Molecular characterisation and expression analysis of Interferon gamma in response to natural Chlamydia infection in the koala, Phascolarctos cinereus
Mathew, M, Pavasovic, A, Prentis, PJ, Beagley, KW, Timms, P & Polkinghorne, A 2013, Gene, vol. 527, pp. 570-577.
Transcriptome analyses of koalas have identified the sequence of the genes encoding koala interferon gamma (IFNγ), a cytokine involved in antimicrobial responses. By comparing tissues from cohorts of koalas with and without active Chlamydia infections using quantitative real time PCR (qPCR), significantly different expression patterns of IFNγ between both groups were observed.
Efforts to understand the koala IFNγ response have been hampered by a lack of information about the sequences of key cytokines in koalas. The aim of this study was to identify the complete sequence of koala IFNγ and understand the functional characteristics of koala IFNγ during chlamydial infection. The full koala IFNγ coding sequence was found to be 495 base-pairs (bp) long and encode a protein that is 165 amino acids long. Phylogenetic analyses with multiple sequence alignment and phylogenetic tree construction using IFNγ sequences from different hosts revealed that the koala IFNγ has a 77 – 89.5% sequence similarity to other marsupials, while maintaining 60 – 63% sequence similarity to other mammals and 59.7% similarity to the platypus. It was also observed that the koala IFNγ was distantly related to that of avian and piscine species. Peripheral blood mononuclear cells from ten koalas were sampled and stimulated using UV-inactivated Chlamydia pecorum for 12, 24 and 48 hours to determine if there was a difference in the expression pattern of koala IFNγ during chlamydial infection. Using a koala-specific IFNγ qPCR assay developed in this study from the full koala IFNγ sequence, koalas with active chlamydial infection were seen to have significantly higher expression of IFNγ compared to koalas that did not have clinical signs of chlamydial infection, with one koala having a seven-fold increase of expression of IFNγ at 12 hours post-simulation.
Conventional methodologies have been of limited use when it comes to discerning the koala immune response. As shown in this study, the koala IFNγ sequence has low sequence similarity to eutherian mammals, which include model organisms such as mice, and thus impede efforts using homologous sources to design assays for characterisation of the koala immune response. Here, using an assay developed specifically from the sequence of koala IFNγ, it was found that expression of IFNγ significantly increased in C. pecorum infected koalas, indicative of the Th1 immune response being activated. It should be noted that this was a preliminary study, and thus the authors suggest a longitudinal study be conducted in koalas to assess the role and expression levels of IFNγ in C. pecorum infected koalas before any physiological significance can be associated with the observed differential expression of IFNγ in koalas.
In conclusion, this study identified the full coding sequence of IFNγ in koalas and demonstrated the use of an effective koala-specific quantitative assay to evaluate differential expression patterns of IFNγ during C. pecorum infection. This information will benefit further efforts in elucidating possible solutions to relieve the burden of chlamydial disease in koalas.
Summarised by Daniel Chew
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