Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family

The emergence of pathogenic bacteria and fungi resistant to commonly used antibiotics poses a serious threat to public health and necessitates novel treatment approaches in order to control infections. Antimicrobial peptides (AMPs) are one of the central components of the system of innate immunity and due to their non-specific and highly destructive mechanism of killing, pathogens will develop resistance at lower rates than conventional antibiotics.Skin secretions of frogs from the family Pipidae are a rich source of AMPs which show potential for development into therapeutic agents. Until recently, the only representatives of the Pipidae family frogs from which dermal AMPs had been identified were the diploid frog <em>Silurana tropicalis</em>, the tetraploid frog <em>Xenopus laevis</em> and the octoploid frog <em>Xenopus amieti</em>. Therefore, this program of research was undertaken with the aim to isolate, purify and characterize AMPs with therapeutic potential from skin secretions of other polyploid species of African clawed frogs of the Pipidae family. Emphasis is given to the application of the AMPs as markers to elucidate the taxonomic relationships and evolutionary history of the frogs. The study also investigates the effects which polyploidization and interspecies hybridization have had on the multiplicity of AMPs in frog skin secretions. Chapter <strong>2</strong> and Chapter <strong>3</strong> present data from the peptidomic analysis of norepinephrine-stimulated skin secretions of two well-characterized and closely related tetraploid <em>Xenopus</em> species – <em>X. borealis</em> and <em>X. clivii</em>. Multiple peptides with varying degrees of antimicrobial activity were isolated. Structural characterization demonstrated that they were orthologous to magainins, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragments (CPFs) and xenopsin-precursor fragments (XPFs), previously isolated from <em>S. tropicalis</em>, <em>X. laevis</em> and <em>X. amieti</em>. CPF-B1 and CPF-C1 were the most abundant AMPs in the skin secretions of <em>X. borealis</em> and <em>X. clivii</em> respectively. CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) was active against clinical isolates of the hospital-associated pathogens, methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and multidrug-resistant <em>Acinetobacter baumannii</em> (MDRAB) with MIC = 5 µM and showed low hemolytic activity against human erythrocytes (HC₅₀ >200 µM). CPF-C1 (GFGSLLGKALRLGANVL.NH₂) also showed potent activity against a range of Gram-negative bacteria. CPF peptides, therefore, represent promising candidates for development into therapeutic agents for use against these emerging antibiotic-resistant pathogens. The genera <em>Silurana</em> and <em>Xenopus </em>are united in the subfamily Xenopodinae and have a complex evolutionary history. Chapter <strong>4</strong> includes data from the peptidomic analysis of skin secretions from an incompletely characterized tetraploid species termed “<em>S.</em> new tetraploid 1”with chromosome number 2n=40 and the octoploid species <em>X. andrei</em> (2n=72). The species represent model systems in which to study of the fate of duplicated AMP genes following putative allopolyploidization events. Multiple peptides belonging to the PGLa, XPF, and CPF familes were identified. The primary structures of the AMPs from <em>X. andrei</em> indicate aclose phylogenetic relationship between this species and the previously studied <em>X. amieti</em>. Three CPF peptides from “<em>S.</em> new tetraploid 1” showed potent, broad-spectrum antimicrobial activity and are present in high abundance. In contrast, only a single CPF peptide was isolated in low yield from the <em>X. andrei</em> secretions. There is no increase in the multiplicity of the AMPs in skin secretions of “<em>S.</em> new tetraploid 1”and the octoploid <em>X. andrei</em> when compared to the diploid <em>S. tropicalis</em> (2n=20) and the tetraploid <em>X. laevis</em> (2n=36). It is concluded that nonfunctionalization (gene silencing) has been the most common fate of duplicated AMP genes following polyploidization in the <em>Silurana</em> and <em>Xenopus</em> lineages. AMPs constitute a characteristic ‘‘fingerprint’’ of a particular frog species that may be used for an unequivocal taxonomic classification. Two populations of the tetraploid <em>X. muelleri</em>, occupying separate non-contiguous ranges in east and west Africa, are studied in Chapter <strong>5</strong>. Their taxonomic relationship is unclear and it has been proposed that the western population represents a separate species referred to as <em>X. muelleri</em> West while the eastern population retains the original name <em>X. muelleri</em>. A comparison of the primary sequences of AMPs in skin secretions reveals that no orthologous peptide from the two populations of <em>X. muelleri</em> has the same amino acid sequence. Additionally, the <em>X. muelleri </em>secretions, like those from <em>X. clivii</em>, did not contain a PGLa peptide whereas the <em>X. muelleri </em>West secretions contained two members of this family. The data indicate that <em>X. muelleri</em> West is more closely related to <em>X. borealis</em> than to <em>X. muelleri</em> and so provide strong support for the proposal that <em>X. muelleri</em> and <em>X. muelleri</em> West should be described as separate species. In contrast to species in the subfamily Xenopodinae, frogs from the subfamily Pipinae have not been investigated as a source of AMPs. The AMP profile in skin secretions from <em>Hymenochirus boettgeri</em> as a representative of genus <em>Hymenochirus</em> (subfamily <em>Pipinae</em>) is described in Chapter <strong>6</strong>. A novel family of five structurally-related peptides, designated as hymenochirins, was identified. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAV AKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B - 5B have the general structure XKIPX₂VKDTLKKVAKG X₂SX₂AGAX₃.COOH. The most abundant peptide in the secretions was hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ). Synthetic replicates of hymenochirin-1B - 4B possess broad-spectrum antimicrobial activity and relatively weak hemolytic activity and so represent potential candidates for development into therapeutically valuable agents against drug-resistant pathogens. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of <em>S. tropicalis</em> and <em>X. laevis</em> consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. The F1 hybrid frogs <em>X. laevis</em> x <em>X. muelleri</em> represent a model of interspecies hybridization in the Pipidae family that does not result in an increase in ploidy. They arestudied in Chapter <strong>7 </strong>and the data obtained provide an insight into the mode of inheritance of AMPs. A total of 18 different AMPs were isolated from skin secretions of the female hybrids. In addition to the complement of AMPs from the parent species, three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either <em>X. laevis</em> or <em>X. muelleri</em>. Magainin-LM1 differs from magainin 2 from <em>X. laevis</em> by a single amino acid substitution (Gly¹³®Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among <em>Xenopus</em> species to increase protection against pathogenic microorganisms in the environment. The thesis is completed by a general discussion in Chapter <strong>8 </strong>of theresults and conclusions in Chapters <strong>2</strong>-<strong>7</strong>. The potential of AMPs from skin secretions of frogs belonging to the Pipidae family is reviewed from three different aspects: promising candidates for development into therapeutic valuable anti-infective agents; reliable taxonomic and phylogenetic markers; and tools to study the fate of duplicated genes in <em>Xenopus</em> and <em>Silurana</em>. The interspecies <em>Xenopus</em> hybrids are proposed as a suitable model to perform future studies on the mode of inheritance of skin AMPs.