INTRODUCTION

Bacillus sphaericus is an aerobic, Gram-positive, spore-forming bacterium which is widespread in soil and aquatic environments. Some strains of B. sphaericus produce protein toxins which are lethal to mosquito larvae (1, 2). The best studied mosquitocidal strains of B. sphaericus are divided into a high toxicity group (e.g.. 2362, 2297, and IAB59) and a low toxicity group (e.g.. SSII-1, 31-2 and Kellen Q) (1). The high toxicity but not the low toxicity strains encode 51.4- and 41.9-kDa proteins, which together form a binary toxin expressed at high levels during sporulation. Most mosquito pathogenic B. sphaericus tested also harbor a 100-kDa toxin gene (mtx) and a 31.8-kDa toxin gene (mtx2)¹ (3, 4).

Toxin production in the low toxicity strain B. sphaericus SSII-1 begins in the vegetative phase of growth before the onset of sporulation (5, 6). The mtx2 gene of this strain encodes a polypeptide of 292 aa (Mtx2) with a molecular mass of 31.8 kDa, which is detected in the vegetative phase of growth (4). Mtx2 is unrelated to the binary and 100-kDa toxins but has regions of significant homology with the 33-kDa  toxin of Clostridium perfringens and the 31.68-kDa cytotoxin of Pseudomonas aeruginosa (4). In this study, we demonstrate that the Mtx2 toxins from six strains of B. sphaericus have few sequence differences, but some of these toxins exhibit major differences in larvicidal activities against two species of mosquitoes. The results permitted the design and assay of hybrid toxins and the identification of aa residues in Mtx2 that are unusual determinants of larvicidal activity and mosquito host range.

EXPERIMENTAL PROCEDURES

Plasmid pTH26 (7), a derivative of plasmid pGEX-1 (8), was used for the initial cloning of the mtx2 genes. Plasmid pTH82 was used for the construction of the mutant mtx2 genes: SSII-1 (T279A)²; SSII-1 (K224T,T279A); 31-2 (A279T); 31-2 (T224K,A279T); IAB59 (D31H); IAB59 (R50K); 31-2 (P67S); 2297 (T171K); 2362 (K40Q); SSII-1 (S37I); and SSII-1 (S67P). pTH82, which contains the SSII-1 mtx2 gene derived from genomic DNA, was previously constructed from plasmids pS35BP2 and pTH81 as follows. Plasmid pS35 (4) was digested with BamHI so as to remove a ~3-kb BamHI fragment and self-ligated to give pS35B. Plasmid pS35B was digested with PstI so as to remove a ~3-kb PstI fragment and self-ligated to give pS35BP2. Separately, B. sphaericus SSII-1 genomic DNA was amplified by PCR with oligonucleotides TT119 and TT120 (see below) and a ~0.8-kb NcoI/ClaI fragment was cloned into the large vector fragment of pTH26 (7) to give pTH81. The large HindIII/AflII vector fragment of pTH81 was ligated to the ~0.9-kb HindIII/AflII fragment of pS35BP2 (containing the mtx2 gene) to give pTH82. Taq polymerase and [³⁵S]dATP were from Amersham International, plc. Glutathione-Sepharose was purchased from Pharmacia Biotech Inc., and reduced glutathione was from Sigma. Dried yeast powder was purchased from Life Technologies, Inc.

B. sphaericus SSII-1 was a gift from E. W. Davidson, Arizona State University, Tempe. Strains Kellen Q and 31 were obtained from A. A. Yousten, Virginia Polytechnic Institute and State University, Blacksburg. Strain 1593M was a gift from J. Szulmajster, C.N.R.S., Gif sur Yvette, and all other B. sphaericus strains were obtained from H. de Barjac, Pasteur Institute, Paris. All B. sphaericus strains were grown either in L-broth or NYSM medium (5).

The protein coding regions of mtx2 from eight different strains of B. sphaericus (31-2, IAB59, Kellen Q, 2297, 2362, 1691, 1593M, and 2317.3) was amplified by PCR from genomic DNA (9) using synthetic oligonucleotides based on the sequence of B. sphaericus SSII-1 mtx2 (4). The sequences of these oligonucleotides were: 5-CCCCCCATGGATCCAATGAAAAGGACCAAATTACTTTTTTATATT-3 (TT119) for the 5 (upstream) primer, and 5-CCCGACGTCATCGATGCATGCCTTAAGTTATTTAAAAGAAATTTCTTTAACATCTATTA-3 (TT120) for the 3 (downstream) primer. Artificial BamHI, NcoI, ClaI, SphI, and AflII restriction enzyme sites were incorporated into these sequences for cloning purposes. The 0.8-kb PCR products were digested with NcoI and ClaI, and cloned into the NcoI and ClaI sites of the large (vector) fragment of plasmid pTH26 (7), which was propagated in Escherichia coli DH5. Another set of recombinants were generated in the same way, and both sets were completely sequenced to ensure mutations were not artefactually introduced into the mtx2 genes during the PCR. Cloning and sequencing were carried out in the standard manner (10). Protein sequence analysis and alignments were performed using DNASTAR software.

Mutagenesis was carried out as described in the Sculptor^TM in vitro mutagenesis system (Amersham). A 0.8-kb BamHI-SphI restriction fragment of pTH26 containing the appropriate mtx2 gene was subcloned into the BamHI-SphI site of bacteriophage M13mp19. Recombinant M13 phage were propagated in E. coli TG1, and single-stranded DNA was purified (10). The mutagenic primers (mutation underlined), M1, 5-ATCATGATACATCAAACA-3 (SSII-1-S37I); M3, 5-CTTCTGGGAAGTAATTTC-3 (SSII-1-K224T); M4, 5-ACTACAATTATGAAAATAAT-3 (IAB59-D30H); M5, 5-GATATTGATAAAAAATTGAT-3 (IAB59-R50K); and M6, 5-AGCATCAAAAACAAGGTGTA-3 (2362-K40Q) were annealed to single-stranded templates of the mtx2 genes cloned in M13mp19, and mutants were identified directly by dideoxynucleotide DNA sequencing. All mutant genes were fully resequenced to ensure unwanted mutations had not arisen. Finally, all mutant genes were cloned from M13mp19 back into pTH82 (7) by inserting the BamHI-SphI gene fragment into the large vector fragment of BamHI-SphI-digested pTH82.

To construct the 31-2 (A279T) and SSII-1 (T279A) mutant mtx2 genes, the 31-2 mtx2 gene cloned in pTH26 (see above) and the SSII-1 mtx2 genomic DNA cloned in plasmid pTH82 were each digested with NdeI and PstI, and the large (vector) fragments and small (gene) fragments were exchanged, then ligated together. To construct the 31-2 (T224K,A279T) and SSII-1 (K224T,T279A) mutant mtx2 genes, the same strategy was used except that the above plasmids harboring the 31-2 and SSII-1 mtx2 genes were double digested with AccI and PstI.

The construction of genes expressing Mtx2 fusion proteins in E. coli DH5 (in which the N-terminal 15 aa of Mtx2 were removed) was performed as follows. To construct mtx2 from SSII-1 (K224T) and SSII-1 (K224T,T279A), the plasmids containing the SSII-1 (K224T) or SSII-1 (K224T,T279A) mtx2 gene (cloned in pTH26) were digested with NcoI and HindIII, blunt-ended with Klenow, gel-purified, and self-ligated. To construct mtx2 from 31-2 and 31-2 (T224K,A279T), the mtx2 genes cloned in pTH26 were PCR amplified with primer TT120 (see above) and primer 5-CATGCCATGGGATTGTTTGTAAACGGAAGTATTTATACG-3 (W3), and the amplified genes were eluted from an agarose gel, digested with NcoI and ClaI, and cloned in the large vector fragment of NcoI-ClaI-digested pTH26. The construction of mtx2 from SSII-1 has been described (4).

Synthesis and purification of Mtx2 fusion proteins was carried out essentially by a published procedure (8), except that E. coli DH5 were grown at 30 °C for 2 h after induction of Mtx2 synthesis.

Polyclonal antibodies against recombinant Mtx2 were raised in a previous study (4) and used to perform Western blotting as described (4) to detect recombinant Mtx2 homologs expressed in recombinant E. coli.

All plasmids expressing Mtx2 fusion proteins were transformed into E. coli DH5 for larvicidal assay. The toxic activities of different E. coli clones synthesizing the Mtx2 fusion proteins (or purified Mtx2 fusion proteins) (4) were measured on laboratory-reared 1st instar larvae of Culex quinquefasciatus and Aedes aegypti mosquitoes. Recombinant E. coli cultures were harvested, washed once with water, resuspended in 0.1 the original volume with water, and the A₆₀₀ was measured. The cultures were adjusted to the desired range of concentrations with water and added to 10 1st instar larvae in 1 ml of water with 50 µl of a 100 mg/ml suspension of yeast. Assays using the desired range of concentrations of Mtx2 fusion proteins were performed in a similar manner, except that 50% PBS was used throughout. All assays were performed at least twice, and each concentration of bacteria (or purified Mtx2 fusion protein) was assayed in triplicate. Surviving larvae were counted at 24 h. The LC₅₀ values were calculated from the average mortality observed at 24 h by using the CA Cricket Graph III program. Mortality of control larvae fed with yeast suspension alone, with 50% PBS, or with E. coli transformed with plasmid vector alone was less than 10% in all assays.

RESULTS

Using PCR primers based on the sequences at the extremities of the region coding for the mtx2 gene from B. sphaericus SSII-1 (4), 0.8-kb PCR products were generated from the genomic DNA of eight strains of B. sphaericus, namely 31-2, IAB59, Kellen Q, 2297, 2362, 1691, 1593M, and 2317.3. The amplified DNAs were cloned in a plasmid vector, pTH26 (7), and two independent clones of each mtx2 gene were completely sequenced to exclude the possibility of PCR-derived mutations.

The predicted aa sequences of the eight Mtx2 homologs were compared with each other and with the known sequence of B. sphaericus SSII-1 Mtx2, and aa variations were found at 10 positions between aa 31 and aa 279 (Fig. 1). The Mtx2 homologs from B. sphaericus 2362, 1691, 1593M, and 2317.3 were identical to each other (Table I). Overall, there were between four and eight aa substitutions when the Mtx2 sequences from strains 31-2, IAB59, Kellen Q, 2297, and 2362 were compared in a pairwise fashion with the prototype Mtx2 sequence from SSII-1 (Table I). Mtx2 from SSII-1 was unique in having Ser³⁷, Ser⁶⁷, Lys²²⁴, and Thr²⁷⁹, while Phe²³⁹ was only found in Mtx2 from Kellen Q, and Thr¹⁷¹ was unique to Mtx2 from strain 2297 (Table I).

Table I. Amino acid differences among Mtx2 proteins in B. sphaericus strains The sequences were compared with the sequence of Mtx2 from strain SSII-1 (Fig. 1). SSII-1 31-2 IAB59 Kellen Q 2297 2362 (1691, 1593M and 2317.3) His31  a D D D   His35       L   Ser37 I I I I I Gln40         K Lys50   R R R   Ser67 P P P P P Lys171       T   Lys224 T T T T T Tyr239     F     Thr279 A A A A A a indicates identity with SSII-1 Mtx2.

In order to compare the mosquitocidal activities of the six Mtx2 homologs, the mtx2 coding regions were fused in frame to GST (see ``Experimental Procedures''). Recombinant E. coli cells were incubated with isopropyl--D-thiogalactopyranoside to induce the synthesis of GST-Mtx2, and cell extracts were examined by Western blotting using a polyclonal antiserum to Mtx2 from B. sphaericus SSII-1. All recombinant cells produced a protein of ~58 kDa, the expected size of GST-Mtx2 (Fig. 2). The synthesis in E. coli of Mtx2 from B. sphaericus strains 2362 and Kellen Q was lower than that of Mtx2 from SSII-1, 31-2, IAB59, and 2297, but expression of all fusion proteins did not vary by more than 2.7-fold (Fig. 2).

Intact E. coli cells were fed to larvae of the mosquito C. quinquefasciatus, and larvicidal activities of the various recombinant E. coli clones harboring Mtx2 fusion proteins were quantitated (Table II), taking into account the different levels of Mtx2 fusion proteins (Fig. 2 legend). All clones exhibited significant toxicity to C. quinquefasciatus, and E. coli synthesizing Mtx2 from strains SSII-1 and 2362 were the most toxic. E. coli synthesizing Mtx2 from SSII-1 were ~7 to ~14 times more toxic than E. coli synthesizing Mtx2 from strains 31-2, IAB59, 2297, and Kellen Q (Table II). E. coli synthesizing Mtx2 from SSII-1 were ~7 times more toxic to C. quinquefasciatus larvae than clones synthesizing Mtx2 from strain 31-2 (Table II). Surprisingly, the opposite result was obtained when E. coli synthesizing Mtx2 from SSII-1 and 31-2 were assayed against larvae of the mosquito, A. aegypti. Mtx2 from strain 31-2 was about 100-fold more toxic to A. aegypti than Mtx2 from SSII-1 (Table III). SSII-1 and 31-2 Mtx2 differ in four aa positions, namely 37, 67, 224, and 279 (Table I), and any or all of these aa could contribute to the significant differences in the toxicity and mosquito host range of these toxins.

Table II. Mosquito larvicidal assays of E. coli cells synthesizing GST-Mtx2 fusion proteins against C. quinquefasciatus larvae Source of toxin protein (B. sphaericus strain) LC50 at 24 h cells/mla ()b >2.50  × 109c SSII-1 1.08  × 106 31-2 7.77  × 106 2362 2.00  × 106 IAB59 1.48  × 107 Kellen Q 9.10  × 106 2297 7.59  × 106 a Variation between replicates within one assay ± 10%. Values are means of two or more assays and are normalized taking into account the variation in yield of GST-Mtx2 fusion proteins (Fig. 2). b () indicates control bacteria lacking mtx2 gene were added. c 2.00 × 109 cells/ml is nontoxic.

Table III. Mosquito larvicidal assays of E. coli cells synthesizing mutant GST-Mtx2 fusion proteins against C. quinquefasciatus and A. aegypti larvae Source of toxin protein (B. sphaericus strain) LC50 at 24 h C. quinquefasciatus A. aegypti cells/mla SSII-1 1.08  × 106 5.34  × 108 31-2 7.77  × 106 4.20  × 106 SSII-1 (T279A) 1.93  × 105 2.10  × 108 31-2 (A279T) 8.71  × 106 5.75  × 106 SSII-1 (K224T) >2.00  × 109b 6.57  × 106 SSII-1 (K224T,T279A) >2.50  × 109 3.75  × 106 31-2 (T224K,A279T) 1.48  × 105 5.38  × 108 a Variation between replicates within one assay ± 10%. Values are means of two or more assays and are normalized taking into account the variation in yield of GST-Mtx2 fusion proteins (Fig. 2). b 2.00 × 109 cells/ml is nontoxic.

Single or double amino acid substitutions were introduced by site-directed mutagenesis into selected Mtx2 homologs to test the contribution of individual aa to toxicity and host range. Changes were made to render the Mtx2 proteins more or less like Mtx2 from strain SSII-1 (Table I). None of the single substitutions in Mtx2, including IAB59 (D31H or R50K), 31-2 (P67S), 2297 (T171K), 2362 (K40Q), SSII-1 (S37I), and SSII-1 (S67P) (Table I) had any detectable effect on toxicity toward C. quinquefasciatus larvae (data not shown).

However, substitutions at aa positions 224 and 279 (creating hybrids between SSII-1 and 31-2 Mtx2) significantly affected toxicity to C. quinquefasciatus and A. aegypti larvae. The T279A mutation in SSII-1 Mtx2 resulted in a modest ~5-fold increase in toxicity to C. quinquefasciatus larvae, whereas the SSII-1 (K224T) mutant and the double mutant SSII-1 (K224T,T279A) were completely nontoxic to these larvae (Table III). The reverse mutations 31-2 (A279T) and 31-2 (T224K,A279T) were generated so that the 31-2 Mtx2 mutants more closely resembled Mtx2 from SSII-1. 31-2 (A279T) was about as toxic to C. quinquefasciatus larvae as the parental 31-2 Mtx2 protein, but 31-2 (T224K,A279T) was at least 50-fold more toxic than 31-2 Mtx2 (Table III) and at least 7-fold more toxic than SSII-1 Mtx2. These results emphasize that K224 is particularly important in the toxicity of Mtx2 proteins to C. quinquefasciatus larvae.

Completely opposite results were obtained when the aa 224 and 279 mutants were assayed against A. aegypti larvae. SSII-1 (T279A) was about as toxic to these larvae as the weakly toxic SSII-1 Mtx2 protein, but SSII-1 (K224T) and the double mutant SSII-1 (K224T,T279A) were ~50-150-fold more toxic than the parental SSII-1 Mtx2. The excellent A. aegypti toxicity of 31-2 Mtx2 was not significantly affected by the A279T mutation, but the double mutant 31-2 (T224K,A279T) was only weakly toxic to A. aegypti due to a drop in larvicidal activity of ~100-fold compared with 31-2 (A279T) (Table III).

Assays against C. quinquefasciatus and A. aegypti larvae were performed with purified Mtx2 fusion proteins in which the N-terminal 15 amino acids containing the putative signal sequence of Mtx2 were deleted from GST-Mtx2 (Fig. 3) to exclude the possibility that the different larvicidal activities of E. coli expressing SSII-1 and 31-2 Mtx2 were due to differences in toxin solubility or stability in the bacteria. Table IV shows that SSII-1 Mtx2 was over 4-fold more toxic than 31-2 Mtx2, and that the 31-2 (T224K,A279T) mutant was about 17-fold more toxic than parental 31-2 Mtx2 to C. quinquefasciatus larvae. Both SSII-1 mutants with the K224T substitution were nontoxic to C. quinquefasciatus (Table IV). Conversely, purified toxins with T224 were the most toxic to A. aegypti, and toxins with K224 (SSII-1 and 31-2 (T224K,A279T)) were much less toxic to these larvae (Table IV). These results mirror the larvicidal activities of these toxins in live E. coli (Table III), suggesting that the different toxicities of the recombinant bacteria are due to variations in the aa sequences of the Mtx2 homologs, particularly in residue 224. 

SDS-PAGE analysis of GST-Mtx2 fusion proteins purified from

Table IV. Mosquito larvicidal assays of purified Mtx2 fusion proteins against C. quinquefasciatus and A. aegypti larvae Source of toxin protein (B. sphaericus strain) LC50 at 24 h C. quinquefasciatus A. aegypti µg/mla SSII-1 0.93 14.50 SSII-1 (K224T) >30 2.89 SSII-1 (K224T,T279A) >20b 3.67 31-2 3.90 3.91 31-2 (T224K,A279T) 0.23 16.36 a Variation between replicates within one assay ± 10%. Values are means of two or more assays using equivalent amounts of GST-Mtx2 fusion proteins based on SDS-polyacrylamide gel electrophoresis analysis (Fig. 3). b >20 µg/ml is nontoxic.

DISCUSSION

The Mtx2 homologs from six strains of B. sphaericus differ in only a few aa positions, and some of the homologs were found to be more toxic to mosquito larvae than others. These observations allowed us to design mutagenesis experiments which pinpointed Lys²²⁴ as an important aa in the toxicity of Mtx2 to C. quinquefasciatus larvae. The SSII-1 (T279A) mutant was 5-6-fold more toxic to C. quinquefasciatus larvae than SSII-1 Mtx2, but the double mutant SSII-1 (K224T,T279A) was inactive, showing that, although Ala²⁷⁹ is more favorable than Thr²⁷⁹ in the context of SSII-1 Mtx2, Lys²²⁴ is an overriding determinant of larvicidal activity. Why did 31-2 Mtx2 exhibit significant toxicity to C. quinquefasciatus despite the absence of Lys²²⁴? The highly active 31-2 Mtx2 toxin differs from the inactive SSII-1 (K224T,T279A) mutant in having Ile³⁷ and Pro⁶⁷, suggesting that one or both of these aa contributes to the toxicity of 31-2 Mtx2 and compensates for the absence of Lys²²⁴. This assumption is strengthened by the observation that the 31-2 (T224K,A279T) mutant, which is identical to SSII-1 Mtx2 except for the presence of Ile³⁷ and Pro⁶⁷, was over 7-fold more toxic to C. quinquefasciatus larvae than SSII-1 Mtx2. Therefore, it was surprising that the single substitutions 31-2 (P67S), SSII-1 (S37I), and SSII-1 (S67P) had no effect on toxicity. Further reciprocal constructs (e.g. 31-2 (I37S), SSII-1 (S37I,K224T), and SSII-1 (S67P,K224T)) are needed to resolve the issue of the relative importance of Ile³⁷ and Pro⁶⁷ in the context of 31-2 Mtx2.

When E. coli synthesizing Mtx2 from SSII-1, 31-2, and their respective mutants were assayed against A. aegypti larvae, even greater differences in toxicity were observed. However, in complete contrast to the results of larvicidal assays against C. quinquefasciatus, SSII-1 Mtx2 was found to be ~100-fold less toxic to A. aegypti than was 31-2 Mtx2; and surprisingly, the difference was also due to aa position 224. Parental or mutant Mtx2 proteins with Thr²²⁴ were always ~50-150-fold more toxic to A. aegypti than their counterparts with Lys²²⁴ (Table III).

How can a single aa substitution in a toxin substantially increase toxicity to one species of mosquito and virtually abolish toxicity to another species of mosquito? Although this is a new phenomenon, there are earlier studies on lepidopteran and dipteran toxins which are instructive and allow us to speculate on the possible mechanism of action of aa 224 (11, 12, 13, 14, 15, 16). Important genetic determinants of mosquito host range in the binary toxin from B. sphaericus have been localized to aa positions 99 and 104 in the 41.9-kDa subunit, but other aa in the 51.4-kDa subunit also contribute to toxicity (16). This is analogous to aa 224 in Mtx2 playing a major role in mosquito host range and aa 37 and/or 67 playing an accessory role. Several other studies have also concluded that one or a very few aa are major determinants of larvicidal activity and host range in a variety of toxins active against Lepidoptera and/or Diptera (11, 12, 13, 14, 15). However, in many cases insect specificity could not easily be attributed to particular aa, as different toxin segments were found to determine specificity by interacting in an undefined manner (11, 12, 13). Nevertheless, it is evident that different and sometimes overlapping segments of many toxins carry determinants of specificity for different insects (11, 12, 13).

In one interesting study, Ile⁵⁴⁵ of a dual specificity larvicidal protein from Bacillus thuringiensis aizawai ICI was found to be essential for toxicity to A. aegypti larvae, but not to larvae of the caterpillar Pieris brassicae; conversely, the single substitution I568T abolished toxicity to P. brassicae larvae but not to A. aegypti larvae (12). Together, the results suggested that the I568T mutation destroyed proteolytic cleavage activation of the 130 × 10³ M_r protoxin to a known ~55 × 10³ M_r Lepidoptera-active toxin, while the I545P mutation inhibited proteolytic activation at a different site of the protoxin and prevented the formation of a known ~53 × 10³ M_r Diptera-active toxin (12).

Although proteolytic cleavage by larval gut proteases activates many insecticidal toxins (1, 2, 12), it is unclear whether this also occurs in Mtx2 as the mechanism of action of the recently discovered Mtx2 family of mosquitocidal toxins is unknown (4). However, it is worth speculating that K224 in SSII-1 Mtx2 may be a major site of proteolytic cleavage activation by C. quinquefasciatus gut trypsin-like proteases (1, 2), and that other as yet unknown neighboring site(s) in 31-2 Mtx2 (which has Thr²²⁴) are exposed for cleavage activation by the compensating aa, Ile³⁷ and Pro⁶⁷. By analogy with the dual specificity larvicidal toxin from B. thuringiensis aizawai ICI, we would have to postulate that in the A. aegypti larval gut a different protease is responsible for cleavage activation of Mtx2 at a distinct site from the one cleaved in the C. quinquefasciatus gut, and that the T224K mutation in 31-2 Mtx2 adversely affects substrate specificity or denies access to the putative A. aegypti protease.

An alternative and perhaps more plausible hypothesis is simply that there are subtle (but vital) aa differences in the C. quinquefasciatus and A. aegypti gut receptors in the domains which interact with position 224 on the surface of Mtx2. For example, a crucial electrostatic interaction might form only with Thr²²⁴ in A. aegypti, while in C. quinquefasciatus a similar crucial interaction might form only with Lys²²⁴. Position 224 is predicted to lie at the peak of 1 of 11 predicted hydrophilic regions in Mtx2, which is consistent with a surface location. Regardless of the mechanism, it is clear that aa position 224 in the Mtx2 family of mosquitocidal toxins is an important and unusual determinant of toxicity and mosquito host range.

Finally, it is worth noting that some of the mutant Mtx2 toxins were significantly more toxic to C. quinquefasciatus larvae than their natural counterparts (SSII-1 (T279A) and 31-2 (T224K,A279T)). This approach may, therefore, be helpful in the design of exceptionally potent Mtx2 toxins.