Skip to main content

Advertisement

Springer Nature Link
Log in

Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity

  • Research Article
  • Published:
Genes & Genomics Aims and scope Submit manuscript

Abstract

Background

Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of β-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established.

Objective

In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis.

Methods

RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation.

Results

Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity.

Conclusions

To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  • Arakane Y, Muthukrishnan S (2010) Insect chitinase and chitinase-like proteins. Cell Mol Life Sci 67:201–216

    Article  CAS  Google Scholar 

  • Arakane Y, Zhu Q, Matsumiya M, Muthukrishnan S, Kramer KJ (2003) Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect Biochem Mol Biol 33:631–648

    Article  CAS  Google Scholar 

  • Badariotti F, Lelong C, Dubos MP, Favrel P (2007a) Characterization of chitinase-like proteins (Cg-Clp1 and Cg-Clp2) involved in immune defence of the mollusc Crassostrea gigas. FEBS J 274:3646–3654

    Article  CAS  Google Scholar 

  • Badariotti F, Thuau R, Lelong C, Dubos MP, Favrel P (2007b) Characterization of an atypical family 18 chitinase from the oyster Crassostrea gigas: evidence for a role in early development and immunity. Dev Comp Immunol 31:559–570

    Article  CAS  Google Scholar 

  • Badariotti F, Lelong C, Dubos MP, Favrel P (2011) Identification of three singular glycosyl hydrolase family 18 members from the oyster Crassostrea gigas: Structural characterization, phylogenetic analysis and gene expression. Comp Biochem Physiol B Biochem Mol Biol 158:56–63

    Article  Google Scholar 

  • Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, Brys L, Gomez J, De Baetselier P, Timmermans M (1998) Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J Biol Chem 273:24948–24954

    Article  CAS  Google Scholar 

  • Bhambri A, Dhaunta N, Patel SS, Hardikar M, Bhatt A, Srikakulam N, Shridhar S, Vellarikkal S, Pandey R, Jayarajan R et al (2018) Large scale changes in the transcriptome of Eisenia fetida during regeneration. PLoS ONE 13:e0204234

    Article  Google Scholar 

  • Edwards CA, Fletcher KE (1988) Interactions between earthworms and microorganisms in organic-matter breakdown. Agric Ecosyst Environ 24:235–247

    Article  Google Scholar 

  • Garcia-Fraga B, da Silva AF, Lopez-Seijas J, Sieiro C (2014) Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli. Appl Microbiol Biotechnol 98:2133–2143

    Article  CAS  Google Scholar 

  • Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512

    Article  CAS  Google Scholar 

  • Kim D, Kim J-S, Park I-Y, Kwak H-J, Lee DH, Cho S-J, Park SC (2016) A novel chitinase from the earthworm, Eisenia andrei. Anim Cells Syst 20:48–51

    Article  CAS  Google Scholar 

  • Kim S, Jeon D, Lee JY, Cho SJ, Lim Y, Eyun SI, Park SC, Seo YJ (2020) Upregulation of cellulase activity and mRNA levels by bacterial challenge in the earthworm Eisenia andrei, supporting the involvement of cellulases in innate immunity. Biochem Biophys Res Commun 521:15–18

    Article  CAS  Google Scholar 

  • Kucerova L, Broz V, Arefin B, Maaroufi HO, Hurychova J, Strnad H, Zurovec M, Theopold U (2016) The Drosophila chitinase-like protein IDGF3 is involved in protection against nematodes and in wound healing. J Innate Immun 8:199–210

    Article  CAS  Google Scholar 

  • Liu M, Chen C, Wu Q-C, Chen J-L, Dai L-S, Chu SH, Liu Q-N (2021) Chitinase involved in immune regulation by mediated the toll pathway of crustacea Procambarus clarkii. Fish Shellfish Immun 110:67–74

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Lu Y, Zen KC, Muthukrishnan S, Kramer KJ (2002) Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase. Insect Biochem Mol Biol 32:1369–1382

    Article  CAS  Google Scholar 

  • Mali B, Mohrlen F, Frohme M, Frank U (2004) A putative double role of a chitinase in a cnidarian: pattern formation and immunity. Dev Comp Immunol 28:973–981

    Article  CAS  Google Scholar 

  • Park IY, Cha JR, Ok SM, Shin C, Kim JS, Kwak HJ, Yu YS, Kim YK, Medina B, Cho SJ, Park SC (2017) A new earthworm cellulase and its possible role in the innate immunity. Dev Comp Immunol 67:476–480

    Article  CAS  Google Scholar 

  • Parle JN (1963) Micro-organisms in the intestines of earthworms. J Gen Microbiol 31:1–11

    Article  Google Scholar 

  • Prochazkova P, Roubalova R, Dvorak J, Navarro Pacheco NI, Bilej M (2020) Pattern recognition receptors in annelids. Dev Comp Immunol 102:103493

    Article  CAS  Google Scholar 

  • Storey KG (1989) Cell lineage and pattern formation in the earthworm. Development 107:519–531

    Article  Google Scholar 

  • Sutherland TE, Logan N, Rückerl D, Humbles AA, Allan SM, Papayannopoulos V, Stockinger B, Maizels RM, Allen JE (2014) Chitinase-like proteins promote IL-17-mediated neutrophilia in a tradeoff between nematode killing and host damage. Nat Immunol 15:1116–1125

    Article  CAS  Google Scholar 

  • Tellam RL, Wijffels G, Willadsen P (1999) Peritrophic matrix proteins. Insect Biochem Mol Biol 29:87–101

    Article  CAS  Google Scholar 

  • Tracey MV (1951) Cellulase and chitinase of earthworms. Nature 167:776–777

    Article  CAS  Google Scholar 

  • Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515

    Article  CAS  Google Scholar 

  • Ueda M, Shioyama T, Nakadoi K, Nakazawa M, Sakamoto T, Iwamoto T, Sakaguchi M (2017) Cloning and expression of a chitinase gene from Eisenia fetida. Int J Biol Macromol 104:1648–1655

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202130202). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A06046235). This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C2007785).

Author information

Author notes

  1. Beom Jun Park, Yoo Bin Yoon and Dong Ho Lee have contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, Chung-Ang University, Seoul, 06974, Republic of Korea

    Beom Jun Park, Yoo Bin Yoon & Soon Cheol Park

  2. Da Vinci College of General Education, Chung-Ang University, Seoul, 06974, Republic of Korea

    Dong Ho Lee

  3. Department of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju, 26493, Republic of Korea

    Chuog Shin

  4. Department EGRA, University of Kisangani, Kisangani, Democratic Republic of the Congo

    Louis Juakali

  5. Department of Biological Sciences and Biotechnology, Chungbuk National University, Chungbuk, 28644, Republic of Korea

    Sung-Jin Cho

Authors
  1. Beom Jun Park
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yoo Bin Yoon
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Dong Ho Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Chuog Shin
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Louis Juakali
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Sung-Jin Cho
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Soon Cheol Park
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding authors

Correspondence to Sung-Jin Cho or Soon Cheol Park.

Ethics declarations

Conflict of interest

No potential conflict of interest was reported by the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

13258_2021_1183_MOESM1_ESM.pptx

Supplementary file1 Supplemental Figure 1. Nucleotide and deduced amino acid sequences of the top 3 chitinase-related genes upregulated by B. subtilis challenge in E. andrei. (A) The open reading frame of EaChi2 (Trinity ID: DN2491_c0_g1_i4; Genbank accession number: MZ615707) consists of 1653 nucleotides encoding 551 amino acids. (B) The partial open reading frame of EaChi3 (Trinity ID: DN8211_c0_g1_i1; Genbank accession number: MZ615705) consists of 1506 nucleotides encoding 502 amino acids, and (C) the open reading frame of EaChi4 (Trinity ID: DN3614_c0_g1_i1; Genbank accession number: MZ615704) consists of 1290 nucleotides encoding 430 amino acids. The stop codon is indicated by an asterisk (*). Residue numbers for nucleotides and amino acids are indicated in the top and bottom row. Putative signal peptide sequence, glycol_18 domain, chitin-binding peritrophin-A domain and PGST-rich domain are indicated in black, green, blue, and orange boxes respectively. Amino acid residues P, G, S, T in the PGST-rich domain are highlighted in black. The two red boxed sequences in catalytic domain represent the chitin-binding and chitin-catalyzing domains, respectively. (PPTX 202 KB)

13258_2021_1183_MOESM2_ESM.pptx

Supplementary file2 Supplemental Figure 2. Temporal expression analysis of earthworm chitinase mRNAs using real-time qRT-PCR after challenge with B. subtilis.The data, obtained from three independent experiments, are expressed as mean ± SEM. *Indicates statistical significance (p < 0.01) compared with the control group. Differences between groups were assessed using a student’s t-test. (PPTX 69 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Park, B.J., Yoon, Y.B., Lee, D.H. et al. Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity. Genes Genom 43, 1497–1502 (2021). https://doi.org/10.1007/s13258-021-01183-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13258-021-01183-z

Keywords