Article

# Molecular Characterization of Two Novel Antibacterial Peptides Inducible upon Bacterial Challenge in an Annelid, the Leech Theromyzon tessulatum

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## Abstract

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.

## Supplementary resources (2)

... Most medicinal leech species, such as Poecilobdella javanica and Hirudo medicinalis, are ectoparasites that feed on the blood of vertebrates, including human [2,3]. Leeches [4][5][6][7] or their secretions are used for medical procedures, such as clearing of pooled blood after certain surgical procedures and extracting anticoagulants and antibacterial peptides [8][9][10], which was like the venom secretions not only as preying tool of venous animals but also for drug discovery [11,12]. These processes comprise medicinal leech therapy (MLT). ...
... Leech medical therapy was one of oldest medicinal application in centuries BC [13,14]. Previous studies focused on its application and evaluation [36][37][38][39], medicinal cases [40,41], isolation and identification of bioactive molecules [10,[42][43][44], possible effects [18], function and mechanisms [45]. The application of leech therapy has a long history, but it is only recently started to be clarified to its effect mechanisms [1,46]. ...
... The determination the profiles of leech salivary secretions is indeed to its medicinal application. Compared to the traditional proteomic analysis with 2D-SDS-PAGE, HPLC, and SELDI-MS/MS [27][28][29][30]10], our methods were easy to determine widely profiles of proteins under low concentration of secretions. Just the existence of the difference in genes expression suggests the treatment efficiency can be different depending on the various sucking leeches. ...
Article
Medicinal leeches have been widely utilized in medical procedures for thousands of years. The application of leeches depends on the components of leech saliva secretions and active molecules, but many components of the secretions are not well characterized due to their low concentration and abundance. Determination of the profiles of leech salivary secretions is important to its medicinal application. In this study, we performed an in-depth proteomic analysis of leech salivary glands and deduced 434 full-length protein sequences from combined leech proteome and transcriptome databases. After integrating data from both datasets, forty-four proteins and two hundred twenty-one transcripts of bioactive molecules were involved in leech sucking pathways. Using gene expression analysis, we found that two-thirds of bioactive genes played key roles in leech bite processes and were associated cave-dwelling habitats. Our results indicate that the treatment efficiency can differ depending on the sucking leech species. Moreover, combining high-throughput proteomic and transcriptomic analyses is effective for the determination of wide profiles of proteins that are present at low concentrations in secretions. These findings highlight the extensive diversity of bioactive molecules and provide a new foundation for performing novel investigations and discovering future pharmacological agents or targets in leech medicinal therapy. Significance: Medicinal leech therapy has been used for many centuries depending on the components of leech saliva secretions and active actions, but many components of the secretions were less known due to its low concentration and abundance. Determination of the profiles of leech salivary secretions is important to its medicinal application. Hereby, the molecular information provided by proteomic and transcriptomic analysis can be used to develop a more thorough understanding of leech sucking pathway and medicinal application. It provided a new foundation for performing novel investigations and discovering future pharmacological agents or targets in leech medicinal therapy.
... The relatively low antimicrobial activities of lumbricin-like AMPs suggest that the microbial clearance is not the main biological function of this molecule. In 2004, the first member of the macin family (theromacin) was characterized in leeches [44]. Despite their different disulfide arrays, macins and invertebrate defensins share the CSαβ motif also characteristic of the members of the scorpion toxin-like superfamily [6]. ...
... Macins are cationic cysteine-rich AMPs. Members of this family of peptides have been first described in leeches (Theromyzon tessulatum and Hirudo medicinalis) [43,44], and later in Hydra vulgaris [43,118] and in the mollusks Hyriopsis cumingii [80] and Mytilus galloprovincialis [119]. Both leeches belong to the "Clitellata" class: T. tessulatum is a shallow water rhynchobdellid leech, ectoparasite of aquatic birds [120]; H. medicinalis, a gnathobdellid leech, is an ectoparasite of mammals which lives in stagnant freshwater and streams [121]. ...
... Both leeches belong to the "Clitellata" class: T. tessulatum is a shallow water rhynchobdellid leech, ectoparasite of aquatic birds [120]; H. medicinalis, a gnathobdellid leech, is an ectoparasite of mammals which lives in stagnant freshwater and streams [121]. Tt-theromacin (Tt-T) in T. tessulatum [44], Hm-neuromacin (Hm-N) and Hm-theromacin (Hm-T) in H. medicinalis [43], have several functions that includes bacterial killing, symbiostasis in the gut, immune defense, and regeneration of the damaged nerve cord. Their primary structure is highly conserved with the presence of a signal peptide (except for Hm-Theromacin), four disulfide bridges [122], and a fifth intramolecular disulfide bond (C31:C73) in theromacins ( Figure 10) [118]. ...
Article
Full-text available
Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms. In metazoans, they act as host defense factors by eliminating microbial pathogens. But they also help to select the colonizing bacterial symbionts while coping with specific environmental challenges. Although many AMPs share common structural characteristics, for example having an overall size between 10–100 amino acids, a net positive charge, a γ-core motif, or a high content of cysteines, they greatly differ in coding sequences as a consequence of multiple parallel evolution in the face of pathogens. The majority of AMPs is specific of certain taxa or even typifying species. This is especially the case of annelids (ringed worms). Even in regions with extreme environmental conditions (polar, hydrothermal, abyssal, polluted, etc.), worms have colonized all habitats on Earth and dominated in biomass most of them while co-occurring with a large number and variety of bacteria. This review surveys the different structures and functions of AMPs that have been so far encountered in annelids and nematodes. It highlights the wide diversity of AMP primary structures and their originality that presumably mimics the highly diverse life styles and ecology of worms. From the unique system that represents marine annelids, we have studied the effect of abiotic pressures on the selection of AMPs and demonstrated the promising sources of antibiotics that they could constitute.
... Among the most abundant transcripts shown in Table 2, a putative antimicrobial peptide is also present. Group 15, including four clones in four different clusters, is similar to the theromacin, a recently described antimicrobial from T. tessulatum (Tasiemski et al., 2004) and represents 4% of secreted proteins (Fig. 2). All of them are full-length clones and are represented by Set 2 at Table 3, which were h i g h l y s i m i l a r t o t h a t an t i m i cr o b i a l p e p t i d e (gbjAAR12065.1j). ...
... Antimicrobial substances were also described only recently from coelomic liquid of the leech, T. tessulatum. Theromacin and theromyzin are peptides with 75 and 86 amino acids, respectively, which exhibit an activity directed against Gram positive bacteria (Tasiemski et al., 2004). ...
... -The carbonic anhydrase family of proteins was the most abundant in this tissue and it was described for the first time in leeches, although we supposed that this protein family has an important role during the feeding process of this hematophage. -A putative antimicrobial was described for the first time in salivary complexes of leeches; it is similar to the theromacin, an antimicrobial recently described in coelomic liquid from T. tessulatum (Tasiemski et al., 2004), yet we supposed that this putative secreted substance, present in the salivary complexes, is probably essential to the host interaction process, mainly with a role in the animal innate response. -Through comparative analysis, we can suggest that the coagulation inhibitors present a profile very characteristic of each hematophagous animal class and although the feeding processes are similar to other bloodfeeding organisms, the involved proteins are certainly others. ...
Data
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... The macins are a family of peptides that have not usually been included in analyses of defensins and defensinlike peptides, but clearly have the CS-αβ fold. Macins were originally described from annelids [39,40] and have been reported from the cnidarian Hydra magnipapillata [40,41], the mussels Hyriopsis cumingii [42] and Mytilus galloprovincialis [43], and the giant African land snail, Achatina fulica [44]. The addition of a fourth bond formed by C1 and C7 as seen in hydramacin (Fig. 1a, l) [41] may be a defining characteristic of macins. ...
... To the best of my knowledge, antimicrobial activities of mytimacins from mussels have not been published yet. Other macins (hydramacin, neuromcain, theromacin, and mytimacin-AF) have shown primarily antibacterial activity, with antifungal testing being rather limited [39][40][41]44]. ...
... Rodríguez de la Vega and Possani point out that the lack of defensins reported from basal taxa (such as annelids and merastomatans) and sister groups (including crustaceans, cephalopods, gastropods, and spiders) complicates establishing invertebrate defensins as orthologs [84]. More recently, complete defensin sequences have been reported from five spider species [85] and the gastropod Haliotis discus [86].While sequences that look like typical arthropod or mollusk defensins have not been reported from annelids, macins have been reported from the annelids Hirudo medicinalis [40] and Theromyzon tessulatum [39], as well as from the gastropod Achatina fulica [44]. ...
Article
Full-text available
Background: "Invertebrate defensins" belong to the cysteine-stabilized alpha-beta (CS-αβ), also known as the scorpion toxin-like, superfamily. Some other peptides belonging to this superfamily of defensive peptides are indistinguishable from "defensins," but have been assigned other names, making it unclear what, if any, criteria must be met to qualify as an "invertebrate defensin." In addition, there are other groups of defensins in invertebrates and vertebrates that are considered to be evolutionarily unrelated to those in the CS-αβ superfamily. This complicates analyses and discussions of this peptide group. This paper investigates the criteria for classifying a peptide as an invertebrate defensin, suggests a reference cysteine array that may be helpful in discussing peptides in this superfamily, and proposes that the superfamily (rather than the name "defensin") is the appropriate context for studying the evolution of invertebrate defensins with the CS-αβ fold. Methods: CS-αβ superfamily sequences were identified from previous literature and BLAST searches of public databases. Sequences were retrieved from databases, and the relevant motifs were identified and used to create a conceptual alignment to a ten-cysteine reference array. Amino acid sequences were aligned in MEGA6 with manual adjustments to ensure accurate alignment of cysteines. Phylogenetic analyses were performed in MEGA6 (maximum likelihood) and MrBayes (Bayesian). Results: Across invertebrate taxa, the term "defensin" is not consistently applied based on number of cysteines, cysteine spacing pattern, spectrum of antimicrobial activity, or phylogenetic relationship. The analyses failed to reveal any criteria that unify "invertebrate defensins" and differentiate them from other defensive peptides in the CS-αβ superfamily. Sequences from various groups within the CS-αβ superfamily of defensive peptides can be described by a ten-cysteine reference array that aligns their defining structural motifs. Conclusions: The proposed ten-cysteine reference array can be used in addition to current nomenclature to compare sequences in the CS-αβ superfamily and clarify their features relative to one another. This will facilitate analysis and discussion of "invertebrate defensins" in an appropriate evolutionary context, rather than relying on nomenclature.
... Compared to cationic AMPs, anionic peptides are relatively small and include only a few reported groups of molecules that are found in different vertebrate and invertebrate species. Several anionic peptides such as dermcidin [42], PvHCt [43], maximin H5 [44], theromyzin [45], SCY1 [15] and stylicin [46] are reported, including three small ones rich in glutamic and aspartic acids [47]. The mode of action of the anionic peptides is still not clearly understood. ...
... The anionic amphibian peptide Maximin H5 is active only against Gram-positive bacteria (Staphylococcus aureus) [44] whereas the anionic annelid's (Theromyzon tessulatum) peptide theromyzin shows limited bacteriostatic activity against a Gram-positive bacterium (M. luteus) [45]. In our study, we observed that the recombinant SCY2 had antimicrobial activity against several Gram-positive bacteria, including M. luteus, C. glutamicum, B. subtilis and M. lysodeikticus. ...
... Parmi les peptides cycliques, il existe un peptide cationique isolé de la sangsue (Theromyzon tessulatum) et nommé Théromacine, de 75 AA dont 10 résidus cystéine formant ainsi cinq ponts disulfure (figure 12). Cette structure unique n'a été jusqu'à présent retrouvée dans aucune autre espèce, et joue un rôle important dans l'activité biologique de la molécule : la réduction du nombre de ponts disulfure entraîne la perte de son activité antibactérienne [Tasiemski et al. 2004]. ...
... Un de ces peptides, possèdant 47 AA, a été identifié dans la sueur humaine : la Dermcidine [Schittek et al. 2001]. Le premier peptide anionique des invertébrés a été trouvé chez la sangsue (Theromyzon tessulatum), la Théromyzine, peptide de 86 AA [Tasiemski et al. 2004]. Chez les amphibiens, le premier peptide anionique a été découvert chez Bombina maxima, la Maximin H5, contenant 3 résidus aspartiques et aucun AA basique [Lai et al. 2002]. ...
Thesis
Découverts au début du 20ème siècle, les Peptides AntiMicrobiens (PAM) sont depuis considérés comme des effecteurs clés de l'immunité innée. Cathélicidines, défensines, magainines... autant de noms différents qui soulignent la diversité, la complexité et l'importance croissante donnée aux PAM. A ce jour plus de 1500 ont été découverts, et ce dans un très grand nombred'organismes, très différents : insectes, plantes, mammifères.Ces PAM présentent aussi des activités très diverses : antibactérienne, antifongique, antivirale ou antiparasitaire.Ce travail a pour but de présenter : les grandes familles de PAM, leur variété de structure, les mécanismes d'action supposés (principalement antibactérien et antiviral) et leur rôle dans l'immunité. Il met également l'accent sur le développement clinique et une possible utilisation en thérapeutique de ces PAM, soulignant l'intérêt que ces derniers pourrait représenter, pour répondre à l'urgence de trouver de nouvelles molécules antibactériennes. En effet, l'apparition de bactéries multirésistantes aux antibiotiques nécessite la recherche de nouveaux anti-infectieux ; les PAM pourraient alors y trouver toute leur place.
... This implies that arenicins may be secreted into the gut lumen, although this requires further investigation. Similarly, AMPs of another annelid worm-theromicin and theromyzin from a leech Theromyzon tessulatum-were shown to be expressed both in intestinal and epidermal cells (Tasiemski et al., 2004). ...
... 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 betadefensins in skin and other borders) (De Smet and Contreras, 2005). Another remarkable fact is that there is no detectable antimicrobial activity in coelomic fluid, whereas in other investigated annelids (Thremyzon or Nereis) AMPs are secreted by producing cells to participate in host defense as humoral factors (Tasiemski et al., 2004(Tasiemski et al., , 2007. Signaling cascades, regulating AMP expression are thoroughly investigated in mammals and insects, but still unexplored in annelids. ...
Article
Full-text available
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 Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues-coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defense against infections, which implies their important role as key components of both epithelial and systemic branches of host defense.
... Hedistin is an antimicrobial peptide that was isolated from the CF of the Nereidid Nereis (Hediste) diversicolor. Hedistin is a bromotyrosine containing antibacterial peptide (molecular mass: 2,223 Da) which is constitutively expressed in natural killer like type 3 granulocytes and released upon immune challenge (Tasiemski et al. 2004). In aqueous solutions, hedistin exists as a random coil, however once attached to phospholipid bilayers, secondary structures are induced, leading to the formation of helix-turn-helix structures. ...
... Theromyzin and theromazin are both exclusively synthesized in so-called Large Fat Cells and released into the CF. Once secreted, the peptides may exert their action directly in the CF, or are taken up into gut skin epithelia to challenge bacterial infections (Tasiemski et al. 2004). ...
Chapter
The coelomic cavity is part of the main body plan of annelids. This fluid filled space takes up a considerable volume of the body and serves as an important site of exchange of both metabolites and proteins. In addition to low molecular substances such as amino acids and glucose and lactate, the coelomic fluid contains different proteins that can arise through release from adjacent tissues (intestine) or from secretion by coelomic cells. In this chapter, we will review the current knowledge about the proteins in the annelid coelomic fluid.
... At present, four species of Macin family members have been discovered, namely theromaicn (Tasiemski et al., 2004), hyramacin (Jung et al., 2009), mytimacin (Marco et al., 2012), and neuromacin (Sascha et al., 2012). Among them, theromaicn (TM) is an antimicrobial peptide that has been proven to have antimicrobial effects and plays a vital role in the body's immune system. ...
... Among them, theromaicn (TM) is an antimicrobial peptide that has been proven to have antimicrobial effects and plays a vital role in the body's immune system. It has been reported in species such as Theromyzon tessulatum (Tasiemski et al., 2004), Hirudo medicinalis (Schikorski et al., 2008), and Hyriopsis cumingii (Xu et al., 2010). ...
... This implies that arenicins may be secreted into the gut lumen, although this requires further investigation. Similarly, AMPs of another annelid worm-theromicin and theromyzin from a leech Theromyzon tessulatum-were shown to be expressed both in intestinal and epidermal cells (Tasiemski et al., 2004). ...
... 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 betadefensins in skin and other borders) (De Smet and Contreras, 2005). Another remarkable fact is that there is no detectable antimicrobial activity in coelomic fluid, whereas in other investigated annelids (Thremyzon or Nereis) AMPs are secreted by producing cells to participate in host defense as humoral factors (Tasiemski et al., 2004(Tasiemski et al., , 2007. Signaling cascades, regulating AMP expression are thoroughly investigated in mammals and insects, but still unexplored in annelids. ...
Article
Full-text available
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 Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues - coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defence against infections, which implies their important role as key components of both epithelial and systemic branches of host defence.
... These enzymes also support antimicrobial activity. 9,10,14 7,9,14,15 gelin, 9,14 factor Xa inhibitor, 9,10,14 destabilase, 9,14,[26][27][28] new leech protein-1, whitide, and whitmanin 29 Antimicrobial effect Destabilase, 9,14,[26][27][28] chloromycetyn, 9,10,14 theromacin, theromyzin, and peptide B 30,31 LDTI, leech-derived tryptase inhibitor. ...
Article
Full-text available
Complementary medical methods have a long history, but modern medicine has just recently focused on their possible modes of action. Medical leech therapy (MLT) or hirudotherapy, an old technique, has been studied by many researchers for possible effects on various diseases such as inflammatory diseases, osteoarthritis, and after different surgeries. Hirudo medicinalis has widest therapeutic usage among the leeches, but worldwide, many different species were tested and studied. Leeches secrete more than 20 identified bioactive substances such as antistasin, eglins, guamerin, hirudin, saratin, bdellins, complement, and carboxypeptidase inhibitors. They have analgesic, anti-inflammatory, platelet inhibitory, anticoagulant, and thrombin regulatory functions, as well as extracellular matrix degradative and antimicrobial effects, but with further studies, the spectrum of effects may widen. The technique is cheap, effective, easy to apply, and its modes of action have been elucidated for certain diseases. In conclusion, for treatment of some diseases, MLT is not an alternative, but is a complementary and/or integrative choice. MLT is a part of multidisciplinary treatments, and secretes various bioactive substances. These substances vary among species and different species should be evaluated for both treatment capability and their particular secreted molecules. There is huge potential for novel substances and these could be future therapeutics.
... Of particular relevance to the present study, melanin production has repeatedly been demonstrated as an effector mechanism in bivalve immunity (Luna-Acosta et al. 2017). Theromacin is an antimicrobial protein, originally identified in a leech, which is active against Gram-positive bacteria (Tasiemski et al. 2004). A similar gene has been reported for bivalves, which presumably has the same function (Xu et al. 2010). ...
Article
Full-text available
Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation. Electronic supplementary material The online version of this article (10.1007/s12192-018-0910-5) contains supplementary material, which is available to authorized users.
... Another peptide Theromyzin has 86-amino acid linear peptide and it constitutes the first anionic antimicrobial peptide observed in invertebrates. Both these peptides exhibit activity against Gram-positive bacteria [29]. ...
Article
Antimicrobial peptides appear to be ever-present and multipotent components of the innate immune defense mechanism used by both prokaryotic and eukaryotic organisms. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. AMPs have been isolated from a wide variety of animals, both includes vertebrates and invertebrates, and plants as well as from bacteria and fungi. Antimicrobial peptides interact with microbes initially it emphasize the binding of lipids. These peptides exhibit broad-spectrum activity against a wide range of microorganisms includes Grampositive and negative bacteria, protozoa, yeast, fungi and viruses. A few no of peptides have also been found to be cytotoxic to sperm and tumour cells. In this current review we discuss about the antimicrobial peptides isolated from marine invertebrates, and these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.
... La théromacine perd son activité antibactérienne lorsqu'on réduit le nombre de ponts disulfure. Ces derniers jouent donc un rôle important dans l'activité biologique de cette molécule (Tasiemski et al., 2004). ...
Article
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The chemical communication systems constitute an essential element in the establishment of intra- or inter-species relationships in marine environment, weaving a dense network of relations between individuals, in ecosystem. The invertebrates lacking of an immune system and usually sessile produce this type of bioactive metabolites playing a crucial role in the answer to the environmental pressures like the predation and the defence against potentially pathogens organisms. The aim of this work was to identify antimicrobial peptides in commercially bivalve and gastropod marine molluscs. Thus, the search for antimicrobial molecules from peptidic nature was undertaken in acid extracts of bivalve molluscs Cerastoderma edule, Ruditapes philippinarum, Ostrea edulis, and gastropods Crepidula fornicata, Buccinum undatum and Littorina littorea and from Crassostrea gigas hemolymph. The extracts were pre-purified by Solid Phase Extraction C18 (SPE) and elution was ensured by three successive steps of 10%, 40% and 80% of ACN-0.1% TFA. The antibacterial activities were assayed by determination of the CMI against a panel of target bacteria including Gram+ and Gram- bacteria. In parallel, antiviral activities were assayed in vitro against Herpes simplex virus type 1 and Vero cells by cell viability. The species C. edule, L. littorea and C. gigas proved to be the most effective and non cytotoxic species. A partial characterization of the activity detected in these species allowed determining the protenaceous nature of the active molecules. The purification of antimicrobial peptides realised on the C. gigas hemolymph led us to the identification of a peptide which structure lets foresee a bacterial origin. The hypothese of an association between C. gigas and bacteria led us from non axenic oysters culture to search for antagonist bacteria in C. gigas hemolymph and has conduced to isolate 2 Vibrio spp. and 3 Pseudoalteromonas spp. The Pseudoalteromonas spp. hCg 5 strain, allowed to partially characterized an active compound. Whole of these results suggests that the bacteria associated with the immune system could play an essential function of defence in bivalves.
... Finally, we also used a biochemical approach dedicated to the research of such molecules. This classical procedure for the identification of cationic AMPs was successful in numerous animal models 184,185,[197][198][199] but confirmed the absence of additional peptides in B. glabrata (G. Mitta, personal communication). ...
Data
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Supplementary Figures, Supplementary Notes and Supplementary References
... In the crop, bacteria are phagocytosed by circulating hemocytes (Figure 1B). A. veronii expresses a type 3 secretion system (T3SS) that is critical for avoiding this phagocytosis (Silver et al., 2007a). In addition, a number of antimicrobial peptides have been identified in H. medicinalis and related hirudiniform leeches (Tasiemski et al., 2004(Tasiemski et al., , 2015Schikorski et al., 2008) including salivary lectins (Min et al., 2010), theromyzin (TMZ), theromacin (TMC), allograft inflammatory factor-1 (AIF-1), neuromacin (NMC), lumbricin (LUMB). TMC and NMC are active against both Gram-positive and -negative bacteria through pore-forming and aggregateforming mechanisms respectively (Jung et al., 2012). ...
Article
Full-text available
Digestive-tract microbiota exert tremendous influence over host health. Host-symbiont model systems are studied to investigate how symbioses are initiated and maintained, as well as to identify host processes affected by resident microbiota. The medicinal leech, Hirudo verbana, is an excellent model to address such questions owing to a microbiome that is consistently dominated by two species, Aeromonas veronii and Mucinivorans hirudinis, both of which are cultivable and have sequenced genomes. This review outlines current knowledge about the dynamics of the H. verbana microbiome. We discuss in depth the factors required for A. veronii colonization and proliferation in the leech crop and summarize the current understanding of interactions between A. veronii and its annelid host. Lastly, we discuss leech usage in modern medicine and highlight how leech-therapy associated infections, often attributable to Aeromonas spp., are of growing clinical concern due in part to an increased prevalence of fluoroquinolone resistant strains.
... After dehydration, animals were embedded in paraplast and 7 μ m sections were cut, mounted on poly-L-lysine-coated slides, and stored at 4 °C until use. Paraffin sections were re-hydrated and non-specific background staining was blocked as previously described 33 . Samples were then incubated with rabbit LUMB antibodies (1:200), rabbit TMC antibodies (1:800) or mouse TMZ antibodies (1:800) diluted in the AB solution (PBS containing 1% BSA 0.05% Triton 1% NGS 1% NDS 1% Ovalbumin) overnight at RT. ...
Article
The medicinal leech has established a long-term mutualistic association with Aeromonas veronii, a versatile bacterium which can also display free-living waterborne and fish- or human-pathogenic lifestyles. Here, we investigated the role of antibiotics in the dynamics of interaction between the leech and its gut symbiont Aeromonas. By combining biochemical and molecular approaches, we isolated and identified for the first time the antimicrobial peptides (AMPs) produced by the leech digestive tract and by its symbiont Aeromonas. Immunohistochemistry data and PCR analyses evidenced that leech AMP genes are induced in the gut epithelial cells when Aeromonas load is low (starved animals), while repressed when Aeromonas abundance is the highest (post blood feeding). The asynchronous production of AMPs by both partners suggests that these antibiotic substances (i) provide them with reciprocal protection against invasive bacteria and (ii) contribute to the unusual simplicity of the gut microflora of the leech. This immune benefit substantially reinforces the evidence of an evolutionarily stable association between H. verbana and A. veronii. Altogether these data may provide insights into the processes making the association with an Aeromonas species in the digestive tract either deleterious or beneficial.
... It serves as important model systems for understanding the structure, function, development, regeneration and repair of nervous systems (Macagno et al., 2010;Hibsh et al., 2015). They directly (Derganc and Zdravic, 1960;Gross and Apesos, 1992;Gideroglu et al., 2003;Herlin et al., 2017) or their secretions are used for medical procedures, such as clearing of pooled blood after certain surgical procedures and extracting of anticoagulants and antibacterial peptides (Seymour et al., 1990;Tasiemski et al., 2004;Zavalova et al., 2006). Recently, a new blood-sucking leech species Haemadipsa cavatuses was found living off bat blood exclusively in caves all lifelong (Yang, 2009). ...
Article
The medicinal leeches have been widely utilized in medical procedures for thousands of years. Recently, there were more and more transcriptomes of leech published online including the medicinal leech (Hirudo medicinalis) and some other leeches. However, leech's genetic backgrounds are still largely unknown. In this report, transcriptomes of three phylogenetically close leeches (Poecilobdella javanica, Whitmania pigra, and Haemadipsa cavatuses) were established by RNA-seq technique for studying their genetic mechanisms of environmental adaption. Over 110 million high-quality reads were generated and assembled into unique transcriptome (reads = 200 bp). 27,138 out of de novo assembled transcripts (41.77%) were assigned to one or more GO terms. Additionally, the transcripts were detected in 217 predicted KEGG pathways. The enriched genes were involved in protein metabolism, GPCRs and pathogen-resistant pathways. The results showed that the great variations existed in gene expression of olfactory transduction pathway among three leech species. The comparisons of leech species hinted at the underlying mechanism of leeches adapting well in various environments. Our study will provide useful rationales for future studies of leeches and other annelid species.
... Neuromacin is a relative of theromacin, a cysteine-rich AMP first identified from the body fluid 376 of the leech Theromyzon tessulatum (Tasiemski et al., 2004) and later in Hirudo (Hm- 377 theromacin) (Tasiemski and Salzet, 2010). Theromacin is synthesized by the large fat cells and 378 the blood cells while neuromacin is produced by the epithelial cells of the gut, the neurons and 379 the microglial cells of the leech CNS ( Boidin-Wichlacz et al., 2012;Tasiemski et al., 2015;380 Tasiemski et al., 2004). ...
Article
An important question that remains unanswered is how the vertebrate neuroimmune system can be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining responses in mammals probably lies in the conflation in the central nervous system (CNS), of the innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy for understanding the relation between neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose CNS is well described and regeneration competent. The medicinal leech offers such opportunity. If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and conduction of signals through the damaged region is restored within a few days, even when the nerve cord is removed from the animal and maintained in culture. When the mammalian spinal cord is injured, regeneration of normal connections is more or less successful and implies multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the leech lesioned nerve cord is even more successful under septic than under sterile conditions suggesting that a controlled initiation of an infectious response may be a critical event for the regeneration of normal CNS functions in the leech. Here are reviewed and discussed data explaining how the leech nerve cord sensu stricto (i.e. excluding microglia and infiltrated blood cells) recognizes and responds to microbes and mechanical damages.
... Compared to other metazoans, the AMP arsenal of B. glabrata is restricted to a gene family of macins , with one macin known to be upregulated in B. glabrata after exposure to S. mansoni miracidia (Ittiprasert et al., 2010). This particular macin sequence was most similar to theromacin originally described from the leech Theromyzon tessulatum (Tasiemski et al., 2004). Similarly, just two macin-like AMP sequences were recorded for P. acuta. ...
Article
The freshwater snail Physella acuta was selected to expand the perspective of comparative snail immunology. Analysis of Physella acuta, belonging to the Physidae, taxonomic sister family to Planorbidae, affords family-level comparison of immune features characterized from Biomphalaria glabrata, the model snail often used to interpret general gastropod immunity. To capture constitutive and induced immune sequences, transcriptomes of an individual Physella acuta snail, 12 h post injection with bacteria (Gram-/+) and one sham-exposed snail were recorded with 454 pyrosequencing. Assembly yielded a combined reference transcriptome containing 24,288 transcripts. Additionally, genomic Illumina reads were obtained (∼15-fold coverage). Recovery of transcripts for two macin-like antimicrobial peptides (AMPs), 12 aplysianins, four LBP/BPIs and three physalysins indicated that Physella acuta shares a similar organization of antimicrobial defenses with Biomphalaria glabrata, contrasting a modest AMP arsenal with a diverse set of antimicrobial proteins. The lack of predicted transmembrane domains in all seven Physella acuta PGRP transcripts supports the notion that gastropods do not employ cell-bound PGRP receptors, different from ecdysozoan invertebrates yet similar to mammals (vertebrate deuterostomes). The well-documented sequence diversification by Biomphalaria glabrata FREPs (immune lectins comprising immunoglobulin superfamily domains and fibrinogen domains), resulting from somatic mutations of a large FREP gene family is hypothesized to be unique to Planorbidae; Physella acuta revealed just two bonafide FREP genes and these were not diversified. Furthermore, the flatworm parasite Echinostoma paraensei, confirmed here to infect both snail species, did not evoke from Physella acuta the abundant expression of FREP proteins at 2, 4 and 8 days post exposure that was previously observed from Biomphalaria glabrata. The Physella acuta reference transcriptome also revealed 24 unique transcripts encoding proteins consisting of a single fibrinogen-related domain (FReDs), with a short N-terminal sequence encoding either a signal peptide, transmembrane domain or no predicted features. The Physella acuta FReDs are candidate immune genes based on implication of similar sequences in immunity of bivalve molluscs. Overall, comparative analysis of snails of sister families elucidated the potential for taxon-specific immune features and investigation of strategically selected species will provide a more comprehensive view of gastropod immunity.
... Currently, several AMPs have been characterized from different leech species, including theromyzin, theromacin, neuromacin and hydramacin-1 [21]. Linear, anionic theromyzin displayed potent activity against Gram-positive bacteria, but no activity was found towards the Gram-negative bacteria [22]. The macins, which comprise cysteine-rich theromacin, neuromacin and hydramacin-1, are antimicrobially active against Gram-positive and Gramnegative bacteria [23]. ...
Article
The rise of antibiotic resistance has necessitated the development of alternative strategies for the treatment of infectious diseases. Antimicrobial peptides (AMPs), components of the innate immune response in various organisms, are promising next-generation drugs against bacterial infections. The ability of the medicinal leech Hirudo medicinalis to store blood for months with little change has attracted interest regarding the identification of novel AMPs in this organism. In this study, we employed computational algorithms to the medicinal leech genome assembly to identify amino acid sequences encoding potential AMPs. Then, we synthesized twelve candidate AMPs identified by the algorithms, determined their secondary structures, measured minimal inhibitory concentrations against three bacterial species (Escherichia coli, Bacillus subtilis, and Chlamydia thrachomatis), and assayed cytotoxic and haemolytic activities. Eight of twelve candidate AMPs possessed antimicrobial activity, and only two of them, 3967 (FRIMRILRVLKL) and 536-1 (RWRLVCFLCRRKKV), exhibited inhibition of growth of all tested bacterial species at a minimal inhibitory concentration of 10 μmol. Thus, we evidence the utility of the developed computational algorithms for the identification of AMPs with low toxicity and haemolytic activity in the medicinal leech genome assembly.
... La théromacine, isolée de la sangsue Theromyzon tessulatum est un peptide cationique de 75 résidus acides aminés contenant 10 cystéines associées en 5 ponts disulfures qui lui confèrent une structure tout à fait originale que l'on n'a retrouvée jusqu'à présent dans aucune autre espèce. La théromacine perd son activité antibactérienne lorsqu'on réduit le nombre de ponts disulfure (Tasiemski et al., 2004). ...
Thesis
The aim of this work was studying the immune responses at molecular and cellular level of mussels exposed to different types of contaminants (metals and chemicals) by studying the transcriptomic of some genes used as biomarkers like MgBD3 (Antimicrobian peptide), lysozyme, catalase and Superoyxde Dismutase), using RT-PCR. In this work, we tried to understand how aquatic species, bivalves here, "react" in polluted environment polluted by cadmium, Chrysene and TiO2 nanoparticles by studying some of their responses at the molecular level, face to these stresses, these responses are multiple, simultaneous or cascading, and always complex. We have tried throughout this study to understand the resistance mechanisms in these organisms. We are also committed to reproduce the environmental pollution conditions by using during the bivalve laboratory exposure, the same concentrations of those encountered in the natural study sites. The results obtained in this work show that: - These contaminants affect the immune parameters that respond differently. - These parameters are all in a functional relationship with or control the generation of reactive oxygen species. - There is a threshold concentration responsible for the type variation the biomarkers response (increase or decrease) but must be determined and validated. - Responses against contaminants influence the generation of reactive oxygen species in the same way. - The response intensity against heavy metals and PAHs varies between these two. - Nanoparticles affect the immune response of bivalves.
... Despite the lack of information regarding bivalve's mytimacins, it is possible that these proteins are closely related to the mucus produced to protect different tissues, as it happens in land invertebrates. [65,91,92]. ...
Article
Antibacterial research is reaching new heights due to the increasing demand for the discovery of new substances capable of inhibiting bacteria, especially to respond to the appearance of more and more multi-resistant strains. Bivalves show enormous potential for the finding of new antibacterial compounds, although for that to be further explored, more research needs to be made regarding the immune system of these organisms. Beyond their primary cellular component responsible for bacterial recognition and destruction, the haemocytes, bivalves have various other antibacterial units dissolved in the haemolymph that intervene in the defense against bacterial infections, from the recognition factors that detect different bacteria to the effector molecules carrying destructive properties. Moreover, to better comprehend the immune system, it is important to understand the different survival strategies that bacteria possess in order to stay alive from the host's defenses. This work reviews the current literature regarding the components that intervene in a bacterial infection, as well as discussing the enormous potential that freshwater bivalves have in the discovery of new antibacterial compounds.
... They are known to be active against both Gram-positive and Gram-negative bacteria. Theromacin with 75 amino acid, cationic peptides with 10 C residues in a disulfide array and theromyzin with 86 amino acid linear peptide have been reported to be expressed by the large fat cells of intestinal epithelia and epidermis and secreted in the coelomic liquid by T. tessulatum (Tasiemski et al. 2004). Neuromacin has been reported of strongly permeabilize the cytoplasmic membrane of Bacillus megaterium (B. ...
Article
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The earthworm immune system is robust and comprises of the coelom cytolytic factor (CCF), lysenin and antimicrobial peptides (AMPs, Ghosh in Environ Sci Pollut Res 25: 6196, 2018) have been reported from organisms belonging to Phylum Annelida including earthworms (Ghosh in Int J Pept Res Ther, http://doi.org/10.1007/s10989-019-09970-9, 2019) which reveal structural diversity. Leeches have been known of their medical importance and known to produce AMPs (Hung et al. in J Proteomics 103:216–226, 2014; Salzet Curr Med Chem 12(6):3055–3061, 2005), but information on their detailed structures were not observed in published literature. In this study we have conducted insilico studies to understand the (i) Physicochemical properties (ii) phylogenetic relation from peptide sequence (iii) molecular modelling studies by insilico approaches which reveals overall diversity in their structure despite their similar functional role. This report highlights the properties of all AMPs in leeches with known sequences and the importance of application of insilico tools, performing a mutation analysis affecting protein stability, to highlight the significance of K(8) amino acid of the AMP 536_2 from Hirudo medicinalis in maintaining protein stability and functional importance.
... When they reach the cytoplasm, they may attach to ribosomes or inhibit ribonuclease activity (Jezȯwska-Bojczuk and Stokowa-Sołtys, 2018). Some anionic AMPs, such as theromyzin from Theromyzon tessulatum (Tasiemski et al., 2004), require zinc as a functional cofactor and it was found that the complex with zinc has stronger antimicrobial activity . ...
Article
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Antibiotics are essential drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. Antibiotic resistance is a serious challenge and has led to the need for new alternative molecules less prone to bacterial resistance. Antimicrobial peptides (AMPs) have aroused great interest as potential next-generation antibiotics, since they are bioactive small proteins, naturally produced by all living organisms, and representing the first line of defense against fungi, viruses and bacteria. AMPs are commonly classified according to their sources, which are represented by microorganisms, plants and animals, as well as to their secondary structure, their biosynthesis and their mechanism of action. They find application in different fields such as agriculture, food industry and medicine, on which we focused our attention in this review. Particularly, we examined AMP potential applicability in wound healing, skin infections and metabolic syndrome, considering their ability to act as potential Angiotensin-Converting Enzyme I and pancreatic lipase inhibitory peptides as well as antioxidant peptides. Moreover, we argued about the pharmacokinetic and pharmacodynamic approaches to develop new antibiotics, the drug development strategies and the formulation approaches which need to be taken into account in developing clinically suitable AMP applications.
... In the liquid growth inhibition assay, the purified neuromacin and theromacin were active against gram-positive bacteria. However, no antibacterial activity was found against gramnegative bacteria (Schikorski et al., 2008;Tasiemski et al., 2004). Mytimacin-AF exhibited broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria and fungus (Zhong et al., 2013). ...
Chapter
Antimicrobial peptides (AMP), also known as host defense peptides or alarmins, are among the first lines of defense against infection in many organisms. Marine organisms have proved to be a rich source of AMPs, and several uniquely structured marine AMPs have been isolated using biochemical, in silico and genetic approaches. Hemolymph is the main source of AMPs in marine invertebrates, although other tissues may also contain these peptides. Based on their three-dimensional structure, AMPs may be classified into peptides with ˛-helix structures, peptides with ˇ-sheets and cysteine residues, peptides enriched for modified and rare amino acids, and peptides with ring structure amino acids. This chapter describes AMPs of each structure, in terms of their antibacterial, antiviral, antifungal, insecticidal, nematicidal, and immunomodulatory functions. It is hoped that the diversity of marine AMPs may facilitate the identification of effective substitutes to existing antibiotics, thereby helping to minimize the development of antibiotic-resistant pathogen strains in response to the continuous application of a single antibiotic therapy.
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Les activités métallurgiques entraînent l’accumulation d’éléments traces métalliques dans les couches superficielles des sols, où l’on peut observer des concentrations largement supérieures au fond pédogéochimique et aux normes en vigueur. La contamination des sols est une menace pour la santé publique et la présence de grandes quantités d’éléments traces métalliques peut générer un stress susceptible d’affecter les organismes exposés à des sols pollués. Les Annélides Oligochètes vivent en contact étroit avec ces sols pollués et sont parmi les organismes vivants présentant une sensibilité exacerbée aux métaux. Très peu de choses sont connues quant à l’identification et la mise en place des mécanismes de réponse à ces métaux au niveau moléculaire. En exploitant la conservation phylogénique observée entre espèces, nous avons été capables de cloner et de caractériser un ensemble de biomarqueurs potentiels à partir des coelomocytes de l’Annélide Oligochète Eisenia fetida, une espèce modèle recommandée en écotoxicologie. Deux approches ont été mises en place. Premièrement, une approche qualifiée de ciblée, consistant à identifier tous les effecteurs parmi des protéines fortement conservées pour lesquels une variation lors d’une exposition métallique était reportée dans la littérature. Deuxièmement, nous avons entrepris une approche, qualifiée de globale, consistant en la construction de banques soustractives pour identifier chez Eisenia fetida les gènes dont l’expression est affectée lors d’une exposition à un mélange complexe de métaux, représentatif d’un site naturel fortement contaminé. Ces deux approches ont permis l’identification de 4 candidats biomarqueurs de pollution métallique.
Chapter
Antimicrobial peptides (AMP ), also known as host defense peptides or alarmins, are among the first lines of defense against infection in many organisms. Marine organisms have proved to be a rich source of AMPs, and several uniquely structured marine AMPs have been isolated using biochemical, in silico and genetic approaches. Hemolymph is the main source of AMPs in marine invertebrates, although other tissues may also contain these peptides. Based on their three-dimensional structure, AMPs may be classified into peptides with α-helix structures, peptides with β-sheets and cysteine residues, peptides enriched for modified and rare amino acids, and peptides with ring structure amino acids. This chapter describes AMPs of each structure, in terms of their antibacterial, antiviral, antifungal, insecticidal, nematicidal, and immunomodulatory functions. It is hoped that the diversity of marine AMPs may facilitate the identification of effective substitutes to existing antibiotics, thereby helping to minimize the development of antibiotic-resistant pathogen strains in response to the continuous application of a single antibiotic therapy.
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The objective of this study was to determine the inhibitory of earthworm (Lumbricus rubellus) extract (ECT) and encapsulated earthworm extract (ECT-t) as poultry feed additive against some pathogenic bacteria. Earthwom extract was prepared by dekokta method with water at 90 ºC then encapsulated by spray drying with maltodextrin as filler. In vitro antibacterial activity was performed using dilution method against Escherichia coli, Staphylococcus aureus, Salmonella pullorum, and Pseudomonas aeruginosa. The optical density results showed that started from ECT level 0.26% inhibited (P
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Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino–alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.
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Invertebrates are a significant source of antimicrobial peptides because they lack an adaptive immune system and must rely on their innate immunity to survive in a pathogen-infested environment. Various antimicrobial peptides that represent major components of invertebrate innate immunity have been described in a number of investigations over the last few decades. In freshwater invertebrates, antimicrobial peptides have been identified in arthropods, annelids, molluscs, crustaceans, and cnidarians. Freshwater invertebrate species contain antimicrobial peptides from the families astacidin, macin, defensin, and crustin, as well as other antimicrobial peptides that do not belong to these families. They show broad spectrum activities greatly directed against bacteria and to a less extent against fungi and viruses. This review focuses on antimicrobial peptides found in freshwater invertebrates, highlighting their features, structure-activity connections, antimicrobial processes, and possible applications in the food industry, animal husbandry, aquaculture, and medicine. The methods for their synthesis, purification, and characterization, as well as the obstacles and strategies for their development and application, are also discussed.
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Peptide antibiotics are produced by bacterial, mammalian, insect or plant organisms in defense against invasive microbial pathogens. Therefore, they are gaining importance as anti-infective agents. There are a number of antibiotics that require metal ions to function properly. Metal ions play a key role in their action and are involved in specific interactions with proteins, nucleic acids and other biomolecules. On the other hand, it is well known that some antimicrobial agents possess functional groups that enable them interacting with metal ions present in physiological fluids. Some findings support a hypothesis that they may alter the serum metal ions concentration in humans. Complexes usually have a higher positive charge than uncomplexed compounds. This means that they might interact more tightly with polyanionic DNA and RNA molecules. It has been shown that several metal ion complexes with antibiotics promote degradation of DNA. Some of them, such as bleomycin, form stable complexes with redox metal ions and split the nucleic acids chain via the free radicals mechanism. However, this is not a rule. For example blasticidin does not cause DNA damage. This indicates that some peptide antibiotics can be considered as ligands that effectively lower the oxidative activity of transition metal ions.
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Some vital components of marine shellfish are documented as important source for both nutritional and pharmacological applications. These bioactive compounds are multifunctional in nature and they function as anti-inflammatory, antimicrobial, anticancer, antidiabetic, anti-hypertensive and so on. Shellfish derived molecules such as lectins, glycoproteins, agglutinins and various endogenous free amino acids and fatty acids display potent immunomodulatory functions associated with human health. In addition, many synthetic immunomodulating agents are commercially available in the market, but they also cause some side effects to the patients. However, the immunomodulating substances derived from natural origin are safer and also cheaper in cost. Shellfish derivatives regulating biological activities and immune modulations are so far underexplored. Therefore, in this review, we discuss the immunomodulatory properties of shellfish derived compounds to emphasize the therapeutic potential in human diseases.
Patent
Methods are provided for isolating and using a whole-saliva leech extract. The methods can include feeding a phagostimulatory agent to a leech; inducing a regurgitation in the leech, the inducing including placing the leech in an environment having a temperature of less than about 0° C.; and, collecting an unrefined, whole saliva in the regurgitation of the cooled leech. The methods can include revitalizing the leech by warming it at a temperature ranging from about 5° C. to about 40° C. Stable, lyophilized, whole-saliva extracts of a leech are also provided, the extract having a stable activity when stored for use at a temperature below about −20° C., the extract maintaining at least 70% of the activity for at least 6 months. The extracts can be used to treat solid tumors, treat liquid tumors, treat diabetes, treat a viral disease, treat a parasitic disease, treat an antibacterial disease, or serve as an anti-oxidant.
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Immune responses of invertebrate animals are mediated through innate mechanisms, among which phagocytosis, encapsulation, production of ROS and antimicrobial peptides. Although polychetes represent an evolutionary interesting group closely related to presumable common ancestor of other celomates, their immune mechanisms still remain scarcely investigated. Here we discuss immune responses of the polychete Arenicola marina, the lugworm. Besides an overview of diversity of celomocytes and cellular responses, we present the synopsis on antimicrobial peptides arenicins: their structure, function and therapeutic potential.
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Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as ‘next generation antibiotics’ due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.
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Copepods, including Apocyclops royi, are small aquatic crustaceans and one of the important foods for fish and shellfish larvae. However, studies of the host-pathogen interactions and understanding of infectious disease in copepods are still very limited, yet they are likely to be a significant factor in the sustainable development of copepod aquaculture. In the present study, we performed de novo RNA sequence analysis of A. royi-TH (a Thai isolate of A. royi), which yielded 4.80 Gb bases of clean data and a total of 29,786 unigenes. Annotation was then performed by comparison against seven functional databases, yielding 17,617 (NR: 59.15%), 2,969 (NT: 9.97%), 15,023 (Swissprot: 50.44%), 14,543 (KOG: 48.82%), 15,077 (KEGG: 50.62%), 6,763(GO: 22.71%), and 15,841 (InterPro: 53.18%) unigenes. In comparison to the components of the shrimp Toll pathway, LGBP, Spätzle, Toll receptors, MyD88, Pelle, TRAF6, Dorsal, and cactus homologs were successfully identified in A. royi-TH. Additionally, a novel antimicrobial peptide (Theromacin-like) was characterized in A. royi (ArTM-like). The ArTM-like ORF was 279 bp and predicted to encode for 92 amino acid residues, with a mature peptide of 75 amino acids and a molecular mass of 8.56 kDa. The genomic organization of the ArTM-like gene consisted of three exons and two introns. Expression analysis indicated that ArTM-like mRNA was abundantly expressed in copepodid and adult stages as an immune responsive gene after infection with the pathogenic Vibrio parahaemolyticus-(AHPND)-causing strain. Altogether, the knowledge obtained in this study will provide a basis for future functional studies of the molecular mechanisms in copepod immunity that may eventually be applied for disease prevention in copepod aquaculture.
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Haemostasis is the prevention of blood fluidity in vertebrates and is the first stage of wound healing. Haematophagous animals use the blood of vertebrates as their sole source of nutrition and have evolved many salivary constituents to counteract the haemostatic response of their prey. These animals and their saliva have been studied for many years, with some applications in medicine. The purpose of this study is to compare the salivary constituents of leeches (Hirudinae), ticks (Argasidae and Ixodidae) and vampire bats (Desmodontinae) and to consider their evolutionary origin. Salivary constituents include plasminogen activators (PAs), anticoagulants (activated factor X, FXa; inhibitors), vasodilators, platelet aggregation inhibitors (PAgI) and thrombin inhibitors. The animals studied all tend to possess an anticoagulant and a form of apyrase (PAgI) to assist with blood feeding. Ticks and vampire bats have a form of PA but the leech does not. The vampire bat has a PAgI but no vasodilator. The animals studied are from taxonomically unrelated groups but exploit similar mechanisms of action to facilitate their haematophagy. Given that the haematophagous lifestyle of these animals developed much later than their common ancestors, we conclude that their mechanisms for haematophagy have arisen by convergent evolution. Some molecules, e.g. serine proteases found in invertebrate saliva, are probably derived from a common ancestral gene. The possible paths that have led to evolution of vampire bat salivary components are considered. Further research into the homology of these salivary constituents is required to give insight into how these animals adapted to haematophagy and their further therapeutic potential.
Chapter
The concept of minimally processed and natural foods is gaining popularity among the different group of consumers, and the globalization of food markets, unique new manufacturing processes, and demand for nutritional food without preservatives has enforced research for natural antimicrobials. Food outbreaks caused by Listeria monocytogenes, Escherichia coli O157 and eradication of Bacillus and Clostridium spores have emerged as major challenges faced by the modern food industry. The antimicrobial compounds have a broad range of biologically active molecules of multiple applications. These molecules could be of multiple origins, and mainly consist of carbohydrates, proteins (cationic or anionic), lipids, essential oils or secondary metabolites (saponins, flavonoid, thymols, linalool, citral, tanins, eugenols, terpene, polyphenols, phytophenols, phenolic acids etc.). Many of the naturally occurring antimicrobials are commercialized however; the efficacy, consumer acceptance, and regulation are still a matter of extensive research. In the present chapter, identification, characterization, the mechanism of action of selected natural antimicrobial peptides and polyphenols and their potential applications in food safety and preservation have been discussed.
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Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism’s medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia.
Article
The novel antimicrobial gene Hirudomacin (Hmc), with a 249-bp cDNA, encodes a mature protein of 61 amino acids and a 22-amino acid signal peptide. Hmc exhibits the highest similarity, at 90.1%, with macin family members found in the salivary gland of the leech Hirudo nipponica Whitman. A mature Hmc protein concentration of 219 μg/mL was detected using the Bradford method. The mature Hmc protein is 6862.82 Da and contains 8 cysteine residues. Antimicrobial assays showed a minimum bactericidal concentration and 50% lethal dose of 1.56 μg/mL and 0.78 μg/mL, respectively, for Staphylococcus aureus and 0.39 μg/mL and 0.195 μg/mL, respectively, for Bacillus subtilis. Transmission electron microscopy revealed membrane integrity disruption in S. aureus and B. subtilis, which resulted in bacterial lysis. The level of Hmc mRNA in the salivary gland during three blood meal stages indicated a remarkable trend of increase (P < .05), and western blotting demonstrated that among the three blood meal stages, expression of the mature Hmc protein was highest in both the salivary gland and intestine at the fed stage (P < .05). Immunofluorescence further showed the mature Hmc protein to be localized outside the cell nucleus, with the signal intensity in the salivary gland peaking at the fed stage (P < .05). In conclusion, the mature Hmc protein exhibits broad-spectrum antimicrobial effects against gram-positive and gram-negative bacteria, and a blood meal upregulates Hmc gene and protein expression in H. nipponica.
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Antimicrobial peptides (AMPs) play fundamental roles in the innate immunity of invertebrates. Mytimacin-4 is a kind of AMP gene previously sequenced from Mytilus galloprovincialis based on an identified EST sequence in our lab. In the present study, the tissue distribution and antimicrobial activities of mytimacin-4 were further investigated. A qRT-PCR analysis revealed that mytimacin-4 transcripts were constitutively expressed in all of the tested tissues of M. galloprovincialis, with the highest expression level in the posterior adductor muscle. After challenge by Vibrio anguillarum, the expression level of mytimacin-4 gene was significantly increased at 24 h (P < 0.05) in the mantle and increased at 48 h (P < 0.05) in the posterior adductor muscle. This finding suggested that mytimacin-4 transcripts were inducible upon pathogen infection. A minimal inhibitory concentration (MIC) assay indicated that recombinant mytimacin-4 protein had potent antimicrobial activities against gram-positive and gram-negative bacteria. Among the tested microorganisms, mytimacin-4 protein exhibited strong inhibition activities against Bacillus subtilis and Vibrio parahaemolyticus with MICs of 0.315 μM and 0.62 μM, respectively. This study provides for the first time direct evidence of antimicrobial action of mytimacin-4 in M. galloprovincialis.
Article
In the present study, a macin was cloned and characterized from clam Venerupis philippinarum (designed as VpMacin). The full-length cDNA of VpMacin was of 579 bp, encoding a peptide of 87 amino acids with the predicted molecular weight of 9.7 kDa. Analysis of the conserved domain suggested that VpMacin was a new member of the macin family. In non-stimulated clams, VpMacin transcripts exhibited different tissue expression pattern, and highly expressed in the tissues of gills and hepatopancreas. Generally, the temporal expression of VpMacin transcripts was significantly induced in hemocytes of clams post Vibrio anguillarum challenge. Moreover, the recombinant VpMacin protein (rVpMacin) showed obvious antimicrobial activities against Gram-positive and Gram-negative bacteria. After incubated with 40 μM rVpMacin, all detected Escherichia coli could be killed within 60 min. Membrane integrity analysis revealed that rVpMacin could increase the membrane permeability of bacteria and then resulted in cell death. Overall, our results suggested that VpMacin had an important function in host defense against invasive pathogens.
Article
Antimicrobial peptides (AMPs) are molecular factors in innate immunity and are believed to play a key role in invertebrate host defence. We identified theromacin (TM) from an Asian polychaeta, Perinereis linea, using de novo RNA-seq analysis. TM, a typical AMP of invertebrates, is a cysteine-rich AMP with five disulfide bonds consisting of ten cysteine residues. In gene expression analysis, TM genes were constantly upregulated after lipopolysaccharide (LPS) stimulation. In contrast, after peptidoglycan (PGN) stimulation, it was upregulated initially and downregulated after 12 hours. We synthesized a peptide based on the macin AMP in the TM amino acid sequence. The synthetic peptide showed antibacterial activity against some Gram-positive and Gram-negative bacteria. Therefore, the AMPs of P. linea might have broad roles in host defence and exhibit different degrees of activity.
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The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.
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With advancements in genomics, there has been substantial reduction in the cost and time of genome sequencing and has resulted in lot of data in genome databases. Antimicrobial host defense proteins provide protection against invading microbes. But confirming the antimicrobial function of host proteins by wet-lab experiments is expensive and time consuming. Therefore, there is a need to develop an in silico tool to identify the antimicrobial function of proteins. In the current study, we developed a model AniAMPpred by considering all the available antimicrobial peptides (AMPs) of length $\in$[10 200] from the animal kingdom. The model utilizes a support vector machine algorithm with deep learning-based features and identifies probable antimicrobial proteins (PAPs) in the genome of animals. The results show that our proposed model outperforms other state-of-the-art classifiers, has very high confidence in its predictions, is not biased and can classify both AMPs and non-AMPs for a diverse peptide length with high accuracy. By utilizing AniAMPpred, we identified 436 PAPs in the genome of Helobdella robusta. To further confirm the functional activity of PAPs, we performed BLAST analysis against known AMPs. On detailed analysis of five selected PAPs, we could observe their similarity with antimicrobial proteins of several animal species. Thus, our proposed model can help the researchers identify PAPs in the genome of animals and provide insight into the functional identity of different proteins. An online prediction server is also developed based on the proposed approach, which is freely accessible at https://aniamppred.anvil.app/.
Article
In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens.
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To isolate and purify anti-fungal active substances from immunized housefly (Musca domestica), low dose of Candida albicans was injected into the larvae of the housefly to induce the appearance of potent anti-fungal active substances in the hemolymph. This purification work was performed by the routine isolation and purification processes of protein, namely, solid phase extraction (SPE), SDS-PACE electrophoresis, HPLC purification. Three 4-16 kDa peptides which exhibited antifungal activity against Candida albican and other fungi were isolated from induced hemolymph. Consequently, further anti-fungal activity study showed that these three peptides were different either in molecular weight or in anti-fungal activity. All isolated substances were proved to be active and resistant to high-temperature. It was deduced that these peptides isolated from induced housefly were novel members of the insect defensin family and they were inducible.
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DNA sequence analysis dictates new interpretation of phylogenic trees. Taxa that were once thought to represent successive grades of complexity at the base of the metazoan tree are being displaced to much higher positions inside the tree. This leaves no evolutionary “intermediates” and forces us to rethink the genesis of bilaterian complexity.
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Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.
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Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.
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Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.
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Previously, we reported antibacterial activity in the body fluid of the nematode Ascaris suum (Kato, Y. (1995) Zool. Sci. 12, 225-230). The antibacterial activity is due to a heat-stable and trypsin-sensitive molecule that was designated as ASABF (A. suum antibacterial factor). In the present study, the purification, determination of primary structure, and cDNA cloning of ASABF were carried out. The mature peptide of ASABF is a basic peptide consisting of 71 residues and containing four intramolecular disulfide bridges. The amino acid sequence of a precursor for ASABF, deduced from a cDNA clone, indicates that flanking peptides both at the N terminus and at the C terminus are eliminated by processing. ASABF exhibits potent antibacterial activity particularly against Gram-positive bacteria. ASABF has several features that resemble those of insect/arthropod defensins, whereas the statistical significance of the similarity is not observed on comparison of amino acid sequences. A search of data bases revealed ASABF homologues in Caenorhabditis elegans.
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Variations in the number of galanin receptor (Gal-R1)-expressing cells and levels of Gal-R1 messenger RNA (mRNA) were determined in the preoptic area in intact female rats throughout the phases of the estrous cycle and compared with those in the male. Female and male Wistar rats were fixed by perfusion with 4% paraformaldehyde. Cryostat sections were hybridized with a 35S-labeled antisense Gal-R1 riboprobe. The number of Gal-R1 mRNA-expressing cells was lower in the rostral preoptic area than in the medial preoptic area. During the estrous cycle, the highest number of Gal-R1 mRNA-expressing cells in the rostral preoptic region was detected at 0800 h on proestrus, whereas in the medial preoptic area, the maximum number was observed at 1800 h on estrus. Gal-R1 mRNA levels in individual cells were low during diestrus and increased at estrus in both areas. In the male, the number of mRNA-expressing cells and the hybridization signal were significantly lower than those in females during estrus. The results demonstrate that Gal-R1 gene expression in the preoptic area varies during the estrous cycle and is low in males. Short term treatment of ovariectomized rats with estradiol plus progesterone caused significantly decreased preoptic Gal-R1 mRNA levels compared with those after treatment with estrogen only. These observations suggest that in the preoptic area, expression of Gal-R1 is influenced by progesterone. The variation in Gal-R1 expression is likely to influence the extent to which galanin can influence the preoptic cells implicated in the control of neighboring GnRH cells.
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When viewed from the perspective of host defence, the mammalian digestive tract presents many challenges. At the outset food contains various micro-organisms that could thrive in the rich growth medium of digested nutrients. Because a large surface area is required for the absorption of nutrients, there are abundant potential sites for microbial attachment and invasion in the long digestive tube with its many folds, villi and microvilli. The requirements of efficient nutrient absorption also place limits on the barrier components of host defence. Indeed, unlike the skin, vaginal epithelium or oral mucosa, intestinal mucosa is comprised of only a single layer of epithelial cells. This delicate barrier is further threatened by the effects of acid, bile salts, and hydrolytic enzymes required for digestion. Although some of these molecules are toxic to microbes, and hence may help to control microbial proliferation and survival, these agents may also subtly damage the mucosa and compromise its barrier functions. The potential for cumulative damage may explain why this epithelium is rapidly and continually replaced throughout the lifetime of mammals. As a result of the need for epithelial renewal, stem cell proliferation and differentiation are critical for normal function of the gastrointestinal tract. Damage to, or parasitisation of stem cells would have severe consequences for the maintenance of the normal digestive epithelium. A continuous microbial threat is also posed by a wide array of colonising microbes throughout the gut, with especially large numbers in the mouth and colon. Infectious complications that often follow treatment with antibiotics suggest that colonising symbionts contribute to host defence against pathogens. Yet, in the absence of effective defence mechanisms, even symbiotic microbes can multiply rapidly and overwhelm the mammalian host. Despite the presence of microbes, normal digestive tract surfaces generally show little evidence of inflammation, the coordinated pattern of vasodilatation, …
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Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger(heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.
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Two genes encoding the ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptide, abf-1 and abf-2, were identified in Caenorhabditis elegans. Recombinant ABF-2 exhibited potent microbicidal activity against Gram-positive and Gram-negative bacteria, and yeasts. The tissue-specific distribution estimated by immunofluorescence staining and transgenic analysis of a gfp fusion gene (where GFP corresponds to green fluorescent protein) suggested that ABF-2 contributes to surface defence in the pharynx. abf-1 contains a single intron at a conserved position, suggesting that asabf and abf originated from a common ancestor. Both transcripts for abf-1 and abf-2 were detected as two distinct forms, i.e. spliced leader (SL)1-trans-spliced with a long 5'-untranslated region (UTR) and SL-less with a short 5'-UTR. A polycistronic precursor RNA encoding ABF-1 and ABF-2 was detected, suggesting that these genes form an operon. An 'opportunistic operon' model for regulation of abf genes, including the generation of short SL-less transcripts, is proposed. In conclusion, C. elegans should have an immune defence system due to the antimicrobial peptides. C. elegans can be a novel model for innate immunity. Furthermore, the combination of biochemical identification in Ascaris suum and homologue hunting in C. elegans should be a powerful method of finding rapidly evolved proteins, such as some immune-related molecules in C. elegans.
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During the past year, dramatic progress has been achieved in our understanding of Drosophila immune reactions. The completion of the Drosophila genome sequencing project, microarray analysis and the use of genetic screens have led to the identification of several new genes required to combat microbial infection, filling in some important gaps in the understanding of innate immunity. At the same time, this insect was used as a model for the study of host-pathogen interactions. The recent major advances on the mechanisms by which this insect defends itself against intrusion of pathogens are discussed in this review.
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Recently, invertebrate models have been widely used for the study of innate immunity. Nematodes are novel potential candidates because of the experimental advantages of Caenorhabditis elegans. However, whether nematodes have active immune responses is still ambiguous. Previously, we reported ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides in the parasitic nematode Ascaris suum and the genetic model nematode C. elegans. Further screening of a cDNA library and an expressed-sequence-tag database search detected five novel members of ASABF (ASABF-beta, -gamma, -delta, - epsilon and -zeta) in A. suum. The transcripts for ASABF-alpha, -beta, -gamma, and -delta clearly increased in the body wall, and also in the intestine for ASABF-delta, 4 h after injection of heat-killed bacteria into the pseudocoelom (body cavity), suggesting that these peptides are inducible in the acute phase of immune response. These results also suggest that the nematodes can recognize bacteria in the pseudocoelomic fluid and evoke an active immune response.
Article
Two genes encoding the ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptide, abf-1 and abf-2, were identified in Caenorhabditis elegans. Recombinant ABF-2 exhibited potent microbicidal activity against Gram-positive and Gram-negative bacteria, and yeasts. The tissue-specific distribution estimated by immunofluorescence staining and transgenic analysis of a gfp fusion gene (where GFP corresponds to green fluorescent protein) suggested that ABF-2 contributes to surface defence in the pharynx. abf-1 contains a single intron at a conserved position, suggesting that asabf and abf originated from a common ancestor. Both transcripts for abf-1 and abf-2 were detected as two distinct forms, i.e. spliced leader (SL)1-trans-spliced with a long 5′-untranslated region (UTR) and SL-less with a short 5′-UTR. A polycistronic precursor RNA encoding ABF-1 and ABF-2 was detected, suggesting that these genes form an operon. An ‘opportunistic operon’ model for regulation of abf genes, including the generation of short SL-less transcripts, is proposed. In conclusion, C. elegans should have an immune defence system due to the antimicrobial peptides. C. elegans can be a novel model for innate immunity. Furthermore, the combination of biochemical identification in Ascaris suum and homologue hunting in C. elegans should be a powerful method of finding rapidly evolved proteins, such as some immune-related molecules in C. elegans.
Article
Variations in the number of galanin receptor (Gal-R1)-expressing cells and levels of Gal-R1 messenger RNA (mRNA) were determined in the preoptic area in intact female rats throughout the phases of the estrous cycle and compared with those in the male. Female and male Wistar rats were fixed by perfusion with 4% paraformaldehyde. Cryostat sections were hybridized with a ³⁵S-labeled antisense Gal-R1 riboprobe. The number of Gal-R1 mRNA-expressing cells was lower in the rostral preoptic area than in the medial preoptic area. During the estrous cycle, the highest number of Gal-R1 mRNA-expressing cells in the rostral preoptic region was detected at 0800 h on proestrus, whereas in the medial preoptic area, the maximum number was observed at 1800 h on estrus. Gal-R1 mRNA levels in individual cells were low during diestrus and increased at estrus in both areas. In the male, the number of mRNA-expressing cells and the hybridization signal were significantly lower than those in females during estrus. The results demonstrate that Gal-R1 gene expression in the preoptic area varies during the estrous cycle and is low in males. Short term treatment of ovariectomized rats with estradiol plus progesterone caused significantly decreased preoptic Gal-R1 mRNA levels compared with those after treatment with estrogen only. These observations suggest that in the preoptic area, expression of Gal-R1 is influenced by progesterone. The variation in Gal-R1 expression is likely to influence the extent to which galanin can influence the preoptic cells implicated in the control of neighboring GnRH cells.
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Electrospray ionization mass spectrometry (ESI-MS) was used to investigate metal ion interactions with salivary peptides histatin 3 (H3) and histatin 5 (H5). Conformational changes of these peptides in the presence of metal ions were studied using circular dichroism spectroscopy. H3 and H5 formed high affinity complexes with Cu2+ and Ni2+ and, to a lesser extent, with Zn2+. Both peptides show the potential for multiple binding sites for Cu2+ and Ni2+ and only a single strong binding site for Zn2+. The binding of a third Cu2+ ion to H3 seems to enable the binding of a fourth ion to H3. The binding of a second and third Ni2+ ion to H5 has a similar effect in enabling the binding of a fourth ion. None of the metal ions examined stabilized a regular secondary structure for either peptide. Subtle changes in overall conformation are seen with the addition of Cu2+ to both H3 and H5. Copyright © 2000 John Wiley & Sons, Ltd.
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Research on the innate immune response of mammals has revealed similarities with the invertebrate immune system. Thus, insects have developed an acute response resembling that seen in humans, implicating similar effectors, receptors and regulation of gene expression. Mussels have developed intracellular phagocytosis resembling that seen in mammalian neutrophils, using cationic antibacterial peptides in phagolysosomes. Leeches, like amphibians, contain antibacterial peptides and immune stimulators that derive from the processing of neuropeptide precursors. This pattern of similarities suggests that the vertebrate innate immune response resembles a patchwork of those responses seen in several invertebrate models.
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The hallmark of the innate immune response of higher insects is the rapid and transient synthesis of a battery of broad spectrum antimicrobial peptides by the fat body. The control of the genes encoding these peptides involves cis-regulatory promoter elements homologous to sequences functional in mammalian acute-phase genes. Study of immune-deficient mutants of Drosophila has indicated that distinct pathways control the antibacterial and antifungal responses in this species. Novel receptors potentially involved in the initiation of the immune response have been recently characterized.
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The developmental analysis of the inducibility of the Drosophila diptericin gene promoter as a response to septic injury shows an important increase in the response during the third larval instar leading to a maximum in late larvae and early prepupae. This increase, or maturation, is temporally correlated with known ecdysone induced events of the salivary gland and we now present evidence, using wild type and mutant larvae, that it does indeed depend upon ecdysone. The response remains minimal in larvae carrying either the temperature sensitive ecdysone deficient late larval lethal allele ecd1, or l(1)t187, a deep orange allele known to be deficient in the ecdysone response. However, experiments with the late larval lethal Broad-Complex mutant l(1)t435 show that the regulation of this response is distinct from the developmental ecdysone regulated hierarchies.
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Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.
Article
A novel antimicrobial peptide was isolated and characterized from the earthworm, Lumbricus rubellus. The antimicrobial peptide was purified to homogeneity by a heparin-affinity column and C18 reverse-phase HPLC, and named lumbricin I. Lumbricin I was a proline-rich antimicrobial peptide of 62 amino acids (15% proline in molar ratio; molecular mass, 7231 Da), whose complete sequence was determined by a combination of peptide sequence and cDNA analysis. The peptide and cDNA sequence analysis revealed that lumbricin I was produced as a precursor form consisting of 76 amino acids, with 14 residues in a presegment and 62 residues in mature lumbricin I. Lumbricin I showed antimicrobial activity in vitro against a broad spectrum of microorganisms without hemolytic activity. In addition, a 29-amino acid peptide, named lumbricin I(6-34), which was derived from residues 6-34 of lumbricin I, showed marginally stronger antimicrobial activity than lumbricin I. Northern blot analysis on total RNA revealed that expression of lumbricin I gene was not induced by bacterial infection, but was constitutively expressed. Furthermore, the expression of lumbricin I gene was specific in adult L. rubellus: Lumbricin I mRNA was detected only in adult L. rubellus, but not in eggs and young L. rubellus.
Article
Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescensof a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.
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Eight families of antimicrobial peptides, ranging in size from 2 to 9 kD, have been identified in plants. These are thionins, defensins, so-called lipid transfer proteins, hevein- and knottin-like peptides, MBP1, IbAMP, and the recently reported snakins. All of them have compact structures that are stabilized by 2-6 disulfide bridges. They are part of both permanent and inducible defense barriers. Transgenic overexpression of the corresponding genes leads to enhanced tolerance to pathogens, and peptide-sensitive pathogen mutants have reduced virulence.
Article
Activation of the innate immune response involves recognition of the infectious agent and the subsequent activation of cellular and humoral reactions. In insects, a number of immunity genes are activated at the level of transcription leading to the synthesis of antimicrobial peptides. Genetic analyses in Drosophila have identified several signal transduction pathways that promote activation of these immunity genes. Recent data suggest that the insect immune system is able to discriminate between a bacterial and a fungal infection, and responds by higher levels of activation of the appropriate peptides to repel the infection. These and other recent data on transcription factors and regulation of antimicrobial genes are integrated into a model to suggest how differential activation of antifungal and antibacterial peptides can occur in response to fungal and bacterial infection.
Article
Antimicrobial peptides appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. During the past 15 years a multitude of these peptides have been isolated largely from insects. In spite of great differences in size, amino acid composition and structure, most of the antimicrobial peptides from insects can be grouped into one of three categories. The largest category in number contains peptides with intramolecular disulfide bonds forming hairpin-like beta-sheets or alpha-helical-beta-sheet mixed structures. The second most important group is composed of peptides forming amphipathic alpha-helices. The third group comprises peptides with an overrepresentation in proline and/or glycine residues. In general, the insect antimicrobial peptides have a broad range of activity and are not cytotoxic. Despite a wealth of information on structural requirements for their antimicrobial activity, the mode of action of these peptides is not yet fully understood. However, some data suggest the existence of two types of mode of action: 1. through peptide-lipid interaction or 2. through receptor-mediated recognition processes. This review presents the main results obtained during the last four years in the field of antimicrobial peptides from insects with a special focus on the proline-rich and cysteine-rich peptides.
Article
We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.
Article
In previous papers, we characterised 3 types of 4-kDa, cysteine-rich, cationic antimicrobial peptides: MGDs (for Mytilus galloprovincialis defensins), mytilins and myticins, which are abundant in the mussel hemocytes. In the present work, we revealed a differential distribution of the cells expressing the different genes. In addition, using confocal and electron microscopy, we confirmed that defensins and mytilins were partially located in different sub-types of circulating hemocytes although the peptides can be located in the same cell, and even in the same granule. We also demonstrated that mytilins exert their microbicidal effect within the cells through the process of phagosome-mytilin granule fusion leading to the co-location of ingested bacteria and mytilins.
Article
The production of antimicrobial peptides is an important aspect of host defense in multicellular organisms. In Drosophila, seven antimicrobial peptides with different spectra of activities are synthesized by the fat body during the immune response and secreted into the hemolymph. Using GFP reporter transgenes, we show here that all seven Drosophila antimicrobial peptides can be induced in surface epithelia in a tissue-specific manner. The imd gene plays a critical role in the activation of this local response to infection. In particular, drosomycin expression, which is regulated by the Toll pathway during the systemic response, is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide genes in Drosophila.
Article
Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.
Article
A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.
Article
Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?
Article
Conformational studies of human salivary peptide, histatin 3 (Hst3), were performed by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy in a membrane-mimicking environment. The structural information that was obtained was used in the design of peptide analogues with improved antifungal activity. In the presence of increasing concentrations of L-alpha-dimyristoylphosphatidylcholine (L-alpha-DMPC) lipid vesicles, a dramatic increase in a minimum at 198 nm is observed in the CD spectra of Hst3. The NMR data of Hst3 in the presence of L-alpha-DMPC lipid vesicles reveal the proximity of residues Y(10) and S(20), indicating the existence of a more compact structure. Peptide analogues were designed on the basis of this observation, which incorporated a disulfide bond to stabilize an extended loop in this region of the sequence. One of these, peptide 4, was 100 times more potent than Hst5 against Saccharomyces cerevisiae cells. Conformational analysis of peptide 4 revealed a looped structure with charged residues protruding on the outside surface, while a combination of aromatic residues and histidines are packed into an internal core.
Article
The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.
Article
Water-membrane soluble protein and peptide toxins are used in the defense and offense systems of all organisms, including plants and humans. A major group includes antimicrobial peptides, which serve as a nonspecific defense system that complements the highly specific cell-mediated immune response. The increasing resistance of bacteria to conventional antibiotics stimulated the isolation and characterization of many antimicrobial peptides for potential use as new target antibiotics. The finding of thousands of antimicrobial peptides with variable lengths and sequences, all of which are active at similar concentrations, suggests a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures. Such a mechanism is in agreement with the "carpet model" that does not require any specific structure or sequence. It seems that when there is an appropriate balance between hydrophobicity and a net positive charge the peptides are active on bacteria. However, selective activity depends also on other parameters, such as the volume of the molecule, its structure, and its oligomeric state in solution and membranes. Further, although many studies support that bacterial membrane damage is a lethal event for bacteria, other studies point to a multihit mechanism in which the peptide binds to several targets in the cytoplasmic region of the bacteria.
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