⅡ-1 (77, 78). DNA hybridization suggested the Mtx1 and Mtx2 toxins do not display any
similarity to each other or to the crystal proteins or any other other insecyicidal proteins. The genes
encoding Mtx are wildly distributed in both high-and low-toxicity strains of B.sphaericus (54) .In contrast
to the crystal toxin genes, Mtx genes are expressed during the vegetative and growth phase, and sequences
resembling vegetative promoters of B.subtilis have been found upstream from each gene (77, 78). Fusions
of the lacZ gene to the mtx promoter also firmed vegetative expression, because β-galactosidase activity
was restricted to the early exponential phase, at least in B.sphaericus (1). However, Mtx proteins possess
short N-terminal leader sequences, which is characteristic of gram-positive bacterial signal peptides (77,
78). The Mtx1 toxin can be further processed by gut proteases into two fragments of 27 and 70-kDa that
correspond to the N- and C-terminal regions, respectively (76). The 70-kDa fragment from Mtx1 posses
three repeated regions ~90amino acids each, the function of which is unknown; the 27-kDa fragment
contains a short region corresponding to a transmembrane domain. The 27-kDa part of Mtx1 shares weak
similarity with the catalytic domain of several ADP-ribosylating toxins (76, 77). Mtx2 has 34 and 29%
similarity to two toxins active against mammalian cells, namely the ε-toxin from Clostrium perfringens
and the cytotoxin from pseudomonas aeruginosa, respectively (78). and Mtx3 has 30 and 27% similarity
to the two toxins, respectively (54). The 35.8kDa protein from B.sphaericus exhibits 38% identity to the
31.8kDa Mtx2 toxin of B.sphaericus SSⅡ-1 (55). The toxicity of Mtx1 has been measured using fusion
proteins synthesized in Escherichia coli : it is highly active against mosquito larvae (76), with an LC50
value comparable to that of the crystal toxin (15ng protein/ml). Deletion analysis suggests that the 27-kDa
fragment from Mtx1 can ADP-ribosylate itself, whereas the 70-kDa fragment is responsible for toxicity to
cultured C.pipiens quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito
larvae (76).
MODE OF ACTION OF THE CRYSTAL TOXIN
The mode of action of B.sphaericus crystal toxin has only been studied in mosquito larvae. However, in
one report, the toxin was active in adults of C.pipiens quinquefasciatus, but not in A.aegypti adults, after
introduced enema into the midgut (74).
Investigations into the mode of action of the B. sphaericus crystal toxin on Culex pipiens, a highly
susceptible species, have demonstrated that following ingestion, the crystal is solubilized under the alkaline
pH in the larval midgut, where the serine- proteases promote the proteolytic activation of the protoxin into
toxin (17, 28, 57). B.sphaericus crystals release the toxin in all species, even in nonsusceptible species such
as A. aegypti. Indeed, some studies report that the differences in solubilization and /or activation of the
crystal toxin (11, 28, 57).
Cytopathology and Physiological Effects
Bti toxins completely break down the larval midgut epithelium, whereas B.sphaericus toxins do not.
Nevertheless, midgut alterations start as soon as 15 min after ingestion of the B.sphaericus spore –crystal
complex (24,32, 49, 73 ). Midgut damages in culex pipiens are the same after ingestion of spore crystals of
either strain 1593 or strain 2297 (24, 32, 73). In contast, the symptoms of intoxication produced by these
two strains differ in other mosquito species. Large vacuoles (and/or cytolysosomes) appear in C.pipiens
midgut cells, whereas large areas of low electron density appear in Anopheles stephensi midgut cells. A
generally occurring symptom is mitochondrial swelling, described for C.pipiens pipiens and A.stephensi, as
well as for A.aegypti when intoxicated with a very high dose of spore crystals (24). The midgut cells,
especially those of the posterior stomach and the gastric caecae, are the cells most severely damaged by the
toxin, BinA (P42) and BinB (P51) can form pores or channels in the membrane(65). In vitro assays proved
that the Bin toxin could permeabilize receptor-free large unilamellar phospholipid vesicles and planar lipid
membranes, by a mechanism of pore formation(65). And the work showed that the BinA is more efficient
than BinB to provoke pore formation, the BinB can improve the ability of BinA to permeabilize the large
unilamellar vesicles. The C-terminal region of the BinA of binary toxin, Particularly the residue 312R for
biological activity against mosquito larvae (42). and singh & Gill (74) also report late damage in neural
tissue and in skeletal muscles.
Ultrastructural effects have been reported in cultured cells of C.pipiens quinquefasciatus within a few
minutes of treatment with soluble and activated B.sphaericus toxin..these alterations consisted mainly of
swelling of mitochondrial cristae and endoplasmic reticula, followed by enlargement of vacuoles and
condensation of the mitochondrial matrix (30).
The physiological effects of B.sphaericus crystal toxin have been poorly documented. Lakshmi Narasu
& Gopinathan (51) report that oxygen uptake by mitochondria isolated from B.sphericus-treated C.pipiens
quinquefasciatus larvae is inhibited, as is the activity of larval choline acetyl transferase in the presence of
toxin. In addition, B.sphericus toxin decreases oxygen uptake by mitochondria isolated from rat liver (51).
Binding to a Specific Receptor in the Brush-Border Membrane
As the differences in susceptibility between mosquito species do not seem related to the ability to solubilize
and /or activate the binary toxin, this variation presumably results from differences at the cellar level.
Indeed, studies report the binging of fluorescently labeled toxin to the gastric caecae and the posterior
stomach only in very susceptible Culex species. P51 does not bind to the gut of A.aegypti, whereas P42
binds weakly and nonspecifically in this species. No regional binding was observed in Anopheles spp. (31,
33, 34, 61). Furthermore, these studies showed that, in C.pipiens quinquefasciatus, only P51 binds specially
to the caecum and posterior stomach, whereas P42 binds nonspecifically throughout the midgut. When the
proteins together, the binding of P42 appears to depend on the binding of P51. in vivo binding studies using
nontoxic deletion mutants of the crystal toxin (61) support the hypotheses that the N-terminal region of P51
is involved in regional binding of this protein in the larval midgut and that the C-terminal region of P51 and
the N- and C-terminal regions of P42 are involved in the interaction of the two components that causes
P42 to bind in the same regions as P51. these authors also report that internalization of toxin only seems to
occur when both components are present. However, further intracellular investigations are required to
elucidate whether one or both components are internalized.
The hypothesis that a specific receptor was involved in the toxin binding was confirmed by in vitro
binding assays using 125I-labeled activated crystal toxin and midgut brush-border membrane fractions
(BBMFs) isolated from either susceptible or nonsusceptible mosquito larvae (59). The interaction of the
binary toxin with C. quinqaefasciatus larvae midgut, investigated through qualitative binding assays of the
fluorescent-labled toxin, demonstrated a distinct and regionalized toxin binding localized in the gastric
caeca and posterior stomach (33, 34). And immunocytochemical localization of the binary toxin confirmed
that again (69). Indeed, direct binding experiments with C.pipiens pipiens BBMFs indicated that the toxin
binds to a single class of specific receptor. A single 60kDa midgut membrane protein is identified as the
binding protein, this protein is anchored in the mosquito midgut membrane via a
glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol
specific-phospholipase C (PI-PLC). And the peptide sequence showed a very high degree of similarity with
enzymes belonging to the а-amylase family (68).
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