Edited by: Paivi H. Torkkeli, Dalhousie University, Canada
Reviewed by: Gregory Shaun Watson, James Cook University, Australia; Tomas Erban, Crop Research Institute, Czech Republic
*Correspondence: Arina L. Maltseva, Department of Invertebrate Zoology, Saint Petersburg State University, Universitetskaya 7/9, Saint Petersburg 199034, Russia e-mail:
This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology.
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Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins—new antimicrobial peptides of the lugworm
Antimicrobial peptides (AMPs) are relatively small (not exceeding 100 amino acids) usually cationic polypeptidic molecules with prominent inhibitory potential against various microbial pathogens. It is well recognized that AMPs constitute an important component of the innate immunity, having a role in both effector and regulatory functions. The fact that AMPs are present in a wide range of organisms including plants, vertebrate and invertebrate animals, protists and prokaryotes (rev. in Boman,
The wide distribution of AMPs makes them an attractive object for comparative immunology. Such studies can provide insights into the mechanisms underpinning the evolution of innate immune responses and also lead to identification of new effective molecular structures which could be developed as new therapeutics. AMPs of invertebrate animals are of particular importance, as their host defense against infections relies entirely on the mechanisms of innate immunity. Historically insects represent a group of invertebrate animals where the immune system has drawn intensive interest of investigators and consequently their AMPs are the most well characterized (Bulet et al.,
Annelids might be considered as a basal group which is closest to a putative common ancestor of other coelomate invertebrates. Therefore, their immunity is a fascinating object of study for comparative immunologists. Besides this, annelids are important players in the benthic communities as they are critical for biomass production in marine and freshwater ecosystems. Paradoxically, the existing knowledge about mechanisms of their host defense is largely based on the studies of Oligochaetes (Cooper et al.,
More specifically, just a few species of annelids were investigated as possible sources of AMPs (rev. in Tasiemski,
Arenicins-1 and -2, identified by our group in
In the present study we addressed the question of the physiological functions of arenicin 1 and 2 in the lugworm body as components of its immune system with a specific focus on the spatial distribution of their localization in the tissues and the pattern of their expression upon infectious stimuli.
Adult lugworms (apprx. 2.5 cm length)
To induce immune responses worms were injected by 107 CFU suspension of heat inactivated
Before dissection animals were anesthetized in 5% MgCl2 in SMW whereupon pieces of body wall, salivary glands, foregut, and midgut tissue were dissected, placed into sterile tubes and fixed for RNA extraction.
RNA was isolated from fixed and homogenized in PureZOL (Cat. No. #732-6890, BioRad Inc, CA, USA) tissues, using the RNAeasy kit (Cat. No. 74104 Qiagen Inc., CA, USA). After DNase I (Cat. No. #EN0521 Fermentas, Thermo Fisher Scientific Inc., MA, USA) treatment (30 min at room temperature) and purification RNA quality was verified with NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). 260/280 and 260/230 nm absorbance ratios did not exceed 2.0 indicating appropriate RNA quality.
Arenicin-1, arenicin-2 and actin primers were custom made by Syntol C° (Moscow, Russia). The sequences were as follows.
Arenicin-1: forward 5′-CTAATCCTGGCCATTTTCTGCG-3′; reverse 5′-CCCTGAGCTGACTGGAAATAG-3′; product 338 bp.
Arenicin-2: forward 5′-GCGAGATCGGCTGGAGAG-3′; reverse 5′-CCCTGAGCTGACCGGAAG-3′; product 254 bp.
Actin: forward 5′-CAAATCATGTTCGAGACCTTC-3′; reverse 5′-GCTGATCCACATCTGTTGG3′; product 714 bp.
PCR was performed with SuperScript™ III One-Step RT-PCR System with Platinum® Taq DNA Polymerase protocol (Cat. No. 12574-018, Invitrogen, Thermo Fisher Scientific Inc., MA, USA) according to manufacturer's instructions. β-Actin gene was used as a control to normalize a template loading. Amplification was carried out at C1000X cycler (BioRad Inc., CA, USA). The PCR products were then sequenced in Resource Center for Molecular and Cellular Technologies of St. Petersburg State University (St.Petersburg, Russia).
Real-time PCR was performed with iQ™ SYBR® Green Supermix (Cat. No. 170-8880, BioRad, CA, USA). Five independent RNA isolations with following cDNA synthesis and RT PCR were done for each experimental case. Reactions were conducted on CFX100 cycler (BioRad, CA, USA). Actin was used as a housekeeping gene for normalization and results were analyzed by the ΔCT method to calculate expression changes,
Recombinant arenicin-2 (Ovchinnikova et al.,
Crude extracts of coelomocytes were obtained by homogenization of coelomocytes (prepared as described earlier) in 5% acetic acid. Particles were sedimented by centrifugation (10,000 g, 40 min, 4°C) and supernatants were aspirated and accepted as crude extracts. Synthetic arenicin-1 was kindly provided by Dr. Nickolai I. Kolodkin (Institute of Highly Pure Biopreparations, Saint Petersburg, Russia). Both crude extract (50 μg of total protein) and arenicin-1 (0.5 μg) were loaded into acidic urea 12.5% PAAG (Panyim and Chalkley,
Lugworms (apprx. 2.5 cm length) were anesthetized in 5% MgCl2 in SMW, fixed in 4% paraformaldehyde (Cat. No. 158127, Sigma-Aldrich, MO, USA), subjected to standard alcohol dehydration and via xylol embedded into paraffin. Serial cross-sections (4 μm) were placed on polylysine-coated slides (Cat. No. P0425, Sigma-Aldrich, MO, USA) and dried at 37°C.
Fresh coelomocytes in suspension were placed on polylysine-coated slides (Cat. No. P0425, Sigma-Aldrich, MO, USA), left to dry out at room temperature and fixed with 4% paraformaldehyde (Cat. No. 158127, Sigma-Aldrich, MO, USA). For
Prepared cross-sections or smears were incubated sequentially in PBST (3 times, 20 min), PBST with diluted sheep serum 1: 5 (1 h) and anti-arenicin AB (1: 50) in sheep-serum or rabbit IgG overnight. Preparations were washed by PBST (3 times, 20 min) and stained by HRP-conjugated secondary AB (1: 500) (Cat. No. #31463, Thermo Scientific, Thermo Fisher Scientific Inc., MA, USA). AB was visualized with DAB (Cat. No. D8001, Sigma-Aldrich, MO, USA). Some preparations were additionally stained by hematoxylin-eosin.
CF was obtained as described earlier. Plasma was separated from cells by centrifugation (400 g, 10 min, 4°C). For component analysis of plasma it was loaded onto HPLC analytical column (C18, D 4.6 mm, length 20 cm, grain 5 μm, pore 300 Å, Vydec, The Nest Group Inc., MA, USA) and separated using Agilent 1260 HPLC system (Agilent Technologies, CA, USA). Elution was performed with a gradient of 0–60% acetonitrile in presence of 0.05% trifluoroacetic acid during 1 h. Absorbance at 220 and 280 nm was used for detection.
For antimicrobial testing native plasma was concentrated (3:1) in VacSpeed System (Labconco, MO, USA). Two variations of test were applied as described in Lehrer et al. (
The presence of arenicins-1 and -2 transcripts were examined in coelomocytes, body wall, salivary glands, foregut, and midgut by semi-quantitative RT-PCR. Arenicins mRNA expression was detected in all types of analyzed samples, however the highest levels of both arenicin-1 and 2 mRNA were observed in coelomocytes (Figure
To mimic the infectious process worms were injected with heat-inactivated laboratory strains of microorganisms into the coelomic cavity. In order to investigate the possible role of different PAMPs/PRRs interaction in mediating the response, worms were challenged with representatives of the three major different lineages of microorganisms (gram-positive and gram-negative bacteria and fungi) in different combinations. At 24 h post microbial challenge, samples of different tissues were collected and arenicins mRNA expression was quantified by Real-Time PCR. There was no change in the levels of arenicin-1 and -2 expression post-stimulation of coelomocytes compared to intact or SMW-injected animals (Figure
Anti-arenicins rabbit pAB were purified by affine chromatography against recombinant arenicin-2. Their specificity was confirmed by Western-blot against crude coelomocyte extract, synthetic arenicin-1 being a positive control (Figure
Paraffin-embedded tissue cross-sections were pAB stained to characterize mature peptide localization. Several compartments including the outermost part of body wall and gut wall proved to be pAB-immunoreactive (Figures
In the gut wall arenicins were expressed only in sparse individual cells (Figure
Next we examined the expression of arenicins in coelomocytes. AMPs translated from individual transcripts (in contrast to proteolytic fragments) usually include signal peptide and undergo co-translational transfer into the endoplasmic reticulum. This implicates their localization in the vesicular apparatus of the cell. In our previous study (Ovchinnikova et al.,
Immunostaining of the preparations of the coelomic fluid smears demonstrated heterogeneity in the morphology of the coelomocyte population which supports previously reported observations (rev. in Vetvicka and Sima,
AMPs stored in cytosolic granules can be liberated from cells via exocytosis. We investigated if arenicins could be found in coelomic fluid plasma (CFP). CFP was fractionated by HPLC and major fractions were analyzed by electrophoresis. Neat, as well as concentrated (1: 10) CFP appeared to be free from protein containing fractions with only one macromolecular component (MW > 10 kDa) in CFP detected. However, its detection was not regular. Notably, neither whole CFP nor any of its fractions showed detectable antimicrobial activity. Injection of microbes into coelomic cavity had no effect on the spectrum of CFP fractions as well as on the antimicrobial activity of CFP. Collectively, these results suggest that CFP does not contain anti-microbial components either under normal condition or after the microbial challenge.
One of the main functions of AMPs is their participation in the process of microbial killing inside the phagolysosome. To test this possibility we immunostained the
In the present study we characterized the tissue distribution of previously described antimicrobial peptide arenicin, represented by two isoforms which differed by a single amino-acid replacement in the mature peptide (Ovchinnikova et al.,
There were three major findings in this study.
Firstly, arenicins are expressed in a wide range of tissues of the lugworm body in addition to coelomocytes where they were initially identified. The list of the tissues expressing arenicins includes several epithelia, which implies involvement of arenicins into both systemic and epithelial branches of immunity.
Secondly, expression of arenicins (resembling that of hedistin from
Finally, although coelomocytes are capable of production and storage of arenicins, their secretion into coelomic fluid was negligible. However, our study did demonstrate that arenicins participate in the pathogen inactivation within the phagolysosome.
Lugworms are common dwellers in muddy habitats of subtidal and intertidal zones. Habitats such as these are abundant in microbes. Lugworms spend their lives engulfing and disgorging the surrounding sediments. Both epithelia—body wall and the gut—are under permanent threat of being attacked by microbial pathogens. Our findings supported this as we detected the expression of arenicins using both PCR and immunohistochemical methods in both critical organism boundaries—the body wall and gut. This characterizes arenicins as key players in the epithelial defense.
The outermost part of the body wall which was positive for arenicin consists of a thin cuticle and one layered epithelium. The principal cell type constituting the surface epithelium in polychaetes is represented by so called “supporting” cells. These cells bear apical microvilli which penetrate cuticle and are responsible for its synthesis (rev. in Gardiner,
Midventrally situated neuronal cord, lying upon circular musculature of the body wall, also proved to be arenicin-positive. The structure of the
The gut is the second principal compartment of the lugworm as it exists in permanent contact with the surrounding mud. Interestingly, our study revealed that arenicins are also expressed in this area. The polychaete midgut usually consist of enterocytes as a dominant population and one or several types of secretory gland cells (rev. Saulnier-Michel,
As in original study (Ovchinnikova et al.,
We further addressed the functional role of arenicins as components of lugworm host defense. We examined the possibility for arenicins to be released into the CF thus providing the first line of defense acting as soluble factors. However, thorough analysis of CF by different biochemical methods did not reveal any traces of arenicins. Furthermore, the analysis of CFP antimicrobial activity demonstrated negative results, suggesting that in coelomic cavity, anti-microbial defense predominantly depends on cell-mediated immunity. Next we proposed that the main function of arenicins would facilitate the process of microbial killing within the phagolysosome. Cytoplasm of
Generally there are several types of coelomocytes in polychaetes, such as granulocytes (syn. amoebocytes), eleocytes and hemocytes. However, the last two are accepted to be absent in Arenicolidae (rev. in Vetvicka and Sima,
Still there is unambiguous interpretation of an interrelation among their subpopulations. Moreover, an evident demonstration of the common hematopoietic area is also lacking. Different derivatives of coelomic peritoneum including extravasal tissue are most often proposed as candidates (rev. in Gardiner,
In granulocytes, immunopositivity was detected in granular apparatus, which was expected, as pre-pro-arenicins include signal peptides. There are two main destinations for cytosolic granules—to be exocytized or to fuse with the other vacuole. Immunohistochemistry of
Coelomocytes were tested for induction of the arenicin gene upon infectious conditions and showed no change in arenicin expression. This result of constitutive arenicins synthesis in coelomocytes is concurrent with that of hedistin—an AMP constitutively expressed in coelomocytes of a benthic polychaete
Active phagocytic cells and epithelia are the most common compartments of AMP expression in metazoans (including mammals and insects). Interestingly, in the present study we revealed expression of the same peptide in both compartments. Usually epithelia and phagocytes produce their own set of AMPs (e.g., in humans—alpha-defensins 1-4 in neutrophils, LL-37 in monocytes, alpha-defensins 5-6 in intestine and different beta-defensins in skin and other borders) (De Smet and Contreras,
Our study demonstrated that arencins are found in the tissues of the lugworm body (coelomocytes, body wall, extravasal tissue and the gut) which provide the first line of defense against infections. This supports the important role of arenicins as key components of both epithelial and systemic branches of host defense. It was established that expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. In coelomocytes, arenicins function as killing agents inside the phagolysosome, and may potentially carry this trait in extravasal tissue. In the gut and body wall epithelia, arenicins are released from producing cells via secretion as they are found in the content of the cuticle and inside the midgut gland cells. This study demonstrates that distribution and functioning of arenicins in the lugworm share some features with AMPs of other annelids but overall their characteristics are quite unique.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The project was supported by the research resource center “Molecular and cell technologies” of St. Petersburg State University, Russia. We thank Nikolai I. Kolokin and Tatiana V. Ovchinnikova for kindly providing synthetic and recombinant arenicins, respectively. We also thank Marina A. Varfolomeeva (StPSU) and Dr. Megan Jackson (QUB) for thorough review of the manuscript. Financial support for this project came from Saint Petersburg State University grant 1.0.140.2010 (PI Andrei I. Granovitch).
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