Cloning and purification of the first termicin-like peptide from the cockroach Eupolyphaga sinensis

 

Zichao Liu^1 2, Kehua Yuan², Ruopeng Zhang³, Xuchen Ren^1 2, Xiaolong Liu^1 2, Shuhua Zhao^4 6, Dingkang Wang^1 2 5

 

1 Key Laboratory of Special Biological Resource Development and Utilization of Universities in Yunnan Province,

2 Key Lab of Aquatic Ecological Restoration of Dianchi Lake in Kunming, Department of Biological Science and Technology, Kunming University, Kunming 650214, China.

2 Department of Oncology, Yan’an Hospital of Kunming City; Yunnan, Cardiovascular Hospital; and Yan’an Hospital of Kunming Medical University, Kunming 650051, China.

3 Department of Obstetrics and Gynecology, Shenzhen Maternal and Child Health Care Hospital, Affiliated to Southern Medical University, Shenzhen 518028, China.

4 Yunnan Key Laboratory of Fertility Regulation and Minority Eugenics, Yunnan Population and Family Planning Research Institute, Kunming 650021, China.

5 Kunming University, Puxin Road 2#, Kunming, Yunnan 650214, China.

6 First Affiliated Hospital of Kunming Medical University, Xichang Road 295#, Kunming, Yunnan 650032, China.

 

ABSTRACT

Background

Termicin is an antimicrobial peptide with six cysteines forming three disulfide bridges that was firstly isolated from the salivary glands and hemocytes of the termite Pseudacanthotermes spiniger. In contrast to many broad-spectrum antimicrobial peptides, termicin is most active against filamentous fungi. Although more than one hundred complementary DNAs (cDNAs) encoding termicin-like peptides have been reported to date, all these termicin-like peptides were obtained from Isoptera insects.

 

Methods

The cDNA was cloned by combination of cDNA library construction kit and DNA sequencing. The polypeptide was purified by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined by Edman degradation and mass spectrometry. Antimicrobial activity was tested against several bacterial and fungal strains. The minimum inhibitory concentration (MIC) was determined by microdilution tests.

 

Results

A novel termicin-like peptide with primary structure ACDFQQCWVTCQRQYSINFISARCNGDSCVCTFRT was purified from extracts of the cockroach Eupolyphaga sinensis (Insecta: Blattodea). The cDNA encoding Es-termicin was cloned by cDNA library screening. This cDNA encoded a 60 amino acid precursor which included a 25 amino acid signal peptide. Amino acid sequence deduced from the cDNA matched well with the result of protein Edman degradation. Susceptibility test indicated that Es-termicin showed strong ability to kill fungi with a MIC of 25 μg/mL against Candida albicans ATCC 90028. It only showed limited potency to affect the growth of Gram-positive bacteria with a MIC of 200 μg/mL against Enterococcus faecalis ATCC 29212. It was inactive against gram-negative bacteria at the highest concentration tested (400 μg/mL Es-termicin showed high sequence similarity with termicins from many species of termites (Insecta: Isoptera).

 

Conclusions

This is the first report of a termicin-like peptide isolated from E. sinensis that belongs to the insect order Blattodea. Our results demonstrate the diversity of termicin-like peptides, as well as antimicrobial peptides in insects.

 

Key words: Eupolyphaga sinensis; Cockroach; Termicin-like peptide; Es-termicin; Antifungal peptide

 

Funding

This work was supported by Key Disciplines (Ecology) Project of Yunnan Education Department and by grants from the Chinese National Natural Science Foundation (31360516), and the Research Foundation of Kunming University (XJL13013 and YJL14002).

 

Received: July 8., 2015.

Revised: January 13, 2016.

Accepted: January 28, 2016.

 

Competing interests

The authors declare that they have no competing interests.

 

Authors’ contributions

ZL conceived and designed the experiments. ZL, KY, RZ, XR and XL performed the experiments. KY, SZ and DW performed the statistical analysis. ZL, SZ and DW wrote the paper. All authors read and approved the final manuscript.

 

doi: 10.1186/s40409-016-0058-7