Research paperIdentification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii)
Introduction
Antimicrobial peptides are gene-encoded proteins with broad-spectrum antimicrobial activity. They form part of the innate immune system and have been identified in a large variety of plants, insects and animals. Online databases now contain peptide sequences of over 800 of these antimicrobial peptides (e.g. http://aps.unmc.edu/AP/main.html). As well as forming part of the innate immune system, they also stimulate an adaptive immune response in vertebrates (Shinnar et al., 2003). More recently antimicrobial peptides have been shown to have potential as alternatives to antibiotics as micro-organisms continue to develop antibiotic resistance. The advantages of using antimicrobial peptides include their broad target range, minimal side effects and low development of resistance by target organisms (Hancock and Lehrer, 1998).
Cathelicidins are a group of antimicrobial peptides found predominantly in mammals and are distinguished by a gene structure composed of 4 exons and 3 introns. Exons 1–3 encode a well conserved pre-propeptide while exon 4 encodes the functional, but variable, antimicrobial peptide (Zanetti et al., 1995, Zanetti, 2005). The name, cathelicidin, is based on a similarity to cathelin, a serine protease inhibitor first identified from pig leukocytes (Ritonja et al., 1989). Most species produce several related cathelicidins, while humans, rats and mice have only one (Zanetti, 2005). The only human cathelicidin, hCAP18/LL37, was first isolated from bone marrow (Agerberth et al., 1995). In all cathelicidins the functional peptide is activated through the proteolytic processing of the pre-propeptide. The signal sequence is removed first by a signal peptidase (Shinnar et al., 2003) with the resulting product stored in the granules of neutrophils. The active antimicrobial peptide is released from the cathelin region by a serine protease in response to a microbial invasion coinciding with the need for the antimicrobial activity at the sight of infection (Tomasinsig and Zanetti, 2005). In hydrophobic solutions most active cathelicidin peptides form an α-helical structure that is thought to form membrane pores in target organisms, thus disrupting metabolic activity (Oren et al., 1999, Turner et al., 1998, Brogden, 2005).
Although predominately stored in the granules of neutrophils, cathelicidin expression has been demonstrated in a range of tissues in the pig (Wu et al., 1999), mouse (Gallo et al., 1997) and rat (Termen et al., 2003). In species producing multiple cathelicidins, these expression patterns appear to differ depending on the type of cathelicidin synthesized (Zanetti, 2005).
Marsupials have become a recent focus of research due to an increased interest in the opportunities afforded by their unique reproductive strategies. They have a short gestation period, ranging from 11 days in the striped-faced dunnart, Sminthopsis macroura, to 35 days in the koala, Phascolarctos cinereus (Tyndale-Biscoe and Renfree, 1987), after which the pouch young undergoes the majority of its physical and immunological development in a non-sterile environment of an external maternal pouch. During this time the young marsupial is apparently dependant on maternal immune defences and behaviour for protection. Studies of the development of immune tissues of marsupial pouch young suggests immunocompetence develops close to midway through pouch residency, for example, at approximately 90 days post-partum in the tammar wallaby, Macropus eugenii (reviewed by Old and Deane, 2000). It has been hypothesised that marsupials have developed specific strategies to fight infection prior to the development of immunocompetence and antimicrobial peptides may form an important component of this system. Multiple cathelicidin genes have been previously identified in 3 marsupials using different methods. These include a single gene in the Australian northern brown bandicoot, Isoodon macrourus, using expressed sequence tags (ESTs) (Baker et al., 2007); 7 genes in the tammar wallaby, M. eugenii, from a mammary gland cDNA library screening protocol (Daly et al., 2008) and 12 genes from the American grey-short tailed opossum, Monodelphis domestica, using in silico sequence database screening (Belov et al., 2007).
The current study reports the identification of a novel eighth cathelicidin gene, MaeuCath8, from the tammar wallaby, M. eugenii. By applying the technique of reverse transcriptase (RT)-PCR and RACE-PCR, the complete cDNA sequence of MaeuCath8 was obtained and used to determine its expression profile in a range of tissues of the marsupial from birth to adulthood.
Section snippets
Collection of tissue
Tissue samples were collected opportunistically from a breeding colony of tammar wallabies (M. eugenii) maintained at the Macquarie University Fauna Park, NSW, Australia. Sampled tissues included blood, skin, liver, lung, kidney, thymus, spleen, bone marrow and gastrointestinal tract (GIT). Samples were collected as either part of routine management of the captive population or surplus from other approved research protocols. Age of the pouch young was estimated based on head length measurements
Identification and analysis of the cathelicidin MaeuCath8
Use of primers to the conserved region of the cathelicidin gene of the northern brown bandicoot (Baker et al., 2007) resulted in the PCR amplification of a 168 bp fragment. Cloning and sequencing of the amplified region confirmed that it had 88% identity to the cathelicidin gene of the northern brown bandicoot. Subsequent RACE-PCR produced a 279 bp product in the 5′ direction and 981 bp product in the 3′ direction resulting in a 1234 bp product covering the full 507 bp length of the expressed tammar
Discussion
Using RACE-PCR a new marsupial cathelicidin, MaeuCath8, has been isolated and sequenced and its expression documented in the developing and adult tissues of the tammar wallaby. The approach used in this study contrasts with the protocols used to isolate the other tammar wallaby cathelicidin genes, MaeuCath1-7, which were detected during sequencing of contigs from a mammary cDNA library of M. eugenii (Daly et al., 2008). Phylogenetic analysis of the propeptide domain of MaeuCath8 showed that it
Acknowledgements
This research was supported by an Australian Research Council Discovery Grant DP0557854 awarded to NAJ and EMD. We would also like to thank Anne Mouland-Claassens for assistance in sample collection.
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2012, Developmental and Comparative ImmunologyCitation Excerpt :Cathelicidins are especially crucial for neonatal survival and defence and have been detected in the skin of neonatal mice at levels much higher than seen in adults (Dorschner et al., 2003). Fourteen cathelicidin genes have been identified in tammar wallaby pouch young (Wang et al., 2011), with increased expression of MaeuCath1 in the skin, lung, jejunum, liver and spleen over the first 25 days postpartum (Daly et al., 2008a) and MaeuCath8 being expressed in the spleen and GIT of the tammar wallaby from 1 day after birth (Carman et al., 2009). Further, the synthesized cathelicidins were found to be effective in killing a broad range of bacteria, suggesting that the antimicrobials play a large role in protecting the pouch young before its adaptive immune system develops (Wang et al., 2011).
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