Page 25 - O. Microbiology

Lycotoxin-Pa2a, A Novel Antibacterial and Antifungal Peptide
derived from the Venom of the Spider Pardosa astrigera
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Yunkyung Kim , Min Kyoung Shin , In-Wook Hwang , Seung-Tae Kim , and Jung-Suk Sung * 1
Yunkyung Kim , Min Kyoung Shin , In-Wook Hwang , Seung-Tae Kim , and Jung-Suk Sung *
1 Department of Life Science, Dongguk University-Seoul, Goyang 10326, Republic of Korea
1Department of Life Science, Dongguk University-Seoul, Goyang 10326, Republic of Korea
2 Life and Environment Research Institute, Konkuk University, Seoul 05029, Republic of Korea
2Life and Environment Research Institute, Konkuk University, Seoul 05029, Republic of Korea
BACKGROUND AIM
- Venom of spiders consists of various bioactive substances including ions, - To secure genetic resources for Pardosa astrigera, a domestic
peptides, proteins. Among them, peptide substances (1-10 kDa) are known as indigenous spider in Korea.
a major component with cytolytic activity and neurotoxicity. - To identify the peptides expected to have physiological and biochemical
- Peptides with a cationic, α-helical structure are usually called antimicrobial function, and discover more toxic substances and their effects from the
peptides (AMPs) due to its cytolytic activity, and found in venom gland of venom of the Pardosa astrigera.
venomous species such as spider species according to several studies. - To explore the potential and industrial utility as biological and industrial
However, there are few researches on venoms and toxin peptides derived materials through functional analysis and further study on the mechanism
from domestic native spiders in Korea. of action
METHODS
- RNA sequencing and de novo assembly was performed to construct the venom-gland transcriptome of the spider Pardosa astrigera.
- Following homology search against known peptide toxins to select toxin-like sequences, structural characterization including cysteine patterns, disulfide bonds, a-
helical conformation and net charge was conducted using several in silico analysis programs.
- Antibacterial activity of Lycotoxin-Pa2a against gram positive bacteria (B. cereus and S.aureus) and gram negative bacteria (E. coli and E.cartovora) was
evaluated using colony forming unit assay. Also, antibacterial activity of the peptide against methicillin-resistant S.aureus was assessed thorugh detection of ATP.
- Antifungal activity of Lycotoxin-Pa2a against F.oxysporum and C. albicans was determined using colony forming unit assay.
RESULTS
- Mature peptide of TBIU041425, a novel toxin-like peptide consists of 66 amino acid residues and has significant similarity with U1-lycotoxin-Ls1b, known peptide
toxin from Lycosa singoriensis.
- TBIU041425 has a net charge of +5.9, and is composed of cysteine-rich N terminal region as well as α-helical C-terminal region. Formation of disulfide bond and
was predicted by DISULFIND. PEP-POLD, HeliQuest and SWISS-MODEL servers were used to figure out and visualize the α-helical conformation of the peptide.
- After homology search and in silico structural analysis, TBIU041425 was selected and named Lycotoxin-Pa2a.
- The peptide inhibited the growth of gram-positive and gram-negative bacteria. Significant growth inhibition was shown on both gram-negative bacteria E. coli and
gram-positive bacteria B.cereus strains on 1 uM. Considerable growth inhibition was found on gram-negative bacteria E. carotovora and gram-positive bacteria
S.aureus strains. In addition, the peptide significantly inhibited the growth of methicillin-resistant S. aureus on 2 uM.
- TBIU041425 showed significant antifungal activity against Fusarium oxysporum in dose-dependent manner. Also, the peptide exhibited considerable antifungal
activity against Candida albicans.

Figure 1. Sequence alignment and structure analysis of TBIU041425. Figure 3. Antibacterial activity of Lycotoxin-Pa2a
against methicillin-resistant S. aureus.
A

B

C
Figure 4. Antifungal activity of Lycotoxin-Pa2a.
A B

Figure 2. Antibacterial activity of Lycotoxin-Pa2a.
A B C D

REFERENCES

- Brogden, K.A. (2005). Antimicrobial peptides: pore
formers or metabolic inhibitors in bacteria? Nature
CONCLUSION ACKNOWLEDGEMENTS Reviews Microbiology 3, 238.
- Garb, J.E. (2014). Extraction of Venom and
- In this study, the transcriptome and homology This work was supported by a grant from the Venom Gland Microdissections from Spiders for
analysis of venom of the spider Pardosa National Institute of Biological Resources (NIBR), Proteomic and Transcriptomic Analyses. Jove-J
astrigera was performed. funded by the Ministry of Environment (MOE) of the Vis Exp.
- Lycotoxin-Pa2a, a novel toxin-like peptide Republic of Korea (NIBR202009201) and the - Grishin, E.V., Volkova, T.M., and Arseniev, A.S.
was identified and properties of the peptide Dongguk University Research Fund of 2019. (1989). Isolation and structure analysis of
was characterized through in silico analysis. components from venom of the spider Argiope
- Lycotoxin-Pa2a exhibited antibacterial and lobata. Toxicon 27, 541-549.
antifungal activity. Contact information - Wang, G., Li, X., and Wang, Z. (2016). APD3: the
- These results suggest the potential of antimicrobial peptide database as a tool for
peptides derived from spider venom for the 252114@naver.com research and education. Nucleic Acids Res 44,
development of new anti-infective agents. +82-31-961-5173 D1087-1093.