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Last updated: 13th August 2010

Characterization of a New Defensin from Cowpea (Vigna unguiculata (L.) Walp.).
Padovan L, Segat L, Tossi A, Calsa T, Ederson AK, Brandao L, Guimarães RL, Pandolfi V, Pestana-Calsa MC, Belarmino LC, Benko-Iseppon AM, Crovella S
Department of Life Sciences, University of Trieste - Via Giorgieri, 1 - 34127 Trieste, Italy.
Protein Pept. Lett. 2010 Mar;17 (3):297-304
Using Phaseoleae defensins available in databases, a putative defensin gene was isolated in cowpea (Vigna unguiculata (L.) Walp.) and cloned from genomic cowpea DNA. The putative mature defensin sequence displays the characteristic defensins residues arrangement, secondary and tertiary structures were predicted and splicing analysis was performed. Using RT-PCR, defensin expression and differences in response to biotic stimuli between infected and non infected plants were tested.
Mechanisms and physiological effects of protamine resistance in Salmonella enterica serovar Typhimurium LT2.
Pränting M, Andersson DI
Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden.
J Antimicrob Chemother. 2010 May;65(5):876-87.
Objectives Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution of bacterial resistance to protamine sulphate and the resulting effects on fitness and physiology, with the objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides. Methods Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance mutations were identified using transposon insertions and DNA sequencing. Peptide susceptibility was determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as log-phase growth rates. Growth-compensated strains were isolated by serial passage through population bottlenecks followed by visual screening for large colonies. Results Protamine-resistant mutants appeared at a rate of 2.3 x 10(-7)/cell/generation. These mutants were 2-20 times more resistant to protamine than the parental strain and less susceptible to several other antimicrobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bacterial fitness. Some mutants could, in the absence of protamine, be evolved to higher fitness by acquiring second-site compensatory mutations. Conclusions Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infections that are difficult to treat and which could be a strategy for bacteria to escape the killing effects of antimicrobial peptides.
Bacteroides fragilis enterotoxin induces human {beta}-defensin-2 expression in intestinal epithelial cells via mitogen-activated protein kinase/I{kappa}B kinase/NF-{kappa}B- dependent pathway.
Yoon YM, Lee JY, Yoo D, Sim YS, Kim YJ, Oh YK, Kang JS, Kim S, Kim JS, Kim JM
Department of Microbiology, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea.
Infect Immun. 2010 May;78(5):2024-33
Enterotoxigenic Bacteroides fragilis (ETBF) produces an approximately 20 kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although spontaneous disappearance of ETBF infection is common, little information is available on regulated expression of antibacterial factors in response to BFT stimulation. This study investigates the role of BFT in human beta-defensin (hBD)-2 induction from intestinal epithelial cells. Stimulation of HT-29 and Caco-2 intestinal epithelial cell lines with BFT resulted in the induction of hBD-2. Activation of a reporter gene for hBD-2 was dependent on the presence of NF-kappaB binding sites. In contrast, suppression of AP-1 did not affect hBD-2 expression in BFT-stimulated cells. Inhibition of p38 mitogen-activated protein kinase (MAPK) using SB203580 and siRNA transfection resulted in a significant reduction in BFT-induced IKK/NF-kappaB activation and hBD-2 expression. Our results suggest that a pathway, including p38 MAPK, IKK, and NF-kappaB activation, is required for hBD-2 induction in intestinal epithelial cells exposed to BFT, and may be involved in the host defense following infection with ETBF.
[Immune effects of four coxsackievirus B3 VP1 DNA fusion vaccines in mice.]
Liu GX, Lan JM, Chuai X, Gao ZY, Jin YH, Zhang YH, Xie LX, Yin CF, Wang YX
Department of Pathogenic Biology, Hebei Medical University, Shijiazhuang 050017, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2010 Feb;26 (2):103-6
AIM: To compare the immunogenicity and protective effects on CVB3 infected mice of four DNA fusion vaccines coupling coxsackievirus B3 (CVB3) VP1 with macrophage-derived chemokine (MDC), C3d3, shiga toxin B subuit (STxB) and mouse beta-defensin-2 (mBD2), respectively. METHODS: BALB/c mice were divided into 6 groups randomly and inoculated in quadriceps at 3-week interval for 3 times with pcDNA3, pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1, respectively. Fourteen days after every inoculation, serum samples were collected and CVB3 specific neutralizing antibodies were determined. Three weeks after the last immunization, the mice were treated in three ways. First, the spleen cells were isolated from 3 mice of each group and specific CTL activities were tested. Second, 3 mice of each group were further challenged with 3LDLD(50); CVB3 and sacrificed 7 days later, and their blood viral titers were evaluated. Third, the rest mice of each group were subjected to intraperitoneal (i.p.) challenge with 5LDLD(50); CVB3 and their survival was observed. RESULTS: The neutralizing antibodies against CVB3 were induced in pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1 groups, and antibody titers correlated with the number of injections (P<0.01). After three immunizations, the antibody titers in pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2 -VP1 groups were higher than the ones in pcDNA3/VP1and pcDNA3/STxB-VP1 groups (P<0.01). The specific CTL activities in both pcDNA3/STxB-VP1 and pcDNA3/mBD2-VP1 groups were significantly stronger than those in the other groups (P<0.01). After CVB3 challenge, the blood viral titers in the pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2-VP1 groups were lower than those in the other groups (P<0.01), and the pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 mice survived longer than the others (P<0.05). CONCLUSION: Both pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 vaccines could induce stronger immune responses, resulting in higher survival rates and better protective effects on CVB3 infection than pcDNA3/STxB-VP1, pcDNA3/mBD2-VP1 and pcDNA3/VP1 vaccines.
Impact of low-frequency pulsed magnetic fields on defensin and CRP concentrations in patients with painful diabetic polyneuropathy and in healthy subjects.
Wróbel MP, Szymborska-Kajanek A, Strzelczyk JK, Karasek D, Rawwash HA, Biniszkiewicz T, Cieslar G, Hajdrowska B, Sieron-Stoltny K, Sieron A, Wiczkowski A, Grzeszczak W, Strojek K
Diabetological Unit of the Department of Internal Medicine, Diabetology and Nephrology, Medical University of Silesia, Zabrze, Poland.
Electromagn Biol Med. 2010 Jun;29(1-2):19-25.
AIM: The aim was to assess whether magnetic field influences defensin and CRP concentrations in patients with diabetic polyneuropathy and in healthy subjects. METHODS: 61 diabetic patients were randomly divided into 2 groups: study group-32 patients exposed to low-frequency magnetic field; and control group-29 patients with sham exposure. Additionally, 20 healthy subjects exposed to low-frequency magnetic field. Exposures were performed during 3 weeks, 5 days in a week. Defensin and CRP concentrations were measured at baseline, after 3 weeks and at the end of the study. RESULTS: There were no significant changes in defensin concentration in patients with diabetes in both the real and sham exposure group. We observed increased concentration of defensin in healthy subjects in week 5 vs. baseline value (P<0.02). CONCLUSIONS: Magnetic field has no impact on defensin concentration in diabetic patients but has positive influence on this parameter in healthy subjects.
Human beta-defensin-2 increases cholinergic response in colon epithelium.
Himmerkus N, Vassen V, Sievers B, Goerke B, Shan Q, Harder J, Schröder JM, Bleich M
Physiologisches Institut der Christian-Albrechts-Universität, Kiel, Germany.
Pflugers Arch. 2010 Jun;460(1):177-86.
The human beta-defensin-2 (hBD-2) is expressed in epithelial cells of skin and respiratory and gastrointestinal tracts. Defensins are arginine-rich small cationic peptides with six intramolecular disulfide bonds and are antimicrobially active against a broad spectrum of pathogens. In addition, they have cytokine-like immunomodulatory properties. We hypothesized that hBD-2 also might influence epithelial cells themselves, thereby altering fluid composition in the gastrointestinal tract. We therefore tested its impact on electrogenic ion transport properties of distal colon in Ussing chamber experiments. Application of hBD-2 did not affect transepithelial voltage or resistance in cAMP-stimulated distal colon. However, it increased cholinergic Ca(2+)-dependent Cl(-) secretion. After 20 min of incubation with hBD-2, the effect of carbachol (CCh) on the equivalent short circuit current (I´(sc)) was enhanced twofold compared to vehicle-treated colon. Modulation of Ca(2+) signaling by hBD-2 was validated by Fura-2 measurements in human colon carcinoma HT29 cells. Twenty-minute incubation with hBD-2 increased the CCh-induced Ca(2+) transient by 20-30% compared to either vehicle-treated cells or cells treated with the defensins hBD-1, hBD-3, or HD-5. This effect was concentration-dependent, with an EC(50) of 0.043 microg/ml, and still present in the absence of extracellular Ca(2+). Also, the ionomycin-induced Ca(2+) transient was increased by hBD-2 treatment. We conclude that hBD-2 facilitates cholinergic Ca(2+)-regulated epithelial Cl(-) secretion. These findings contribute to the concept of a specific interaction of antimicrobial peptides with epithelial function.
How honey kills bacteria.
Kwakman PH, Te Velde AA, de Boer L, Speijer D, Vandenbroucke-Grauls CM, Zaat SA
Department of Medical Microbiology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands,
FASEB J. 2010 Jul;24(7):2576-82.
With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10-20% (v/v) honey, whereas >/=40% (v/v) of a honey-equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 +/- 0.54 mM H2O2 and contained 0.25 +/- 0.01 mM methylglyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, we discovered bee defensin-1 in honey. After combined neutralization of H2O2, MGO, and bee defensin-1, 20% honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H2O2, MGO, and bee defensin-1. Thus, we fully characterized the antibacterial activity of medical-grade honey.
Expression of Acc-Royalisin gene from royal jelly of chinese honeybee in Escherichia coli and its antibacterial activity.
Shen L, Ding M, Zhang L, Jin F, Zhang W, Li D
Department of Food Science and Nutrition, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang, China.
J Agric Food Chem. 2010 Feb 24;58(4):2266-73.
Royalisin is an antibacterial peptide found in Royal Jelly. Two gene fragments of Chinese honeybee (Apis cerana cerana) head, 280 bp cDNA encoding pre-pro-Acc-royalisin (PPAR) of 95 amino acid residues, and 165 bp cDNA encoding mature Acc-royalisin (MAR) of 51 amino acid residues were cloned into the pGEX-4T-2 vector. They were then transformed individually into Escherichia coli for expression. Two expressed fusion proteins, glutathione S-transferase (GST)-PPAR of 36 kDa and GST-MAR of 32 kDa were obtained, which were cross reacted with GST antibody accounting for up to 16.3% and 15.4% of bacterial protein, respectively. In addition, 41% of GST-PPAR and nearly 100% of GST-MAR were soluble proteins. Both lysates of the two purified fusion proteins displayed antibacterial activities, similar to that of nisin against Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus. MAR peptide released from the thrombin-cleaved GST-MAR fusion protein has a stronger antibacterial activity than that of GST-MAR fusion protein.
Antimicrobial Peptide immunity protects human nasal and auricular cartilage against infection.
Warnke PH, Russo PA, Hopfenziz M, Kurz B, Becker ST, Sherry E, Springer I, Sivananthan S
Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland, Australia.
J Craniofac Surg. 2010 Jan;21(1):198-201.
BACKGROUND:: Despite being impervious to surveillance by the adaptive immune system because of its lack of vascularity, infection of the nasal and auricular cartilage after surgery such as rhinoplasty or otoplasty is rare. Why is this so? Our goal was to determine whether the expression of antimicrobial peptides provides a previously unrecognized nonepithelial layer of innate immune defense within the nasal and auricular cartilage. MATERIALS AND METHODS:: Seven samples of nasal septum cartilage and 2 biopsies from auricular cartilage grafts were harvested during rhinoplasty and otoplasty procedures. Ten cadaveric samples of auricular and 9 samples of nasal cartilage were also obtained. Immunohistochemical staining was directed against the human beta-defensin antimicrobial peptides (hBD) 1, 2, and 3. A semiquantitative analysis was performed to measure immunoreactivity. RESULTS:: All 3 human beta-defensins were detected along the perichondral line and within the cartilage matrix in the nasal and auricular samples. Areas with positive immunohistochemical staining were also detected within chondrocyte cytoplasm. CONCLUSIONS:: We provide the first evidence of antimicrobial peptide expression (hBD-1, -2 and -3) within the perichondrium and cartilage matrix layers of the nasal and auricular cartilage. This previously unrecognized innate immune function of perichondrocytes and chondrocytes may explain the resistance of the nasal and auricular cartilage to infection after surgical procedures despite the absence of a vascular system.
Identification and characterization of the parasitic wasp Nasonia defensins: positive selection targeting the functional region?
Gao B, Zhu S
Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Dev Comp Immunol. 2010 Jun;34(6):659-668.
Defensin is a crucial component of innate immunity highly conserved across different insect orders. Here, we report identification and characterization of defensins in the parasitic wasp Nasonia (Hymenoptera: Pteromalidae). In comparison with those in the non-parasitic insect Apis mellifera, two different subtypes of defensins (defensin1 and defensin2) have undergone independent gene duplication to create a mutigene family of five members (named 1-1, 1-2, 2-1, 2-2 and 2-3) in the Nasonia lineage. Such duplication occurred before the divergence of three sibling species (N. vitripennis, N. giraulti and N. longicornis) and the duplicated genes was subsequently subjected to positive selection at the amino-terminal loop and the gamma-core region. RT-PCR identified that only the subtype 1 of defensins were constitutively expressed in the N. vitripennis adult stage and none of the five defensins was expressed in other developmental stages (i.e. the infected Musca domestica pupae). A functional form of 2-2 in N. vitripennis (named navidefensin2-2) was produced in Escherichia coli by an on-column refolding approach. The recombinant peptide presented a typical defensin structure, as identified by CD analysis, and selectively inhibited the growth of two Gram(+) bacteria at low micromolar concentrations. The bioactive surface responsible for antibacterial activity of navidefensin2-2 was identified in the gamma-core region of this molecule. Positive selection targeting the antibacterial region of defensins could be a consequence of evolutionary arms race between Nasonia and its pathogens.