Fig 1.
Multiple sequence alignment of koala cathelicidins with other marsupial and eutherian cathelicidins.
PhciCath4 was not included in the alignment as it contains a premature stop codon and hence is a likely a pseudogene. PhciCath7p to 10p are partial sequences only, as the mature peptide could not be identified. The predicted signal peptide sequence is underlined, followed by two domains; the cathelin domain which contains conserved cysteine residues (boxed), and the antimicrobial domain which encodes the mature peptide and is of variable length and composition. The predicted mature peptide cleavage site is denoted by a star.
Table 1.
Physiochemical properties of predicted mature peptides from full-length koala cathelicidins.
Fig 2.
Full-length koala cathelicidins cluster with other marsupials in the phylogenetic tree, particularly PhciCath5 which clusters with other marsupial cathelicidins that display antimicrobial activity.
Sequences are coloured according to antimicrobial activity against bacteria and/or fungi; blue indicates active, red indicates inactive and black indicates the peptide has not been tested. Koala cathelicidins are denoted by a green circle. Only bootstrap values greater than 70% are shown. Accession numbers for published sequences used in this tree are available in S3 Table.
Fig 3.
Expression of full-length koala cathelicidins in twelve tissue transcriptomes from two individuals [41,42].
Expressed as transcripts per million (TPM).
Table 2.
Koala cathelicidin mature peptide PhciCath5 displays antimicrobial activity against bacteria and fungi from humans and animals, expressed as the minimum inhibitory concentration (MIC).
Fig 4.
PhciCath5 contains two alpha helical regions according to the Deleage and Roux plot (A), as shown by the rise in scores above the 0.99 cutoff. PhciCath5 is also amphipathic according to the Kyte & Doolittle plot (B), with hydrophilic residues spanning the middle of the peptide (negative scores), and hydrophobic residues at the N- and C-terminus (positive scores). The remaining koala cathelicidin mature peptides all contain alpha helical regions (A), and vary in hydropathicity from largely hydrophilic (PhciCath2) to amphipathic (PhciCath1, 3, 5 and 6).
Fig 5.
Activity of koala cathelicidins PhciCath1, 2, 3, 5 and 6 against C. pecorum MarsBar (A) and IPTaLE (B) at 125μg/mL. Expressed as inclusion forming units (IFU) per mL. Treatment of C. pecorum MarsBar and IPTaLE elementary bodies with 125μg/mL of PhciCath5 caused a significant decrease in infectious progeny in both serovars. PhciCath1, 2, 3 and 6 were ineffective against both serovars up to a concentration of 500ug/mL. **** indicates p<0.0001 significance was identified.