Cry protein analysis with mosquitocidal activity and antimicrobial peptide search for the control of pathogens vectorized by mosquitoes

LAZARTE JN; LOPEZ ROCIO P; BERON CORINA; GIL MARIA FLORENCIA; BATTAGLIA, MARINA E.

San Miguel de Tucumán, Tucumán

Congreso; XII Congreso Argentino de Microbiología General SAMIGE; 2017

Asociación Civil de Microbiología General

Bacillus thuringiensis is an entomopathogenic bacteria that produces a parasporal inclusion composedby Cry proteins, toxic against mosquitoes and other insects. When this crystal is ingested by insectlarvae, is solubilized in alkaline gut environment and proteolytically cleaved by gut proteases. Afterbinding to the specific receptors on the brush border membrane of the midgut epithelium, the activatedtoxin would lead to insect death. Recently a novel polycation peptide, BTM-P1 with antimicrobial (AM)activity, was described based on the amino acid sequence of domain I of some Cry protoxins.Aedes aegypti is the principal vector for Zika, chikungunya, yellow fever, and dengue worldwide anditis the main target in vectorial control program. Some Culex species are vector of some encephalitisviruses with relevance in public health. In this context, Cry toxin studies result interesting as a putativealternative for biological control of Aedes and Culex mosquitoes. On the other hand, AM peptides canbe used for the control of human pathogens vectorized by these insects.In this work we analyzed several Cry proteins isolated from a native strain with mosquitocidal activityagainst Ae. aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens and Culex apicinus.The isolated toxins were identified as Cry4-like1, Cry4-like 2, Cry19-like1, Cry19-like2 and Cry24Ca byphylogenetic analysis. We performed the alignment by ClustalW in MEGA Software between all knownCry proteins with mosquitocidal activity and the ones isolated from the native strain and weconstructed a tree using the statistical method UPGMA. Then, we analyzed structural differencesamong native Cry proteins plus other toxins near the native ones according to the previousphylogenetic tree. We focused on: i) number of α-helix, ii) large of β-sheets, iii) loops similarity and iv)conserved motifs of functional importance.Cry native toxins show the typical domains (I, II and III) present in Cry mosquitocidal proteins.Structural analysis revealed that a motif located in α5 is conserved in all native Cry; this motif isinvolved in oligomerization which is necessary for pore formation. Moreover, specific residue located inα4-α5 loop is highly conserved among all native Cry and it is involved in lipid membrane interaction. Inthis context, we hypothesize that native Cry have mosquitocidal activity and Cry4-like1 and Cry4-like2have at least the same efficiency that Cry4Aa and Cry4Ba. Furthermore, Cry proteins will be clonedand expressed in an heterologous system and the toxic activity will be measured by bioassays againstmosquito larvae.In silico analysis shows that domain I of all native Cry toxins have hydrophobic regions, which could beused as templates for the generation of putative AM peptides.In conclusion, native Cry toxins are a promising option as biological agents for mosquito control or forthe control of pathogens vectorized by them.