Number

Ong, Dasi, Shahrudin, Shahriza & Ismail, Mohd Nazri, 2021, Review on antimicrobial peptides from Malaysian amphibian resources: status, research approaches and ways forward, Turkish Journal of Zoology 45 (2), pp. 79-90 : 81-85

Family Number of species

Bufonidae 48

Ceratobatrachidae 2

Dicroglossidae 36

Megophryidae 40

Microhylidae 48

Ranidae 41

Rhacophoridae 61

Ichthyopiidae 12

Total species of Anura 276

Total species of Amphibia 288

3 Frost D (2020). Amphibia Species of the World 6.0, an Online Reference 2020 [online]. Website http://research.amnh.org/ herpetology/amphibia/index [accessed 8 January 2020].

Kinabalu’s highlands (Sabah) to saltwater mangrove swamps. Malaysia holds a high diversity of amphibians; however, only a few studies have explored the potential antimicrobial activity within these species ( Table 2). These studies included two toad species and several frog species from peninsular Malaysia, several more from East Malaysia ( Sarawak), and three Malaysian frog species from other countries (extralimital areas). Examples of the samples studied among Malaysian amphibians are shown in Figure 1.

The skin secretions of Phrynoidis asper , the Asian Giant Toad, have been demonstrated to contain more than 50 types of proteins with a broad-spectrum antimicrobial effect against gram-positive and gram-negative bacteria, including Staphylococcus aureus and Bacillus subtilis ( Dahham et al., 2016) . Parotoid secretions of the common Sunda toad ( Duttaphrynus melanostictus ) have weak inhibitory activity towards microbes (Zahri et al., 2015). Conlon et al. (2008) revealed a structural characterisation of skin secretions of Odorrana hosii and Hylarana picturata from Malaysia that contained eight AMPs. These AMPs belong to the esculentin-1, esculentin-2, brevinin-1, brevinin-2, and nigrocin-2 families. A preliminary study from various frog species from peninsular Malaysian frog skin secretions by Rahman et al. (2016) demonstrated the antimicrobial effects against gram-negative and gram-positive bacteria; unfortunately, the frog species were not specified. Partially purified peptides from East Malaysian ( Sarawak) frog species’ skin secretions showed that some of the frog species contain AMPs and some are more potent than others ( Sabri et al., 2018; Shahabuddin et al., 2018). Brevinin-2 has been identified from protein profiling of a foam nest from Polypedates leucomystax ( Shahrudin et al., 2017)

At least five studies have been performed on extralimital frog species that can also be found in Malaysia ( Table 2) ( Lu et al., 2008; Song et al., 2009; Al-Ghaferi et al., 2010; Wang et al., 2012; Suhyana et al., 2015). Skin secretions of Limnonectes kuhlii , Kuhl’s wart frog, from China revealed that the five novel AMPs thatwere purified and characterised showed strong antimicrobial effects against gram-positive and gram-negative bacteria and fungi (Wang et al., 2012a). The five novel AMPs were temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin- LK1, and gaegurin-LK2 (Wang et al., 2012a). According to Al-Ghaferi et al. (2010), structural characterisation of Hylarana erythraea skin secretions from Vietnam showed AMPs found from the brevinin-1, brevinin-2, esculentin-2, and temporin families. This species’ skin secretions also revealed that they are generally active against gram-positive and gram-negative bacteria, particularly S. aureus and E. coli ( Al-Ghaferi et al., 2010) . Two studies 16 Rhacophoridae Polypedates Peninsular Malaysia foam nest Shahrudin et al. (2017) leucomystax

No. Family Species name Locality Source Reference 1 Bufonidae Phrynoidis asper Peninsular Malaysia skin secretion Dahham et al. (2016) 2 Bufonidae Duttaphrynus melanostictus Peninsular Malaysia paratoid secretion Zahri et al. (2015) 3 Ranidae Odorrana hosii Peninsular Malaysia; East Malaysia skin secretion; skin Conlon et al. (2008); Sabri et al. (2018); Shahabuddin et al. (2018) 4 Ranidae Pulchrana picturata Peninsular Malaysia skin secretion Conlon et al. (2008) 5 Ranidae Pulchrana glandulosa East Malaysia skin secretion Sabri et al. (2018) 6 Ranidae Pulchrana signata East Malaysia skin secretion Sabri et al. (2018) 7 Ranidae Pulchrana baramica East Malaysia skin secretion Sabri et al. (2018) 8 Ranidae Chalcorana raniceps East Malaysia skin secretion Sabri et al., 2018); Shahabuddin et al. (2018) 9 Ranidae Meristogenys jerboa East Malaysia skin secretion Sabri et al. (2018); Shahabuddin et al. (2018) 10 Ranidae Staurois guttatus East Malaysia skin Shahabuddin et al. (2018) 11 Ranidae Hylarana erythraea Vietnam skin secretion Al-Ghaferi et al. (2010) 12 Dicroglossidae Limnonectes leporinus East Malaysia skin Shahabuddin et al. (2018) 13 Dicroglossidae Limnonectes kuhlii East Malaysia; China skin secretion; skin Shahabuddin et al. (2018); Wang et al. (2012) 14 Dicroglossidae Fejervarya cancrivora China skin secretion Lu et al. (2008); Song et al. (2009) 15 Dicroglossidae Fejervarya limnocharis Indonesia skin secretion Suhyana et al. (2015)

on AMPs of Fejervarya cancrivora from China disclosed that this species contains tigerinin-like peptides and cancrin ( Lu et al., 2008; Song et al., 2009). Indonesian frog AMPs from Fejervarya limnocharis suppress the growth of S. pneumoniae multidrug-resistant strain SPN 1307 ( Suhyana et al, 2015).

3. Different approaches to discover the AMPs in Malaysian amphibian species

The methodologies to identify amphibian AMPs include stimulation and collection of secretions, extraction, identification of the peptides and an antimicrobial assay. Many approaches had been considered to study the AMPs from Malaysian amphibian species. A summary of the different approaches is presented in Figure 2.

3.1. Stimulating and collecting secretions

Hormonal stimulation is the most utilised method to collect skin secretions. The skin is an endocrine organ that exhibits hormonal activity. With the introduction of hormone or chemical stimulant, the skin produces positive feedback towards skin secretion production. This type of stimulation is animal-friendly and does not have a long term effect on the individual. Dahham et al. (2016) used a minimal electrical voltage to introduce stress in P. asper (Asian Giant Toad) to stimulate skin secretions. Amphibians produce skin secretions when they feel threatened/stressed ( Hancock and Sahl, 2006).

Zahri et al. (2015) and Shahrudin et al. (2017) directly collected secretions without stimulation because parotoid secretions and foam nests do not need any stimulant for collection. These secretions were collected either by immersing the animals in a low concentration, salt solution (sodium chloride or sodium acetate) or directly rinsing them with deionised water. Centrifugation is essential to sort out these secretions from any contaminants (small organic particles). All of these secretions were frozen and lyophilised. The lyophilisation process is essential to remove water from the secretions for long-term storage and for the convenience of transporting the samples to another place ( Shukla, 2011; Khairnar et al., 2013).

3.2. Peptide extraction

An extraction buffer was the most common method utilised to extract and purify peptides in previous studies. These extraction buffers can either be phosphate ( Lu et al., 2008; Song et al., 2009; Wang et al., 2012a; Suhyana et al., 2015) or Tris-HCl ( Dahham et al., 2016; Shahrudin et al., 2017). The extraction buffer acts as a lysis buffer that improves the peptides’ stability, as they can easily be denatured, damaged or lost during the extraction process. The samples are fractionated either by gel permeation chromatography, solid-phase extraction or gel separation. The fractionation process refers to the isolation and separation process of samples into smaller fractions (quantities) from their original conformation. These smaller fractions increase the sensitivity of peptide identification. All samples are finally analysed by reversedphase high-performance liquid chromatography ( RP-HPLC) before identification of the peptide, except in Suhyana et al. (2015) and Shahriza et al. (2017). RP-HPLC is utilised to separate small fraction samples according to their polarity.

3.3. Peptide identification

Identification and structural characterisation of AMPs are essential to specify their properties. Only two studies have identified AMPs; one focused on AMPs of skin secretions and the other on AMPs from foam nests ( Conlon et al., 2008; Shahrudin et al., 2017). A protein profiling analysis of P. asper skin secretions revealed more than 50 proteins, and some were undiscovered in the available database ( Dahham et al., 2016). Thus, there is a high potential for discovering AMPs in other skin secretions and egg nests (foam and gel) of other amphibian species. However, in East Malaysia ( Sarawak), only partially purified crude peptides have been extracted from frog specimens. These peptides have high potential as AMPs due to their similar molecular weight ranges with previous studies on amphibian AMPs. There is a high chance to uncover new AMPs of amphibian species in East Malaysia (Sabah and Sarawak) as there is a higher species diversity in East Malaysia (Sabah and Sarawak compared to Peninsular Malaysia, 266 vs 183, as of March 2016 ( Inger et al., 2017; Norhayati, 2017).

According to previous studies, mass spectrometrybased protein profiling is more reliable because proteins are very complex biomolecules ( Tyers and Mann, 2003; Kislinger and Emili, 2005). Mass spectrometry refers to an analytical technique that measures the mass to charge (m/z) ratio of ions from a particular compound/mixture/ solution. Most studies that have identified proteins (AMPs) used matrix-assisted laser desorption/ionisation- timeof-flight- mass spectrometry ( MALDI-TOF-MS) instead of liquid chromatography-tandem mass spectrometry ( LC-MS /MS). This is arguablydue to the cost factor, which makes MALDI-TOF-MS more widely available. Furthermore, before the introduction of newer LC-MS / MS technologies, such as Orbitrap and SWATH, MALDI-TOF-MS was the instrument of choice for protein and peptide analyses. Nevertheless, the downside of an MS/ MS system is its inability to distinguish between leucine and isoleucine residues. This problem can be solved by performing genetic comparisons (cDNA) of the host by Edman degradation, as previously done by Wang et al. (2012).

3.4. Antimicrobial assay

The general steps taken for antimicrobial assays are microbial selection, microbial growth/culture, microdilution/series dilution of AMP solution, and AMP solution incubation into a microbial plate (depending on different concentrations), followed by antimicrobial evaluation tests. The antimicrobial assays in most studies were performed against common bacteria and fungus, which include E. coli , S. aureus , Candida albicans (yeast), Methicillin-resistant Staphylococcus aureus (MRSA) , Bacillus cereus , Bacillus subtilis , Shigella dysenteriae, Pseudomonas aeruginosa and Salmonella typhimurium ( Conlon et al., 2008; Wang et al., 2009; Al-Ghaferi et al., 2010; Dahham et al., 2016; Sabri et al., 2018; Shahabuddin et al., 2018).

These assays were evaluated by disc diffusion, minimum inhibitory concentration ( MIC) or minimum bactericidal concentration ( MBC) tests. The MIC is the lowest concentration that an antimicrobial agent is bacteriostatic ( Andrews, 2001). In comparison, MBC indicates the lowest concentration needed for an antimicrobial agent to completely inhibit (kill) a bacterium over a fixed time and under a specific set of conditions ( Andrews, 2001). A range for both MIC and MBC determinations (mg/L) is needed to establish the antimicrobial agent’s bactericidal effect. In this case, the antimicrobial agent refers to the antimicrobial peptide solution.

However, identification of the peptides in their samples indicated the presence of AMPs which have a high potential for inhibiting microbes, especially for resistant strains. Several antimicrobial studies have been performed on the peptides of Malaysian frog species found in other countries, namely Hylarana erythraea from Vietnam and Limnonectes kuhlii and Fejervarya cancrivora from China ( Lu et al., 2008; Al-Ghaferi et al., 2010; Song et al., 2009). These studies indicated that the relationship between AMPs and their antimicrobial activities could be justified for further investigations.