Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to Chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Abstract

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Figure 1. The exon distribution and primer position of koala TNFα (a), IL10 (b) and GAPDH (c) genes when in alignment with other marsupial and human sequences of the corresponding genes.

The forward and reverse primer sequences used in the RT-PCR assays are highlighted in green and red, respectively. The PCR product has been designed such that it spans over an intron-exon junction.

Figure 2. Amino acid sequence alignment of koala, brushtail possum and human TNFα sequences.

(a) Koala TNFα sequence characterisation is done with reference to the human TNFα sequence. Presequence comprising of 76 amino acids and the secreted form of 156 amino acids is indicated by the horizontal red and green lines respectively. The hydrophobic transmembrane region and the intracytoplasmic region are marked out in red and green boxes respectively . Asterisks indicate the receptor binding sites of the protein . (b) Koala IL10 sequence characterisation is done with reference to the human IL10 sequence. Cysteine residues which form intramolecular disulphide bonds in human IL10 are marked in red boxes. Residues important for structural stabilisation are marked in green boxes. The six α-helices of the IL10 monomer are depicted by dotted blue lines .

Figure 3. Phylogenetic tree of (a) TNFα and (b) IL10 nucleotide sequence constructed using Geneious Pro 5.6.5 using Jukes Cantor, UPGMA tree build method with 1000 bootstrap repeat value.

Figure 4. TNFα and IL10 gene expression in koala PBMCs stimulated with UV inactivated C. pecorum in Group I, II and III animals.

Samples were collected at 0, 12, 24 and 48 hours. Results are expressed as fold increased compared to GAPDH.