Human beta-defensin 2 and beta-defensin 3 chimeric peptides reveal the structural basis of the pathogen specificity of their parent molecules.
ABSTRACT: Despite partial sequence identity and structural similarity, human ?-defensin 3 (HBD3) kills Staphylococcus aureus with a 4- to 8-fold higher efficiency than human ?-defensin 2 (HBD2), whereas the activities against Escherichia coli are identical. The design and characterization of HBD2/HBD3 chimeric peptides revealed that distinct molecular regions are responsible for their divergent killing properties. Two of the chimeras killed both E. coli and S. aureus with an even higher efficacy than the wild-type molecules. Moreover, one of these two chimeras maintained its high killing activities in the presence of physiologic salt concentrations. Due to the broad spectrum of their antimicrobial activities against many human multidrug-resistant pathogens, these two designer peptides of human origin represent promising templates for a new class of antibiotics.
Project description:The antimicrobial peptides human beta-defensin-1 (hBD1), hBD2, hBD3, and CAP18 expressed by keratinocytes have been implicated in mediation of the innate defense against bacterial infection. To gain insight into Staphylococcus aureus infection, the susceptibility of S. aureus, including methicillin-resistant S. aureus (MRSA), to these antimicrobial peptides was examined. Based on quantitative PCR, expression of hBD2 mRNA by human keratinocytes was significantly induced by contact with S. aureus, and expression of hBD3 and CAP18 mRNA was slightly induced, while hBD1 mRNA was constitutively expressed irrespective of the presence of S. aureus. Ten clinical S. aureus isolates, including five MRSA isolates, induced various levels of expression of hBD2, hBD3, and CAP18 mRNA by human kertinocytes. The activities of hBD3 and CAP18 against S. aureus were found to be greater than those of hBD1 and hBD2. A total of 44 S. aureus clinical isolates, including 22 MRSA strains, were tested for susceptibility to hBD3 and CAP18. Twelve (55%) and 13 (59%) of the MRSA strains exhibited more than 20% survival in the presence of hBD3 (1 microg/ml) and CAP18 (0.5 microg/ml), respectively. However, only three (13%) and two (9%) of the methicillin-sensitive S. aureus isolates exhibited more than 20% survival with hBD3 and CAP18, respectively, suggesting that MRSA is more resistant to these peptides. A synergistic antimicrobial effect between suboptimal doses of methicillin and either hBD3 or CAP18 was observed with 10 MRSA strains. Furthermore, of several genes associated with methicillin resistance, inactivation of the fmtC gene in MRSA strain COL increased susceptibility to the antimicrobial effect mediated by hBD3 or CAP18.
Project description:Beta-defensins are antimicrobial peptides with an essential role in the innate immune response. In addition beta-defensins can also chemoattract cells involved in adaptive immunity. Until now, based on evidence from dendritic cell stimulation, human beta defensin-3 (hBD3) was considered pro-inflammatory. We present evidence here that hBD3 lacks pro-inflammatory activity in human and mouse primary Mphi. In addition, in the presence of LPS, hBD3 and the murine orthologue Defb14 (but not hBD2), effectively inhibit TNF-alpha and IL-6 accumulation implying an anti-inflammatory function. hBD3 also inhibits CD40/IFN-gamma stimulation of Mphi and in vivo, hBD3 significantly reduces the LPS-induced TNF-alpha level in serum. Recent work has revealed that hBD3 binds melanocortin receptors but we provide evidence that these are not involved in hBD3 immunomodulatory activity. This implies a dual role for hBD3 in antimicrobial activity and resolution of inflammation.
Project description:Balance between the microbiome associated with bladder mucosa and human beta defensin (HBD) levels in urine is a dynamic, sensitive and host-specific relationship. HBD1-possessing both antitumor and antibacterial activity-is produced constitutively, while the inducible production of antibacterial HBD2 and HBD3 is affected by bacteria. Elevated levels of HBD2 were shown to cause treatment failure in anticancer immunotherapy. Our aim was to assess the relationship between microbiome composition characteristic of tumor tissue, defensin expression and HBD levels measured in urine. Tissue samples for analyses were removed during transurethral resection from 55 bladder carcinoma and 12 prostatic hyperplasia patients. Microbiome analyses were carried out with 16S rRNS sequencing. Levels of HBD mRNA expression were measured with qPCR from the same samples, and urinary amounts of HBD1, 2 and 3 were detected with ELISA in these patients, in addition to 34 healthy volunteers. Mann-Whitney U test, Wilcoxon rank sum test (alpha diversity) and PERMANOVA analysis (beta diversity) were performed. Defensin-levels expressed in the tumor did not clearly determine the amount of defensin measurable in the urine. The antibacterial and antitumor defensin (HBD1) showed decreased levels in cancer patients, while others (HBD2 and 3) were considerably increased. Abundance of <i>Staphylococcus</i>, <i>Corynebacterium</i> and <i>Oxyphotobacteria</i> genera was significantly higher, the abundance of <i>Faecalibacterium</i> and <i>Bacteroides</i> genera were significantly lower in tumor samples compared to non-tumor samples. <i>Bacteroides</i>, <i>Parabacteroides</i> and <i>Faecalibacterium</i> abundance gradually decreased with the combined increase in HBD2 and HBD3. Higher <i>Corynebacterium</i> and <i>Staphylococcus</i> abundances were measured together with higher HBD2 and HBD3 urinary levels. Among other factors, defensins and microorganisms also affect the development, progression and treatment options for bladder cancer. To enhance the success of immunotherapies and to develop adjuvant antitumor therapies, it is important to gain insight into the interactions between defensins and the tumor-associated microbiome.
Project description:Human beta defensins (hBDs) may play an important role in the progression of lichen sclerosus (LS), due to their ability to induce excessive stimulation of extracellular matrix synthesis and fibroblast activation. The genetic ability of the individual to produce defensins, the presence of microbes influencing defensin production, and the sensitivity of microbes to defensins together regulate the formation of an ever-changing balance between defensin levels and microbiome composition. We investigated the potential differences in postmenopausal vaginal microbiome composition and vaginal hBD levels in LS patients compared to non-LS controls. LS patients exhibited significantly lower levels of hBD1 (p = 0.0003), and significantly higher levels of hBD2 (p = 0.0359) and hBD3 (p = 0.0002), compared to the control group. The microbiome of the LS patients was dominated by possibly harmful bacteria including Lactobacillus iners, Streptococcus anginosus or Gardnerella vaginalis known to initiate direct or indirect damage by increasing defensin level production. Our observations highlight that correcting the composition of the microbiome may be applicable in supplementary LS therapy by targeting the restoration of the beneficial flora that does not increase hBD2-3 production.
Project description:Antimicrobial peptides can protect the gastric mucosa from bacteria, but Helicobacter pylori (H. pylori) can equally colonize the gastric apparatus. To understand beta-defensin function in H. pylori-associated chronic gastritis, we investigated susceptibility, human beta-defensin mRNA expression, and DNA methylation changes to promoters in the gastric mucosa with or without H. pylori infection. We studied the expression of HBD2 (gene name DEFB4A), HBD3 (DEFB103A), and HBD4 (DEFB104) using real-time PCR in 15 control and 10 H. pylori infection patient gastric specimens. This study demonstrates that H. pylori infection is related to gastric enhancement of inducible HBD2, but inducible HBD3 and HBD4 expression levels remained unchanged. HBD2 gene methylation levels were overall higher in H. pylori-negative samples than in H. pylori-positive samples. We also assessed antimicrobial susceptibility using growth on blood agar. The H. pylori strain Tox+ was susceptible to all defensins tested and their analogs (3N, 3NI). These results show that HBD2 is involved in gastritis development driven by H. pylori, which facilitates the creation of an epigenetic field during H. pylori-associated gastric tumorigenesis.
Project description:BACKGROUND:Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection. MATERIALS AND METHODS:We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy. RESULTS:H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples. CONCLUSION:These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.
Project description:<i>Neisseria meningitidis</i> is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on <i>N. meningitidis</i>. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of <i>N. meningitidis</i> to evade immune defenses.
Project description:Human-?-defensins (HBD1-3) are antibacterial peptides containing three disulphide bonds. In the present study, the effect of Escherichia coli lipopolysaccharide (LPS) on the antibacterial activities of HBD2-3, C-terminal analogues having a single disulphide bond, Phd1-3, and their corresponding myristoylated analogues MPhd1-3 were investigated. The effect of LPS on the activities of linear amphipathic peptides melittin, LL37 and non-ribosomally synthesized peptides, polymyxin B, alamethicin, gramicidin A, and gramicidin S was also examined. The antibacterial activity of HBD 2-3, Phd1-3, and MPhd1-3 in the presence of LPS against E. coli and Staphylococcus aureus was inhibited. While LPS inhibited the antibacterial activity of LL37, the inhibition of melittin activity was partial. The hemolytic activity exhibited by MPhd1, MPhd3, melittin, and LL37 was inhibited in the presence of LPS. HBD2-3, Phd1-3, and MPhd1-3 also showed endotoxin neutralizing activity. The antibacterial and hemolytic activities of polymyxin B, alamethicin, gramicidin A, and gramicidin S were not inhibited in the presence of LPS. Fluorescence assays employing dansyl cadaverine showed that HBD2-3 and defensin analogues bind to LPS more strongly as compared to alamethicin, gramicidin A, and gramicidin S. Electron microscopy images indicated that peptides disintegrate the structure of LPS. The inhibition of the antibacterial activity of native defensins and analogues in the presence of LPS indicates that the initial interaction with the bacterial surface is similar. The native defensin sequence or structure is also not essential, although cationic charges are necessary for binding to LPS. Hydrophobic interaction is the main driving force for association of non-ribosomally synthesized polymyxin B, alamethicin, gramicidin A, and gramicidin S with LPS. It is likely that these peptides rapidly insert into membranes and do not interact with the bacterial cell surface, whereas cationic peptides such as ?-defensin and their analogues, melittin and LL37, first interact with the bacterial cell surface and then the membrane. Our results suggest that evaluating interaction of antibacterial and hemolytic peptides with LPS is a compelling way of elucidating the mechanism of bacterial killing or hemolysis.
Project description:Human beta-defensin 3 (hBD3), an antimicrobial peptide (AMP) expressed in epithelium in response to various stimulations including human papillomavirus infection, has recently been found to be overexpressed in head and neck cancers and exhibit tumorigenic activities. However, the role of hBD3 in cervical cancer remains to be investigated. In this study, we showed by immunohistochemistry that hBD3 expression was elevated in cervical cancer samples of different stages versus the normal tissue, and was positively correlated with the progression of the disease. Overexpression of hBD3 in cervical cancer cell lines promoted cell proliferation by accelerating G1/S progression and enhanced cell migration and invasion in vitro. These oncogenic effects of hBD3 were associated with activation of NF-?B signaling. Using a mouse xenograft model, we further demonstrated that hBD3 overexpression promoted the growth of cervical cancer cells in vivo. Our results suggested that hBD3 is involved in the carcinogenesis and development of cervical cancer, and may serve as a biomarker or therapeutic target of this disease.
Project description:Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human ?-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-?B, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8.