Characterization of Antimicrobial Peptides Isolated from the Processing by-Products of African Catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis>

Antimicrobial peptides (AMPs) as components of innate immunity system have been isolated from fish and other species. In this study, the crude proteins extracted with gradient ammonium sulfate precipitation technique from the processing by-products of African catfish Clarias gariepinus (C. gariepinus) were purified by size-exclusion chromatography and all the four obtained fractions, Clarias antimicrobial peptides I(CAP-I), CAP-II, CAP-III and CAP-IV, showed antimicrobial activity. Among of these fractions, CAP-IV showed the highest antimicrobial activity against Staphylococcus aureus, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli by agar diffusion plate test and the diameter of inhibition zone was 8.34, 9.27, 6.76, 6.13 mm, respectively. The molecular weight of main peptides of CAP-IV was around 4.1 KD by SDS-PAGE analysis. CAP-IV showed antimicrobial activity against both gram-negative and gram-positive bacterial pathogens at minimum inhibitory concentrations (MICs) ranging from 105 to 420 μg/mL. The antimicrobial activity of CAP-IV was stable at wide pH range, 3–11 and was also heat-stable when temperature was below 80 °C. Freeze-thawing treatment also only had slight effects on the antimicrobial activity of CAP-IV. Besides, CAP-IV was not sensitive to the hydrolysis by pepsin and trypsin, except for protease K. These results suggest that CAP-IV isolated from C. gariepinus is potential to be developed as a new antimicrobial peptide and may partially explain the high disease resistance of African catfish C. gariepinus.     

Characterization of Antimicrobial and Antioxidative Peptides Synthesized by <Emphasis Type="Italic">L. rhamnosus</Emphasis> C6 Fermentation of Milk

Lactobacillus rhamnosus C6 was used for milk fermentation with the aim of synthesizing antimicrobial and antioxidant peptides rich preparations. The proteolysis was checked for an incubation period of 72 h to check the extent of both bioactivities in fermentate. The 36 h incubated fermentate showed higher inhibition zone diameter against E. coli ATCC 25922 as well as antioxidant activity. Ultrafiltrate was further purified by solid phase extraction and then subjected to reverse phase chromatography. Among 12 fractions collected, higher activity containing fractions were sequenced through LC–MS and characterized. Total 49 peptide sequences identified including 13 novel sequences rich in proline with helix forming ability. Higher antimicrobial activity containing fractions have potent previously reported Casicidin-17 peptides along with a series of proline rich peptides. Antioxidant rich peptides profile contains 21 peptide of smaller sequence of mainly 9–12 amino acids with lower molecular weight. This study demonstrates the capacity of L. rhamnosus C6 to release antioxidative and antimicrobial peptide by proteolysis of milk proteins through peptide profiling and characterization.     

Isolation Purification and Characterization of Antimicrobial Peptides from <Emphasis Type="Italic">Cuminum cyminum</Emphasis> L. Seeds

In this work, antimicrobial peptides from Cuminum cyminum L. seeds were isolated and purified for the first time by 50% ethanol extraction, C18 reverse phase column chromatography and ion exchange chromatography for separation different peptides fraction. Then isolated fractions were characterized by Gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography and the peptides components and molecular weights were determined by liquid chromatography and mass spectrometry. The extracts were tested against some strains of bacteria (E. coli and Staphylococcus aureus) and one strain of fungi (Candida albicans) using well diffusion and broth dilution assays. The extracts from C. cyminum L. seeds demonstrated a high degree of activity (some antibacterial effect) against the bacteria strains and аntifungal activity against the Candida albicans. However, the study indicates that the crude peptide extracts from C. cyminum L. seeds have promising antimicrobial and antioxidant activities that can be harnessed as leads for potential bioactive compounds.     

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> activity

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.     

Antimicrobial peptides produced by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp.: structure,classification and bioactivity: a mini review

Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Identification and Characterization of Novel Antimicrobial Peptide from <Emphasis Type="Italic">Hippocampus comes</Emphasis> by In Silico and Experimental Studies

Antimicrobial peptides (AMPs) have attracted attentions as a novel antimicrobial agent because of their unique activity against microbes. In the present study, we described a new, previously unreported AMP, moronecidin-like peptide, from Hippocampus comes and compared its antimicrobial activity with moronecidin from hybrid striped bass. Antibacterial assay indicated that gram-positive bacteria were more sensitive to moronecidin and moronecidin-like compared with gram-negative bacteria. Furthermore, both AMPs were found to exhibit effective antifungal activity. Comparative analysis of the antimicrobial activity revealed that moronecidin-like peptide has higher activity against Acinetobacter baumannii and Staphylococcus epidermidis relative to moronecidin. Both moronecidin-like and moronecidin peptides retained their antibacterial activity in physiological pH and salt concentration. The time-killing assay showed that the AMPs completely killed A. baumannii and S. epidermidis isolates after 1 and 5 h at five- and tenfold above their corresponding MICs, respectively. Anti-biofilm assay demonstrated that peptides were able to inhibit 50% of biofilm formation at sub-MIC of 1/8 MIC. Furthermore, moronecidin-like significantly inhibited biofilm formation more than moronecidin at 1/16 MIC. Collectively, our results revealed that antimicrobial and anti-biofilm activities of moronecidin-like are comparable to moronecidin. In addition, the hemolytic and cytotoxic activities of moronecidin-like were lower than those of moronecidin, suggesting it as a potential novel therapeutic agent, and a template to design new therapeutic AMPs.     

A New Broad-Spectrum Peptide Antibiotic Produced by <Emphasis Type="Italic">Bacillus brevis</Emphasis> Strain MH9 Isolated from Margalla Hills of Islamabad,Pakistan

The antimicrobial peptide from a bacterial strain is isolated from soil sample of Margalla Hills of Islamabad, Pakistan. The peptide is found to significantly inhibit the growth of both Gram-positive (Staphylococcus aureus ATCC 6538 and Micrococcus luteus ATCC 10240) and Gram-negative (Escherichia coli ATCC 25922 and Salmonella typhi ATCC 14028) bacteria as compared to gramicidin as standard. The bacterium is identified as Bacillus brevis strain MH9 based on phenotype and phylogenetic analysis. The antibacterial polypeptide was produced optimally at 35 °C after 48 h of growth, precipitated by 50 % ammonium sulphate, and further purified using HPLC. The sequential steps of purification decrease the peptide contents with prominent antibacterial activity. The peptide composed of 11 amino acid was further characterized by FT-IR and NMR. Results suggested that the peptide molecule is a novel antibacterial agent that is effective against both Gram-positive and Gram-negative bacteria. This study may have important implications for new peptide antibiotic that could be a new addition to treat infections.     

Antimicrobial Activities of Novel Peptides Derived from Defensin Genes of <Emphasis Type="Italic">Brassica hybrid</Emphasis> cv Pule

Plant defensins are small and basic antimicrobial peptides characterized by conserved cysteine stabilizing structure with α-helix and triple strand antiparallel β-sheet. In the present study, two novel defensin genes, designated as BhDef1 and BhDef2, was isolated from Brassica hybrid cv Pule, a native unexplored Brassicaceae species found in Thailand. The full-length cDNA of BhDef1 and BhDef2 were 240 and 258 bp encoding a 79 and 85 amino acid residues with 29 and 25 amino acid signal peptide at N-terminal, respectively. The putative BhDef1 and BhDef2 mature proteins showed significant similarity to other Brassicaceae defensins. Their secondary structure comprises of one α-helix and a triple stranded β-sheet stabilized by four disulphide bridges of eight cysteines. BhDef1 and BhDef2 also contain a highly conserved γ-core and α-core motif exhibiting antifungal activity against Colletotrichum gloeosporioides causing anthracnose disease. Six out of eight synthetic BhDef peptide derivatives showed antibacterial activity against both gram-positive bacteria and gram-negative bacteria used in this study. BhDef14, the derivative of BhDef1, showed the highest activity against two test pathogenic bacteria. This activity could probably due to a net positively charge and alpha-helical conformation which are known as the key determinant for the bacterial membrane disruption. To our knowledge, this is the first report on defensin genes isolated from B. hybrid cv Pule. The synthetic peptides designed from their sequences showed antifungal and antibacterial activity.     

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> 607 Isolated from Persimmon

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C₁₈ cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH₂-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Effect of selenium-containing biocomposites based on <Emphasis Type="Italic">Ganoderma</Emphasis> mushroom isolates grown in the presence of oxopropyl-4-hydroxycoumarins,on bacterial phytopathogens

Effect of selenium-containing biocomposites obtained from submerged cultures of macrobasidiomycetes Ganoderma applantum, G. cattienensis, G. colossus G. lucidum, G. neojaponicum, and G. valesiacum, on plant pathogenic bacteria Clavibacter michiganensis ssp. sepedonicus (Cms), Micrococcus luteus, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas fluorescens, Pseudomonas viridiflava, and Xanthomonas campestris was studied. Oxopropyl-4-hydroxychromenones were used as components of the fungal nutrient media. The bacteriostatic and bactericidal activity of the Se-containing and Se-free substances of fungal origin against plant pathogenic bacteria was determined using colony-forming units count, the agar well diffusion method, and by turbidity measurements of bacterial suspensions. The composites produced from the extracellular metabolites of G. cattienensis SIE1302 with 4-hydroxy-3-(3-oxo-1,3-diphenyl propyl)-chromen-2-one (S(45)), and of G. lucidum SIE1303 with 4-hydroxy-3-(3-oxo-1-(3-nitrophenyl)-3-phenylpropyl)-chromen-2-one (S(NO₂)) possessed the most pronounced antibacterial action against Cms. The composites produced from the isolates of G. valesiacum 120702 with S(NO₂) showed the maximal antibacterial activity against Xanthomonas campestris B-610. High antimicrobial effect of G. lucidum 1315 with S(NO₂) against Xanthomonas campestris B-610 and of G. colossus SIE1301 against Pseudomonas fluorescens EL-2.1 was revealed. The pioneering information on the biological activity of coumarin series compounds in their application for producing the substances of fungal origin was obtained.     

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide,Hepcidin (<Emphasis Type="Italic">Le</Emphasis>-Hepc), from <Emphasis Type="Italic">Leiognathus equulus</Emphasis> and Analysis of Its Functional Properties In Silico

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.     

Genes encoding hevein-like antimicrobial peptides WAMPs in the species of the genus <Emphasis Type="Italic">Aegilops</Emphasis> L.

Earlier, in the wheat Triticum kiharae Dorof. et Migusch., a new family of genes coding for the hevein-like antimicrobial peptides WAMPs, involved in the protection of wheat plants against pathogens, was discovered. In the present study, we examined the wamp homologs in plants belonging to ten di-, tetra-, and hexaploid species of the genus Aegilops L., among which there are donors of polyploid wheat genomes, as well as of the resistance genes to the most important wheat pathogens. Using PCR amplification with genomic DNA as a template and primers specific to the sequences of the wheat wamp genes, for the first time, nucleotide sequences of the protein-coding regions of wamp homologs were determined in the species of the genus Aegilops L. The wamp homologs were found in all species studied. It was demonstrated that the WAMP peptide precursors encoded by them differed in single nucleotide substitutions, as well as deletions/insertions of amino acid sequences. The most conserved region of the precursor is the mature peptide region, where, in addition to the variable position 34, deletions of amino acid sequences were found in a number of peptides. To elucidate the role of deletions in the antimicrobial activity of WAMPs, a recombinant WAMP-3 peptide with a deletion in the N-terminal region was produced by expression in E. coli cells, and it was shown that antimicrobial activity of the peptide was preserved. It was demonstrated that all the discovered wamp genes were expressed in seedlings of the studied Aegilops species. The results shed new light on the structural diversity of genes encoding the hevein-like antimicrobial peptides WAMPs.     

Fish Heat Shock Cognate 70 Derived AMPs <Emphasis Type="Italic">Cs</Emphasis>HSC70 A1 and <Emphasis Type="Italic">Cs</Emphasis>HSC70 A2

Heat shock cognate 70 (HSC70) is an important evolutionary conserved protein that plays a major role in maintaining the homeostasis and immunity of many organisms. In this study, a HSC70 from Channa striatus was identified from its cDNA library and characterized using bioinformatics and molecular biology tools. CsHSC70 cDNA was 1953 base pair (bp) in length along with an open reading frame which encoded a polypeptide of 650 amino acid residues. Tissue distribution results showed that CsHSC70 was considerably expressed in gill, to a lesser extent in head kidney, blood, spleen and liver and at low level in other tissues. Using C. striatus gill as cell model, effects of fungal, bacterial and poly I:C stimulant on the mRNA levels of CsHSC70 was examined. We also described the antimicrobial features of two peptides namely CsHSC70 A1and CsHSC70 A2 derived from the N-terminal of CsHSC70 protein. CsHSC70 A1 peptide (40 µg/ml) exhibited potent bactericidal activity against Micrococcus luteus cells. Flow cytometric analysis revealed that the M. luteus cells stained with propidium iodide, upon treated with CsHSC70 A1 at the concentration of 40 µM/ml showed 38% survival compared to its control (99.6%). It seems that CsHSC70 A1 peptide shows antimicrobial activity against M. luteus through membrane disruption. Additionally, scanning electron microscope (SEM) observation confirmed that CsHSC70 A1 peptide treatment completely damaged and destructed the M. luteus cells. Taken together, these findings suggest that CsHSC70 A1 peptide could be a safe and potential therapeutic molecule substitute to antibiotics in various clinical fields.     

In silico design of polycationic antimicrobial peptides active against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Antimicrobial peptides (AMPs) have the potential to become valuable antimicrobial drugs in the coming years, since they offer wide spectrum of action, rapid bactericidal activity, and low probability for resistance development in comparison with traditional antibiotics. The search and improvement of methodologies for discovering new AMPs to treat resistant bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are needed for further development of antimicrobial products. In this work, the software Peptide ID 1.0^® was used to find new antimicrobial peptide candidates encrypted in proteins, considering the physicochemical parameters characteristics of AMPs such as positive net charge, hydrophobicity, and sequence length, among others. From the selected protein fragments, new AMPs were designed after conservative and semi-conservative modifications and amidation of the C-terminal region. In vitro studies of the antimicrobial activity of the newly designed peptides showed that two peptides, P3-B and P3-C, were active against P. aeruginosa Escherichia coli and A. baumannii with low minimum inhibitory concentrations. Peptide P3-C was also active against K. pneumoniae and S. aureus. Furthermore, bactericidal activity and information on the possible mechanisms of action are described according to the scanning electron microscopy studies.     

Antibacterial and antitumor activity of Bogorol B-JX isolated from <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> JX-5

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5 µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5 µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.