The cry toxin operon of Clostridium bifermentans subsp. malaysia is highly toxic to Aedes Larval Mosquitoes

Abstract

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.

FIG 1

Toxicity appears in supernatants within a day of C. bifermentans subsp. malaysia cultures. To analyze the appearance of toxicity in the supernatants, both the pellet and supernatant fractions were monitored for toxicity to fourth-instar Anopheles stephensi larvae. A constant amount of the bacterial culture was assayed. A high level of toxicity in the pellet of the bacterial culture was observed within a short time of culture, while no toxicity was detected in the supernatant until 12 h, when lysis of sporangia occurs (16). Toxicity was monitored at 24, 48, and 72 h.

FIG 2

Toxicity is lost in C. bifermentans subsp. malaysia cultures after 5 days. (A) Toxicity in 1- and 5-day supernatants. Whole cultures were centrifuged to collect supernatants. In 1-day cultures, a high level of toxicity was observed in the supernatant. However, in 5-day cultures no toxicity was observed in 24- and 48-h bioassays. (B) Differential expressions of the protein profiles for 1- and 5-day cultures were observed in the supernatants (bands A and B). Following tryptic digestion, the peptides were sequenced using tandem mass spectrophotometry. Band B of 5-day supernatant was identified as heat shock proteins. In contrast, band A was found in 1-day but not in 5-day supernatants. One protein identified by proteomics was the Cry16A protein in the 1-day supernatant.

FIG 3

The full-length operon expresses both the Cry and hemolysin proteins. (A) Schematic of the Cry operon under the control of the Cyt1A promoter from Bacillus thuringiensis subsp. israelensis. The operon consists of the cry16A, cry17A, cbm17.1, and cbm17.2 genes, and a terminator (TT). Except for cry16A, the native Shine-Dalgarno (SD) sequences were used. For cry16A, the Cyt1A SD was used. (B) Both the Cry16A and Cbm17.1 proteins were expressed when the entire operon was used for expression. Immunoblots using anti-Cry16A and -Cbm17.1 antibodies show that these were expressed at 24 h. (C) The Cry16A protein was also observed in C. bifermentans subsp. malaysia (Cbm) cultures at 18 h but not in C. bifermentans (Cb) cultures. Sup, supernatant.

FIG 4

The full-length Cry operon shows high toxicity to Aedes mosquitoes but not to Anopheles mosquitoes. (A) Toxicity of C. bifermentans subsp. malaysia (Cbm) and the Cry operon to Anopheles mosquitoes. While C. bifermentans subsp. malaysia cultures show high toxicity to Anopheles gambiae mosquitoes, the recombinant B. thuringiensis containing the Cry operon is nontoxic to Anopheles mosquitoes. The toxicity curves for the recombinant B. thuringiensis containing the Cry operon with Anopheles stephensi and Culex quinquefasciatus were similar to those obtained with Anopheles gambiae. (B) Toxicity of C. bifermentans subsp. malaysia and the Cry operon to Aedes mosquitoes. Both C. bifermentans subsp. malaysia and the recombinant B. thuringiensis Cry operon were toxic to Aedes mosquitoes. However, C. bifermentans (Cb) and the recombinant B. thuringiensis pHT315 were nontoxic.

FIG 5

All single genes show no toxicity to Aedes mosquitoes. (A) Each of the toxin genes in the Cry operon (CryO) was expressed under the control of the Cyt1A promoter, the Cyt1A Shine-Dalgarno sequence, and includes a 3′ terminator identical to the Cry operon construct in Fig. 3. (B) The Cry16A toxin was stably expressed, and the Cry17A toxin was expressed but readily degraded in culture even at 24 h. The Cry16A protein was not observed in SDS-PAGE at 24 h (Fig. 3) but was detected by immunoblotting at this time (Fig. 3). (C) None of the single-gene constructs expressed in B. thuringiensis showed any toxicity.

FIG 6

Any gene deletion in the Cry operon results in loss of toxicity. (A) Three gene deletions were made. In the first construct, pCry16, only Cry16A was expressed (Fig. 5), while in the second, CryO/Δ17.1, only cbm17.1 was deleted and in the third, pCry16/17, both cbm17 genes were deleted. The last two were derived from pCryO by restriction with PacI (P) and NcoI (N), respectively. Each of the constructs is under the control of the Cyt1A promoter and includes a 3′ terminator identical to the Cry operon construct in Fig. 3. (B) The Cry16A toxin was observed in the recombinant B. thuringiensis CryO, recombinant B. thuringiensis pCryO/Δ17.1, and recombinant B. thuringiensis pCry16/17 cultures. The recombinant B. thuringiensis CryO construct also shows expression of the Cbm17.1 protein at 48 to 72 h, as observed at 24 h (Fig. 3).

FIG 7

Native PAGE shows that the Cry operon forms a complex. (A) The entire Cry operon (CryO) forms a complex of about 170 kDa in 24-h recombinant B. thuringiensis cultures. This complex included the Cbm17.1 protein as shown in the immunoblot (B). None of the other three constructs (pCry16, pCry16/17, and pCryO/Δ17.1) showed the presence of the complex in recombinant B. thuringiensis cultures.