Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation.

Candida albicans is a commensal organism and opportunistic pathogen that can form biofilms that colonize surfaces of medical devices, such as implants, catheters, and dentures. Compared to planktonic C. albicans cells, cells in biofilms exhibit increased resistance to treatment. Histatin 5 (Hst-5) is an antimicrobial peptide that is natively secreted by human salivary glands and has strong antifungal activity against C. albicans. However, C. albicans produces secreted aspartic proteases (Saps) that can cleave and inactivate Hst-5, limiting its antifungal properties. We previously showed that residue substitutions K11R and K17R within Hst-5 improve its antifungal activity and prevent proteolytic degradation by Saps when treating planktonic C. albicans. Here, we investigated the use of the K11R-K17R peptide as an alternative therapeutic against C. albicans biofilms by assessing its ability to reduce viability of pre-formed biofilms and to inhibit the formation of biofilms and showed that K11R-K17R had improved activity compared to Hst-5. Based on these results, we incorporated K11R-K17R and Hst-5 into polyelectrolyte multilayer (PEM) surface coatings and demonstrated that films functionalized with K11R-K17R reduced the formation of C. albicans biofilms. Our results demonstrate the therapeutic potential of the K11R-K17R Hst-5 variant in preventing and treating biofilms.

Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies.

Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of histatin 1, which are discussed here. Furthermore, we put forward a possible role of histatin 1 in various pathologies and its potential function in clinical applications.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Antimicrobial peptide gene expression in periodontitis patients: A pilot study.

AIM: Antimicrobial peptides (AMPs) are one of the most active components of innate immunity and have characteristics that could place them at the heart of the pathogenesis of periodontal disease. This study investigated differences in the expression of AMP coding genes obtained using a simple harvesting technique, gingival smear, between two groups of patients: chronic periodontitis subjects versus healthy ones. MATERIALS AND METHODS: Twenty-three patients were enrolled in two groups: 12 were diagnosed with moderate or severe generalized chronic periodontitis, and 11 were diagnosed as clinically healthy. Gingival smears were retrieved and studied using reverse transcription-quantitative PCR (RT-qPCR) after mRNA purification. RESULTS: Fifteen gene expressions were obtained using real-time RT-qPCR. Three AMP genes, histatin 3 (HTN3), α-defensin 4 (DEFA4) and lysozyme C (LYZ), presented different expression levels in periodontitis patients compared with healthy subjects. The relative expression level of DEFA4 appeared to be a protective factor against periodontitis. CONCLUSION: Gingival smears studied by RT-qPCR may be used to assess the expression of AMPs coding genes. A lack of expression of DEFA4 could be a potential indicator of periodontitis status.

High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli.

P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.

Practical evaluation of an RNA-based saliva identification method.

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.

The novel Candida albicans transporter Dur31 Is a multi-stage pathogenicity factor.

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine.

Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

BACKGROUND: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue. OBJECTIVES: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system. RESULTS: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of ß -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system. CONCLUSION: Antimicrobial peptides Histatin 1, ß -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Candida albicans flu1-mediated efflux of salivary histatin 5 reduces its cytosolic concentration and fungicidal activity.

Histatin 5 (Hst 5) is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicidal activity of Hst 5 requires intracellular translocation and accumulation to a threshold concentration for it to disrupt cellular processes. Previously, we observed that total cytosolic levels of Hst 5 were gradually reduced from intact cells, suggesting that C. albicans possesses a transport mechanism for efflux of Hst 5. Since we identified C. albicans polyamine transporters responsible for Hst 5 uptake, we hypothesized that one or more polyamine efflux transporters may be involved in the efflux of Hst 5. C. albicans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermidine efflux transporter, indicating that the products of these two genes may be involved in efflux of Hst 5. We found that flu1Δ/Δ cells, but not tpo2Δ/Δ cells, had significant reductions in their rates of Hst 5 efflux and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibilities than did the wild type. We also found that flu1Δ/Δ cells had reduced biofilm formation compared to wild-type cells in the presence of Hst 5. Transcriptional levels of FLU1 were not altered over the course of treatment with Hst 5; therefore, Hst 5 is not likely to induce FLU1 gene overexpression as a potential mechanism of resistance. Thus, Flu1, but not Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans.

Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli.

Human histatin 1 (Hst1), a member of the histatin family, possesses antimicrobial properties. In this study, we applied a previously developed cleavable self-aggregating tag (cSAT) for the expression and purification of histatin 1 to demonstrate its utility for peptide expression and purification. The tag consists of a self-cleavable intein and a self-assembling peptide ELK16 (I-ELK16). First, an active insoluble aggregate of the recombinant histatin 1-Mxe GyrA intein-ELK16 (Hst1-I-ELK16) fusion protein was produced with a yield of 28.9 µg/mg wet cell pellet. The thiol reagent dithiothreitol (DTT) was then used to induce the intein-mediated cleavage and peptide release into the soluble fraction with a yield of 2.06 µg/mg wet cell pellet and a purity of 70%. The peptide was further purified by high performance liquid chromatography. These results were comparable to the yield and purity achieved when the more conventional glutathione transferase (GST) tag was used. The antimicrobial activities of this recombinant histatin 1 were confirmed against three Candida strains. This cSAT technique offers considerable advantages in terms of its simplicity and speed, eliminating the need for an exogenous protease, and reducing the number of chromatography purification steps. This technique should also be useful for the expression and purification of other AMPs.

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening.

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

Sortase A as a tool for high-yield histatin cyclization.

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 µM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.

Histatin-1 is an endogenous ligand of the sigma-2 receptor.

The Sigma-2 receptor (S2R) (a.k.a TMEM97) is an important endoplasmic reticular protein involved in cancer, cholesterol processing, cell migration, and neurodegenerative diseases, including Niemann-Pick Type C. While several S2R pharmacologic agents have been discovered, its recent (2017) cloning has limited biological investigation, and no endogenous ligands of the S2R are known. Histatins are a family of endogenous antimicrobial peptides that have numerous important effects in multiple biological systems, including antifungal, antibacterial, cancer pathogenesis, immunomodulation, and wound healing. Histatin-1 (Hst1) has important roles in epithelial wound healing and cell migration, and is the primary wound healing agent in saliva. Little is understood about the downstream machinery that underpins the effects of histatins, and no mammalian receptor is known to date. In this study, we show, using biophysical methods and functional assays, that Hst1 is an endogenous ligand for S2R and that S2R is a mammalian receptor for Hst1.

Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall V(max)/K(m) ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1. Mathematical models predict that histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 microM, representing 59, 27, and 11% of glandular histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K(13) and K(17) in histatin 1, R(22), Y(24), and R(25) in histatin 3, and Y(10), K(11), R(12), K(13), H(15), E(16), K(17), and H(18) in histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.

A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection.

Messenger RNA profiling for body fluid identification (bfID) is a useful approach to collect contextual information associated with a crime. Current methods require costly fluorescent probes, lengthy amplification protocols and/or time-consuming sample preparation. To simplify this process, we developed a bfID method that has the potential to be rapid in analysis time, inexpensive and fluorescence-free, combining a universal operating procedure with a high-throughout (microwell plate) platform for simultaneous detection of mRNA markers from whole blood, semen, saliva, and vaginal fluid. Full bfID sample preparation and analysis of 23 samples was completed in under 3 h using smart phone optical detection and analysis and show efficacy of the method in a validated blind study. The results provide an efficient, sensitive and specific approach to supplement the current biochemical tests in a forensic laboratory.

Polymorphism of salivary histatin gene and periodontal disease in the Japanese population.

Chronic periodontitis is a widespread and major dental disease. Recent studies have analyzed a possible relationship between polymorphism of several genes and periodontitis. Histatins are salivary polypeptides with fungicidal activities against Candida albicans and yeast and bactericidal activities against Porphyromonas gingivalis and Streptococcus mutans. Histatins are part of the innate defense of the oral cavity. We examined the frequency of the polymorphism codon 23 of the histatin 3 gene (HIS2 allele) in relation to periodontitis in the Japanese population. The subjects were 143 Japanese individuals, of which 63 were healthy control subjects and 80 were periodontal patients. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphism (SNP) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The incidence of polymorphism was analyzed statistically by Fisher's exact test. The results indicated that the gene polymorphism at codon 23 of the histatin 3 gene was not associated with periodontitis in the Japanese population (p = 0.166). Rather, if at all, it appeared to be associated with resistance to periodontitis.

The HTN3-MSANTD3 Fusion Gene Defines a Subset of Acinic Cell Carcinoma of the Salivary Gland.

The spectrum of tumors arising in the salivary glands is wide and has recently been shown to harbor a network of tumor-specific fusion genes. Acinic cell carcinoma (AciCC) is one of the more frequently encountered types of salivary gland carcinoma, but it has remained a genetic orphan until recently when a fusion between the HTN3 and MSANTD3 genes was described in one case. Neither of these 2 genes is known to be implicated in any other malignancy. This study was undertaken to investigate whether the HTN3-MSANTD3 fusion is a recurrent genetic event in AciCC and whether it is a characteristic of one of its histological variants. Of the 273 AciCCs screened, 9 cases showed rearrangement of MSANTD3 by break-apart fluorescence in situ hybridization, 2 had 1 to 2 extra signals, and 1 had gain, giving a total of 4.4% with MSANTD3 aberrations. In 6 of 7 available cases with MSANTD3 rearrangement, the HTN3-MSANTD3 fusion transcript was demonstrated with real-time polymerase chain reaction. Histologically, all fusion-positive cases were predominantly composed of serous tumor cells growing in solid sheets, with serous tumor cells expressing DOG-1 and the intercalated duct-like cell component being CK7 positive and S-100 positive in 6/9 cases. All but one case arose in the parotid gland, and none of the patients experienced a recurrence during follow-up. In contrast, the case with MSANTD3 gain metastasized to the cervical lymph nodes and lungs. In conclusion, we find the HTN3-MSANTD3 gene fusion to be a recurrent event in AciCC with prominent serous differentiation and an indolent clinical course.

Specific metallo-protein interactions and antimicrobial activity in Histatin-5, an intrinsically disordered salivary peptide.

Histatin-5 (Hst-5) is an antimicrobial, salivary protein that is involved in the host defense system. Hst-5 has been proposed to bind functionally relevant zinc and copper but presents challenges in structural studies due to its disordered conformation in aqueous solution. Here, we used circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy to define metallo-Hst-5 interactions in aqueous solution. A zinc-containing Hst-5 sample exhibits shifted Raman bands, relative to bands observed in the absence of zinc. Based on comparison to model compounds and to a family of designed, zinc-binding beta hairpins, the alterations in the Hst-5 UVRR spectrum are attributed to zinc coordination by imidazole side chains. Zinc addition also shifted a tyrosine aromatic ring UVRR band through an electrostatic interaction. Copper addition did not have these effects. A sequence variant, H18A/H19A, was employed; this mutant has less potent antifungal activity, when compared to Hst-5. Zinc addition had only a small effect on the thermal stability of this mutant. Interestingly, both zinc and copper addition shifted histidine UVRR bands in a manner diagnostic for metal coordination. Results obtained with a K13E/R22G mutant were similar to those obtained with wildtype. These experiments show that H18 and H19 contribute to a zinc binding site. In the H18A/H19A mutant the specificity of the copper/zinc binding sites is lost. The experiments implicate specific zinc binding to be important in the antimicrobial activity of Hst-5.

Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst-5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst-5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst-5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site-, residue-, and Sap-dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst-5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C-terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst-5 do not diminish the antifungal activity of Hst-5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst-5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy.

Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32-P113 with enhanced antimicrobial activity in vitro.

Antibiotic-resistant pathogens have become a major public health problem worldwide. New discoveries and strategies as regards antibiotic drug development are urgently in need for curing infected patients. Antimicrobial peptides (AMPs) are short cationic peptides that play important roles in innate immune system with a broad spectrum of antimicrobial activity. Recently, hybrid AMPs have been reported to increase antimicrobial activity, stability, and in vivo half-life. In the present study, a gene encoding for AL32-P113 hybrid peptide consisting of two truncated active forms of human LL-37 and histatin-5 (Hst-5) was commercially constructed, cloned into pTXB-1 commercial plasmid, and expressed in E. coli BL21 (DE3). To increase the yield of target protein expression, IPTG concentration, time and temperature were optimized. The results indicate that AL32-P113-intein fusion protein with 33.7 kDa was expressed mostly in inclusion form and estimated to be 20% of the total protein. After chitin affinity purification, 5.7-kDa of AL32-P113 peptide was separated with an average concentration of 12.1 mg per litre of bacterial culture and over 86% purity. The minimum inhibitory concentration (MIC) was evaluated for antimicrobial activity determination of recombinant AL32-P113 compared to synthetic peptides, LL-37, Hst-5, and L31-P113. The results implied that both hybrid peptides exhibited potent antimicrobial activity against gram-negative bacteria and yeast cells whereas the L31-P113 peptide possessed approximately four times greater antimicrobial activity in gram-positive bacteria than parent LL-37. An increasing of undesired hemolysis of these hybrid peptides toward human red cells was also observed when red blood cell hemolytic assay was performed. Several factors including charge and secondary structure predicted by public software were utilized for explanation of the antimicrobial potency of both hybrid peptides. This study proved that hybrid peptides show broader and more potent antimicrobial ability against pathogens and they could be applied as a therapeutic approach for topical treatment of microbial infection in the future.