Cysteine-rich Antimicrobial Peptides Research Articles

Identification and molecular characterization of two liver expressed antimicrobial peptides (LEAPs) in Amur grayling (Thymallus arcticus grubei Dybowski, 1869)

Antimicrobial peptides (AMPs) are part of the innate immune response against bacteria. Liver expressed antimicrobial peptides (LEAPs) are one of the most important antimicrobial peptides in fish and belong to a family of cysteine-rich antimicrobial peptides with antimicrobial activity against a variety of pathogens. In this study, we cloned and identified two liver expressed antimicrobial peptides (TaLEAP-1, TaLEAP-2) from Thymallus arcticus grubei, functional studies were performed and the two synthesized TaLEAPs peptides were examined in vitro bacterial inhibition. The results showed that the open reading frame lengths of TaLEAP-1 and TaLEAP-2 were 267 bp and 258 bp, respectively. TaLEAP-1 is a β-sheet polypeptide containing 89 amino acids, and TaLEAP-2 is an α-helix with β-sheet polypeptide containing 85 amino acids. Tissue distribution showed that both TaLEAPs were widely expressed in ten tissues of T. arcticus grubei, with the highest expression in the liver. During the bacterial challenge with Aeromonas salmonicida and Aeromonas hydrophila, two LEAPs genes were differentially expressed. TaLEAP-1 has broad-spectrum antibacterial activity against a variety of bacteria. TaLEAP-1 damages bacteria by disrupting cell membranes, showing that TaLEAP-1 has potent antibacterial effects. Cytotoxicity assay results showed that TaLEAP-1 was not toxic and TaLEAP-2 was slightly toxic to RTG-2. The results of this study provide strategies for resistance to bacterial and fungal infections in T. arcticus grubei.

Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-β (IL-1β), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

From Gene to Transcript and Peptide: A Deep Overview on Non-Specific Lipid Transfer Proteins (nsLTPs).

Non-specific lipid transfer proteins (nsLTPs) stand out among plant-specific peptide superfamilies due to their multifaceted roles in plant molecular physiology and development, including their protective functions against pathogens. These antimicrobial agents have demonstrated remarkable efficacy against bacterial and fungal pathogens. The discovery of plant-originated, cysteine-rich antimicrobial peptides such as nsLTPs has paved the way for exploring the mentioned organisms as potential biofactories for synthesizing antimicrobial compounds. Recently, nsLTPs have been the focus of a plethora of research and reviews, providing a functional overview of their potential activity. The present work compiles relevant information on nsLTP omics and evolution, and it adds meta-analysis of nsLTPs, including: (1) genome-wide mining in 12 plant genomes not studied before; (2) latest common ancestor analysis (LCA) and expansion mechanisms; (3) structural proteomics, scrutinizing nsLTPs' three-dimensional structure/physicochemical characteristics in the context of nsLTP classification; and (4) broad nsLTP spatiotemporal transcriptional analysis using soybean as a study case. Combining a critical review with original results, we aim to integrate high-quality information in a single source to clarify unexplored aspects of this important gene/peptide family.

Identification of a Double-β-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

β-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian β-defensin 11 (AvBD11) is unique, with two β-defensin motifs with a broad range of antimicrobial activities. However, a double-sized β-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-β-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to β-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD invivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-β-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

Expression and functional characterization of three β-defensins in grass carp (Ctenopharyngodon idella)

β-defensins (BDs) are a group of cysteine-rich cationic antimicrobial peptides and play important roles in the first line of defense against infection. In this study, the expression and antibacterial activities of three grass carp (Ctenopharyngodon idella) (Ci) β-defensin (BD) peptides were comparatively investigated. Expression analysis reveals that CiBD1-3 were constitutively expressed in tissues, with the highest expression detected in the skin. The CiBD-1 transcripts were more abundant than CiBD-2 and CiBD-3. In the primary head kidney leukocytes, CiBDs were induced by PHA, LPS, poly(I:C) and cytokines such as IL-1β and IFN-γ. In vivo challenge of fish with Aeromonas hydrophila resulted in the up-regulation of CiBDs in the head kidney and hindgut. To determine the biological activities, recombinant CiBD proteins were produced in the HEK293-F cells and purified for the minimum inhibitory concentration assay. It was found that all three recombinant CiBD proteins were effective to inhibit the growth of Gram-negative fish bacterial pathogens including Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare and Klebsiella pneumoniae and Gram-positive Staphylococcus aureus. CiBD-2 and CiBD-3 were more effective than CiBD-1. Our results demonstrate that all the three CiBDs have broad antibacterial activity against fish bacterial pathogens.

Antifungal Activity of Avocado Seed Recombinant GASA/Snakin PaSn.

The avocado fruit (Persea americana) has become a significant fruit in the human diet for its nutritional properties. However, the seed is a source of bioactive molecules and has been poorly utilized. Previously, we reported that the PaSn gene is expressed in the avocado seeds, a cysteine-rich antimicrobial peptide (GASA/Snakin), and demonstrated its antibacterial activity. In this work, we report the recombinant production of PaSn in the Escherichia coli system and evaluate its antifungal activity against plant and human pathogen fungi. The recombinant peptide showed antifungal activity at 200 μg/mL against phytopathogens Colletotrichum gloeosporioides and Fusarium oxysporum and human pathogens Candida albicans and C. glabrata. Our results demonstrate the usefulness of a prokaryotic expression system for avocado antimicrobial peptide production. In conclusion, the snakin PaSn could be helpful in the control of postharvest avocado and other fruits' fungal diseases and human fungal pathogens.

Antimicrobial peptide hepcidin contributes to restoration of the intestinal flora after Aeromonas hydrophila infection in Acrossocheilus fasciatus

Hepcidin is a cysteine-rich antimicrobial peptide that serves an important role in the immunity system of fishes. It exhibits antibacterial, antifungal, antiviral, and antitumor activities. However, the exact role of fish hepcidin in the regulation of the intestinal flora still remains a mystery. In our study, we sequenced and characterized hepcidin from the liver of Acrossocheilus fasciatus. Phylogenetic tree analysis showed that A. fasciatus hepcidin and Gobiocypris rarus hepcidin were the most closely related, and both belonged to the fish HAMP1 cluster. Studies conducted on in vivo tissue distribution showed that the expression of hepcidin was highest in healthy A. fasciatus liver. Aeromonas hydrophila infection was confirmed by the increased expression of pro-inflammatory cytokine genes and bacterial loads in A. fasciatus tissues. After A. hydrophila infection, hepcidin expression significantly increased in the liver, spleen, and head kidney. In vitro antibacterial assays showed that hepcidin exhibits strong broad spectrum antibacterial activity. Furthermore, we examined the regulatory effect of hepcidin on the intestinal flora and found that A. fasciatus hepcidin restored the reduced diversity and compositional changes in intestinal flora caused by A. hydrophila infection. Our results suggest that hepcidin could regulate the intestinal flora in fishes; however, the underlying mechanisms need to be explored in greater detail.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization

BackgroundIncrease of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. ResultsOpen reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. ConclusionFunctional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Structural and functional characterizations and heterogenous expression of the antimicrobial peptides, Hidefensins, from black soldier fly, Hermetia illucens (L.)

Insect defensins are effector components of the innate defense system. Defensins, which are widely distributed among insects, are a type of small cysteine-rich plant antimicrobial peptides with broad-spectrum antimicrobial activity. Here, the cDNAs of the black soldier fly, Hermetia illucens (L.), encoding six defensins, designated herein as Hidefensin1-1, 2, 3, 4, 5, 6. Moreover, Hidefensin1-1, 2, and 5 were identified for the first time by genome-targeted analysis. These Hidefensins were found to mainly adopt α-helix and β-sheet conformation homology as modeled by PRABI, Swiss-Model and ProFunc server. Six conserved cysteine residues that contribute to three disulfide bonds formed the spacing pattern “C-X12-C-X3-C-X9-C-X5-C-X-C”, which play a vital role in the molecular stability of Hidefensins. Phylogenetic analysis revealed that the homology of five Hidefensins (except Hidefensin4) was about 59%–92% compared with other insect defensins, indicating that they are novel antimicrobial peptides genes in black soldier fly. Furthermore, the Hidefensin1-1 was expressed in the Escherichia coli strain BL21(DE3) as a fusion protein with thioredoxin. Results showed that the purified TRX-Hidefensin1-1 exerted strong inhibitory effects against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli. The inhibitory efficacy of TRX-Hidefensin1-1 against Gram-positive bacteria was better than that against Gram-negative bacteria. These results indicated that Hidefensin1-1 has potent antimicrobial activities against test pathogens.

Mass Spectrometric Identification of Antimicrobial Peptides from Medicinal Seeds.

Traditional medicinal plants contain a variety of bioactive natural products including cysteine-rich (Cys-rich) antimicrobial peptides (AMPs). Cys-rich AMPs are often crosslinked by multiple disulfide bonds which increase their resistance to chemical and enzymatic degradation. However, this class of molecules is relatively underexplored. Herein, in silico analysis predicted 80–100 Cys-rich AMPs per species from three edible traditional medicinal plants: Linum usitatissimum (flax), Trifolium pratense (red clover), and Sesamum indicum (sesame). Bottom-up proteomic analysis of seed peptide extracts revealed direct evidence for the translation of 3–10 Cys-rich AMPs per species, including lipid transfer proteins, defensins, α-hairpinins, and snakins. Negative activity revealed by antibacterial screening highlights the importance of employing a multi-pronged approach for AMP discovery. Further, this study demonstrates that flax, red clover, and sesame are promising sources for further AMP discovery and characterization.

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Antimicrobial Peptides: Identification of two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax).

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

Characterization of antibacterial activities and the related mechanisms of a β-defensin in turbot (Scophthalmus maximus)

β-defensins (BDs) are small-molecule cysteine-rich cationic antimicrobial peptides that play an important role in innate immunity against microbial invasion. In this study, a β-defensin (SmBD) was identified and characterized from turbot (Scophthalmus maximus). SmBD has an open reading frame containing 192 bp and encodes a protein of 63 amino acids. It has a predicted signal peptide consisting of 20 amino acids and a mature peptide with six conserved cysteine residues forming three disulfide bonds (C1- C5, C2-C4 and C3-C6). The 3D structure prediction shows an N-terminal α-helix and three reverse parallel β-folds. SmBD showed high sequence identity (70.45% - 95.24%) with β-defensin 2 group members from other teleost species. SmBD had the highest expression in skin of healthy turbot and was significantly up-regulated in mucosal tissues (skin, gill, intestine) after bacteria challenge. SmBD showed antibacterial activity against Vibrio anguillarum, Edwardsiella tarda, Escherichia coli and Streptococcus iniae, and inhibitory effect on the formation of bacterial biofilms. SmBD changed bacterial membrane permeability and affected bacterial morphology, as shown in fluorescence microscopy and scanning electron microscopy analyses, respectively, which indicated that SmBD might exercise antibacterial behaviors via “membrane-attack” mechanism.

Comparative Genomics Reveals 13 Different Isoforms of Mytimycins (A-M) in Mytilus galloprovincialis.

Mytimycins are cysteine-rich antimicrobial peptides that show antifungal properties. These peptides are part of the immune network that constitutes the defense system of the Mediterranean mussel (Mytilus galloprovincialis). The immune system of mussels has been increasingly studied in the last decade due to its great efficiency, since these molluscs, particularly resistant to adverse conditions and pathogens, are present all over the world, being considered as an invasive species. The recent sequencing of the mussel genome has greatly simplified the genetic study of some of its immune genes. In the present work, we describe a total of 106 different mytimycin variants in 16 individual mussel genomes. The 13 highly supported mytimycin clusters (A–M) identified with phylogenetic inference were found to be subject to the presence/absence variation, a widespread phenomenon in mussels. We also identified a block of conserved residues evolving under purifying selection, which may indicate the “functional core” of the mature peptide, and a conserved set of 10 invariable plus 6 accessory cysteines which constitute a plastic disulfide array. Finally, we extended the taxonomic range of distribution of mytimycins among Mytilida, identifying novel sequences in M. coruscus, M. californianus, P. viridis, L. fortunei, M. philippinarum, M. modiolus, and P. purpuratus.

Expression of antimicrobial peptide snakin-1 confers effective protection in rice against sheath blight pathogen, Rhizoctonia solani

Rice sheath blight disease caused by the necrotrophic fungus Rhizoctonia solani, is an economically detrimental fungal disease which significantly affects rice productivity worldwide. Potato snakin-1, a cysteine-rich basic antimicrobial peptide (AMP), is a member of the novel Snakin AMP family. In order to assess the ability of the snakin-1 peptide in protecting rice against the sheath blight disease, we have developed transgenic rice constitutively expressing the snakin-1 peptide. The antimicrobial activity of snakin-1 was evaluated against the sheath blight pathogen Rhizoctonia solani both in vitro and in planta. Crude protein from transgenic rice leaves showed in vitro antifungal activity against Rhizoctonia solani. Moreover, in planta bioassay results also confirmed the same, wherein snakin-1 expressing rice plants showed significantly enhanced protection against the sheath blight disease. This report demonstrates how a member of the Snakin family of antimicrobial peptides has been successfully used to generate sheath blight resistance in rice, without compromising on its agronomic characteristics and at no phenotypic cost.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells

Aims: Defensins are cysteine-rich antimicrobial cationic peptides and BAX is a proapoptotic gene that can cause cell death. This study aimed to investigate the effect of cellular extract of co-cultured Lactobacillus casei on the expression of BAX and human β-defensin 2 (hBD-2) genes in HT29 cells. Methods & Materials: This experimental study was conducted in the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in 2017. The HT29 cell line was obtained from the Pasteur Institute of Iran, and cells were assessed using Microculture Tetrazolium Test (MTT) after culturing. DNA was extracted from the treated cells, and then the DNA ladder assay was carried out. After preparing cDNA, the expression levels of BAX and hBD-2 genes in the HT29 cell line were measured using a real-time Polymerase Chain Reaction (PCR) method. Findings: The results of the MTT assay indicated that Lactobacillus casei inhibited the proliferation of HT29 cells and induced apoptosis in these cells. Results of DAPI staining and DNA ladder assay obtained from treating HT29 cells by Lactobacillus casei showed qualitative changes in cell apoptosis. Moreover, realtime PCR results indicated that Lactobacillus casei bacteria significantly increased the expression of the hBD-2 gene in HT29 colon cancer cells within 12-24 hours (P= 0.023), while BAX gene expression showed no significant change in the first 24 hours (P= 0.37). Conclusion: The extract of Lactobacillus casei can be used to stimulate cancer cells to produce β-defensins, inhibit pathogens, prevent the stimulation of cellular signaling, and fight antibiotic-resistant bacteria.

Cysteine-rich antimicrobial peptides from plants: The future of antimicrobial therapy.

There has been a spurt in the spread of microbial resistance to antibiotics due to indiscriminate use of antimicrobial agents in human medicine, agriculture, and animal husbandry. It has been realized that conventional antibiotic therapy would be less effective in the coming decades and more emphasis should be given for the development of novel antiinfective therapies. Cysteine rich peptides (CRPs) are broad-spectrum antimicrobial agents that modulate the innate immune system of different life forms such as bacteria, protozoans, fungi, plants, insects, and animals. These are also expressed in several plant tissues in response to invasion by pathogens, and play a crucial role in the regulation of plant growth and development. The present work explores the importance of CRPs as potent antimicrobial agents, which can supplement and/or replace the conventional antibiotics. Different plant parts of diverse plant species showed the presence of antimicrobial peptides (AMPs), which had significant structural and functional diversity. The plant-derived AMPs exhibited potent activity toward a range of plant and animal pathogens, protozoans, insects, and even against cancer cells. The cysteine-rich AMPs have opened new avenues for the use of plants as biofactories for the production of antimicrobials and can be considered as promising antimicrobial drugs in biotherapeutics.

Panax notoginseng snakin gene increases resistance to Fusarium solani in transgenic tobacco

Snakins are cysteine-rich plant antimicrobial peptides that are important for plant defense responses to diverse pathogens, and a snakin gene, PnSN1, was cloned from Panax notoginseng to explore the role of snakin in the disease resistance mechanism of P. notoginseng. A gene expression analysis by quantitative real-time PCR revealed that PnSN1 expression was responsive to a Fusarium solani infection and was induced by four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide). A subcellular localization revealed that the PnSN1-GFP fusion protein was present only in the plant cell wall. Moreover, recombinant PnSN1 expressed in Escherichia coli was inhibitory toward the mycelial growth of four plant pathogenic fungi (F. solani, F. oxysporum, F. verticillioides, and Botryosphaeria dothidea), with increasing protein concentrations resulting in greater antifungal activities. The recombinant protein also inhibited F. solani spore germination. Additionally, the overexpression of PnSN1 in the model plant tobacco considerably increased the resistance to F. solani. Furthermore, the transient expression of the PnSN1 RNA interference construct increased the susceptibility of young P. notoginseng leaves to F. solani. These findings confirmed the importance of PnSN1 for the disease resistance of P. notoginseng.