Peptidomic Analysis and Antimicrobial Activity of Serum Peptide from Hevea brasiliensis Clone BPM24.

BACKGROUND: Hevea brasiliensis is severely affected by the fungal disease caused by Phytophthora spp. Significant loss of rubber yield is widespread and extensive use of chemical fungicides has resulted in health and environmental problems. OBJECTIVE: This work aims to extract and identify the latex serum peptides from a disease tolerant clone of H. brasiliensis, and study the inhibitory efficacy against pathogenic bacteria and fungi. METHODS: Serum peptides were extracted from H. brasiliensis BPM24 using mixed lysis solution. Low molecular weight peptides were screened and fractionated by solid-phase extraction and then identified by tandem mass spectrometry. Total and fractionated serum peptides were assayed for bacterial and fungal inhibition using broth microdilution and poisoned food methods. An inhibitory control study in the greenhouse was also performed using susceptible clones for pre and postinfection with Phytophthora spp. RESULTS: Forty-three serum peptide sequences were successfully identified. Thirty-four peptides matched with the proteins associated with plant defense response signaling, host resistance, and adverse environmental factors. The inhibitory study of total serum peptides demonstrated antibacterial and anti-fungal properties. The greenhouse study exhibited disease inhibitory efficacy of 60% for the treatment of Phytophthora spp. in post-infected plants and 80% for pre-treated samples. CONCLUSION: Latex serum peptides from disease tolerant H. brasiliensis revealed several proteins and peptides associated with plant defense and disease resistance. The peptides play a vital role for defense against bacteria and fungi pathogens, including Phytophthora spp. Enhanced disease protection can be obtained when the extracted peptides were applied to the susceptible plants before exposure to the fungi. These findings provided an insight and may pave the way for the development of biocontrol peptides from natural resources.

His180 in the pore-lining α4 of the Bacillus thuringiensis Cry4Aa δ-endotoxin is crucial for structural arrangements of the α4-α5 transmembrane hairpin and hence biotoxicity.

One proposed toxic mechanism of Bacillus thuringiensis Cry δ-endotoxins involves pore formation in target membranes by the α4-α5 transmembrane hairpin constituting their pore-forming domain. Here, nine selected charged and uncharged polar residues in the pore-lining α4 of the Cry4Aa mosquito-active toxin were substituted with Ala. All mutant toxins, i.e., D169A, R171A, Q173A, H178A, Y179A, H180A, Q182A, N183A and E187A, were over-expressed in Escherichia coli as 130-kDa protoxin inclusions at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti larvae revealed that only H178A and H180A mutants displayed a drastic reduction in biotoxicity, albeit almost complete insolubility observed for H178A, but not for H180A inclusions. Further mutagenic analysis showed that replacements of His180 with charged (Arg, Lys, Asp, Glu), small uncharged polar (Ser, Cys) or small non-polar (Gly, Val) residues severely impaired the biotoxicity, unlike substitutions with relatively large uncharged (Asn, Gln, Leu) or aromatic (Phe, Tyr, Trp) residues. Similar to the trypsin-activated wild-type toxin, both bio-active and -inactive H180 mutants were still capable of releasing entrapped calcein from lipid vesicles and producing cation-selective channels with ~130-pS maximum conductance. Analysis of the Cry4Aa structure revealed the existence of a hydrophobic cavity near the critical His180 side-chain. Analysis of simulated structures revealed that His180-to-smaller residue conversions create a gap disrupting such cavity's hydrophobicity and hence structural arrangements of the α4-α5 hairpin. Altogether, our data disclose a critical involvement in Cry4Aa-biotoxicity of His180 exclusively present in the lumen-facing α4 for providing proper environment for the α4-α5 hairpin prior to membrane-inserted pore formation.