Defensin Antimicrobial Peptides Research Articles

Understanding the Dynamics of Human Defensin Antimicrobial Peptides: Pathogen Resistance and Commensal Induction.

Antimicrobial peptides (AMPs), also known as host defense peptides, are petite molecules with inherent microbicidal properties that are synthesized by the host's innate immune response. These peptides serve as an initial barrier against pathogenic microorganisms, effectively eliminating them. Human defensin (HD) AMPs represent a prominent group of peptides involved in the innate immune response of humans. These peptides are primarily produced by neutrophils and epithelial cells, serving as a crucial defense mechanism against invading pathogens. The extensive research conducted has focused on the broad spectrum of antimicrobial activities and multifaceted immunomodulatory functions exhibited by human defensin AMPs. During the process of co-evolution between hosts and bacterial pathogens, bacteria have developed the ability to recognize and develop an adaptive response to AMPs to counterattack their bactericidal activity by different antibiotic-resistant mechanisms. However, numerous non-pathogenic commensal bacteria elicit the upregulation of defensins as a means to surmount the resistance mechanisms implemented by pathogens. The precise mechanism underlying the induction of HD by commensal organisms remains to be fully understood. This review summarizes the most recent research on the expression of human defensin by pathogens and discusses the various defense mechanisms used by pathogens to counter host AMP production. We also mention recent developments in the commensal induction of defensin AMPs. A better knowledge of the pathogens' defensin AMP resistance mechanisms and commensals' induction of AMP expression may shed light on the creation of fresh antibacterial tactics to get rid of bacterial infection.

Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA's ability to selectively interact with specific AMPs to mediate resistance.

Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection.

Common bed bugs, Cimex lectularius, can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis and the Gram-negative (Gr–) bacterium Escherichia coli. We demonstrate, for the first time, sex-specific and immunization mode-specific upregulation of bed bug defensins in response to injection or ingestion of Gr+ or Gr– bacteria. Understanding the components, such as these defensins, of the bed bugs’ innate immune systems in response to pathogens may help unravel why bed bugs do not transmit pathogens to vertebrates.

Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides

The exploitation of conventional antibiotics in conjunction with the adeptness of microbes has led to the emergence of multi-drug-resistant pathogens. This has posed a severe threat to combating life-threatening infectious diseases. Antimicrobial peptides (AMP), which are considered to be the first line of defense in all living organisms, are being developed for therapeutic use. Herein, we determined the NMR solution structure of Rhesus macaque Myeloid Alpha Defensin-4 (RMAD4), a defensin AMP. Additionally, the distinct modes of membrane perturbation for two structurally dissimilar classes of AMPs was studied using biophysical methods namely, Solid-state 31P NMR, DSC and cryo-TEM. The cathelicidin - Bovine myeloid antimicrobial peptide (BMAP-28 (1–18)), which adopts a helical conformation, and the defensin RMAD4 peptide that natively folds to form β-sheets appeared to engage differently with the bacterial membrane. The helical BMAP-28 (1–18) peptide initiates lipid segregation and membrane thinning followed by pore formation, while the β-stranded RMAD4 peptide demonstrates fragmentation of the bilayer by the carpet or detergent-like mechanism of action. Molecular dynamics studies sufficiently corroborated these findings. The structure and mechanism of action of the AMPs studied using experimental and computational approaches are believed to help in providing a platform for the rational design of new competent and cost-effective antimicrobial peptides for therapeutic applications.

Ultrasound-Targeted Microbubble Destruction Enhances Human β-Defensin 3 Activity Against Antibiotic-Resistant Staphylococcus Biofilms

The infection of orthopedic implantation devices with Staphylococcus is a serious concern within the biomaterial community. Treatments are not always successful because of antibiotic-resistant bacteria and serious biofilm infections. Human β-defensin 3 (hBD-3) is considered to be the most promising class of defensin antimicrobial peptides and its effect on antibiotic-resistant Staphylococcus biofilms, combined with ultrasound (US)-targeted microbubble (MB) destruction (UTMD), has not been reported. In the study, we found that biofilm densities, the percentage of live cells and the viable counts of two tested Staphylococcus recovered from the biofilm were significantly decreased in the maximum concentration hBD-3 combined with UTMD compared with those of any other groups. Furthermore, results suggested that UTMD could also enhance 1MIC hBD-3 activity inhibiting the biofilm-associated genes expression of icaAD and the methicillin-resistance genes expression of MecA by simultaneously promoting the icaR expression. UTMD may have great potential for improving antibiotic activity against biofilm infections.

Comparison of the Effects of Human β-defensin 3, Vancomycin, and Clindamycin on Staphylococcus aureus Biofilm Formation

Despite improvements in surgical techniques and implant design in orthopedic surgery, implantation-associated infections are still a challenging problem for surgeons. In 2006, trace quantities of human β-defensin 3 (hBD-3) were found in human bone tissue and bone cells. Human β-defensin 3 is a 45-amino-acid peptide that is considered the most promising class of defensin antimicrobial peptides and may help in the prevention and treatment of implantation-associated infections. Studies of the effectiveness of hBD-3 against Staphylococcus aureus showed that hBD-3 was more potent at low concentrations than other antibiotics. The effect of hBD-3 on S aureus biofilms has not been reported. We studied the effect of hBD-3, vancomycin, and clindamycin on S aureus biofilms and on the survival of the bacteria in the biofilms.Staphylococcus aureus biofilms were examined with confocal scanning laser microscopy. Staining with LIVE/DEAD BacLight viability stain (Molecular Probes Europe BV, Leiden, The Netherlands) differentiated between live and dead bacteria within the biofilms, and extracellular polymeric substances (slime) from the biofilms was evaluated after staining with calcofluor white (Sigma Chemical Company, Rocky Hill, New Jersey). Human β-defensin 3 and clindamycin reduced the S aureus biofilm area. Human β-defensin 3 was significantly more effective against bacteria from the S aureus biofilms than was clindamycin. Vancomycin did not reduce the S aureus biofilm area.

EST-Database Search of Plant Defensins – An Example Using Sugarcane, a Large and Complex Genome

EST (Expressed Sequence Tags) databases are increasing in number and size, especially regarding cultivated plants. Sugarcane is one of the most important tropical and subtropical crops, presenting a complex polyploid genome of hybrid origin, bearing a challenge for the understanding of genetic processes in higher plants. In the present work a general search was carried out on the largest Sugarcane EST Database (SUCEST) that includes 237,954 ESTs aiming to identify defensin antimicrobial peptides - a class of small, basic, cysteine-rich peptides distributed throughout the kingdoms. Using a computational approach 17 new defensin isoforms could be identified. Main steps for the search, characterization and evaluation of the defensin expression profile are presented. Prevalent expression tissues were leaf roll, lateral bark, root apex, base of inflorescence, developing seed, and calli. Bioinformatics and phylogenetic analysis of the primary structure of the sugarcane defensin candidates as well as the 3D structures obtained by comparative modeling support their role as antimicrobials.

A defensin antimicrobial peptide from the venoms of Nasonia vitripennis

Although many antimicrobial components (i.e. antimicrobial peptides) have been found in many social Hymenoptera venoms, no antimicrobial compound is purified and characterized from parasitic Hymenoptera. From the venoms of the ectoparasitic wasp, Nasonia vitripennis, a defensin-like antimicrobial peptide named defensin-NV was purified and characterized. Defensin-NV is composed of 52 amino acid residues including 6 cysteines forming 3 disulfide bridges. Its amino acid sequence is VTCELLMFGGVVGDSACAANCLSMGKAGGSCNGGLCDCRKTTFKELWDKRFG. By BLAST search, defensin-NV showed significant sequence similarity to other insect defensin antimicrobial peptides. Defensin-NV exerted strong antimicrobial activity against tested microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi. The cDNA encoding defensin-NV was cloned from the venom reservoir cDNA library of N. vitripennis. The current work firstly purified and characterized an antimicrobial peptide from parasitic Hymenoptera.

Human β defensin‐1 and ‐2 expression in the gingiva of patients with specific periodontal diseases

beta defensin antimicrobial peptides are important in epithelial innate immunity, and their differential expression is associated with periodontal diseases. The aims of this study were to determine the mRNA expression of human beta defensin-1 and -2 in the gingival tissue of patients with gingivitis, aggressive periodontitis and chronic periodontitis, and to evaluate the relationship between defensin expression and type and/or severity of periodontal destruction. Fifteen patients in each group with gingivitis, aggressive periodontitis and chronic periodontitis, and 10 healthy subjects, were included in the study (n=55). The periodontal status of the subjects was determined by periodontal clinical measurements and radiographical evaluations. Transcriptional levels of human beta defensin-1 and -2 genes in gingival samples were assessed by using the quantitative real-time polymerase chain reaction technique, and the data were evaluated statistically by the relative expression Software Tool 2 for groupwise comparisons. Expression of the human beta defensin-1 gene was lower in gingivitis and aggressive periodontitis groups, and significantly higher in the chronic periodontitis group, than in the control group (p<0.001). Human beta defensin-2 mRNA expression in the gingivitis group was lower than in the control group; however, the difference was statistically significant only in half of the gingivitis patients (p<0.001). Human beta defensin-2 mRNA levels were higher in some chronic periodontitis patients, but lower in the others when compared with the control group (p<0.001). Expression of the human beta defensin-2 gene increased in the aggressive periodontitis group relative to the control group. This study suggests that human beta defensin-1 and -2 genes in the gingival epithelium show differential expression in patients with specific periodontal diseases, and aggressive and chronic periodontitis types demonstrate different gingival beta defensin-1 and -2 expression patterns.

An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides.

Four macrocyclic cystine-knot peptides of 29-31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of approximately 0.2 microM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 microM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 microM. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is salt-dependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their end-to-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics.

Critical Role of Lipid Composition in Membrane Permeabilization by Rabbit Neutrophil Defensins

We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coli whole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry 35, 11888-11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins.

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.