Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species

Abstract

The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells. IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

“…Our data show that this protein was not accumulated to high levels inside cells and did not induce a clear destruction of the cells during intoxication. These data are interesting since we have previously reported that in the mosquito Culex quinquefasciatus the Cyt1Aa toxin also binds to microvilli from all midgut regions, but it also can be observed inside cells, internalized on small vesicles in the cells of caeca and anterior midgut regions, although this distribution pattern requires further analysis [ 39 ].…”

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

“…The Cyt1Aa toxin has a single α-β domain that contains two α-helix surround β-sheet [75]. This toxin interacts directly with phospholipids from the midgut cells; t fore, its action is independent of the presence of specific protein receptors [36,76,77] localization pattern of the Cyt1Aa toxin on the cell microvilli along the whole larvae gut shown by recent studies corroborates its unspecific binding to the cell memb [35,78]. Two models of action were proposed for Cyt1Aa.…”

“…These aggregates of the Cyt toxin on the membrane could function as a Cry toxin receptor, inducing the synergistic effect of these proteins. Cyt1Aa is also involved in the synergy with the Bin toxin from L. sphaericus [ 78 ].…”

“…Recently, the analysis of the molecular mechanism of this synergy showed that it is not based on a specific interaction between the Bin and Cyt toxins. It was demonstrated that the BinA toxin was internalized on the midgut cells in the presence of Cyt1Aa, but not in the presence of a mutant Cyt1AaV122E affected in its oligomerization and pore formation activity, suggesting that the pore formation activity of Cyt1Aa facilitates the transport of BinA into the midgut cells allowing its toxic intracellular effect [ 78 ]. The large pores formed by Cyt1Aa, observed by Tetreau et al [ 89 ], could explain how molecules, such as the BinA subunit, could be internalized into the midgut cells.…”

Molecular and biological features of Culex quinquefasciatus homozygous larvae for two cqm1 alleles that confer resistance to Lysinibacillus sphaericus larvicides

“…4 The binding of the Bin toxin to this receptor mediates its cell internalization, producing cytopathological effects and larval mortality. [5][6][7][8][9] The absence of Cqm1 receptors on the midgut epithelium has been reported as the main responsible factor for C. quinquefasciatus resistance to the Bin toxin. [10][11][12][13] The most common mutations in the cqm1 gene that confer resistance are those causing premature stop codons.…”