Identification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii)

Abstract

Cathelicidins are important components of the innate immune system and have been identified in skin and epithelia of a range of mammals. In this study molecular techniques, including RACE-PCR, were used to identify the full cDNA sequence of a cathelicidin gene, MaeuCath8, from the Australian marsupial, the tammar wallaby, Macropus eugenii. This cathelicidin was not homologous to other such genes previously isolated from a tammar wallaby mammary gland EST library, however, it did contain 4 conserved cysteine residues which characterise the pre-propeptide and had 80% identity with a previously isolated bandicoot cathelicidin. Reverse transcriptase-PCR established the expression profile of MaeuCath8 in a range of tissues, including spleen, thymus, gastrointestinal tract, skin and liver, of the tammar wallaby from birth to adulthood. Expression of MaeuCath8 was observed in spleen and gastrointestinal tract of newborn animals and was observed in most tissues by 7 days post-partum. The results indicate that pouch young could synthesize their own antimicrobial peptides from an early age suggesting that this ability most likely plays a role in protecting the pouch young from infection prior to the development of immunocompetence.

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.