Staphylococcus aureus susceptibility to innate antimicrobial peptides, beta-defensins and CAP18, expressed by human keratinocytes.
ABSTRACT: 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:Antimicrobial peptides, human beta-defensin (hBD), and the 18-kDa cationic antimicrobial protein (CAP18) are components of innate immunity. These peptides have antimicrobial activity against bacteria, fungi, and viruses. Actinobacillus actinomycetemcomitans is a gram-negative facultative anaerobe implicated in the initiation of periodontitis. The innate immunity peptides have antibacterial activity against A. actinomycetemcomitans. We investigated the molecular mechanism of human gingival epithelial cells (HGEC) responding to exposure to A. actinomycetemcomitans. HGEC constitutively express hBD1 and inducibly express hBD2, hBD3, and CAP18 on exposure to A. actinomycetemcomitans. The level of expression varies among clinical isolates. In the signaling pathway for hBD2 induction by the bacterial contact, we demonstrate that the mitogen-activated protein (MAP) kinase and not the NF-kappaB transcription factor pathway is used. We found the outer membrane protein 100 (Omp100; identified by molecular mass) is the component inducing the hBD2 response. Omp100 binds to fibronectin, an extracellular matrix inducing hBD2 via the MAP kinase pathway. Anti-integrin alpha(5)beta(1), antifibronectin, genistein, and PP2 suppress the Omp100-induced expression of hBD2, suggesting that Src kinase is involved through integrin alpha(5)beta(1). The inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and IL-8, produced by HGEC on contact with A. actinomycetemcomitans also stimulate expression of hBD2. Further, neutralizing antibody against TNF-alpha or IL-8 partially inhibits the induction of hBD2 on bacterial contact. Therefore, we found that the induction of the antimicrobial peptides is mediated by a direct response principally through an Omp100-fibronectin interaction, and using secondary stimulation by inflammatory cytokines induced by the bacterial exposure.
Project description:Human beta-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression.Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression.Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1beta, TNFalpha, and interferon-gamma (IFNgamma). In contrast to constitutive expression levels, IFNgamma-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression.The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNgamma inducibility of these beta-defensins and is likely to be more protective in conditions that enhance IFNgamma expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.
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:Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human ?-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.
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:Antimicrobial peptides (AMPs) have recently been proposed as significant immunological factors involved in pathogenesis of coronavirus disease 19 (COVID-19). Human β-defensins (hBDs) are among these AMPs, but the evidence is not well detailed. Therefore, this case-control study analyzed levels of hBD1, hBD2, hBD3 and hBD4 in serum of 103 patients with severe COVID-19 and 105 healthy controls. Most patients were older than 45 years (80.6%), and more than 50% suffered from chronic diseases (cardiovascular and diabetes). Results revealed that median levels of hBD1 and hBD3 did not show significant differences between patients and controls. On the contrary, HBD2 levels were significantly decreased in patients compared to controls (1036 vs. 1289 ng/L; p < 0.001), while HBD4 levels were significantly increased (4.04 vs. 2.43 ng/L; p < 0.001). Receiver operating characteristic curve analysis demonstrated the predictive significance of hBD2 (area under the curve [AUC] = 0.795; 95% confidence interval [CI] = 0.729-0.861; p < 0.001) and hBD4 (AUC = 0.816; 95% CI = 0.756-0.876; p < 0.001) in discriminating between COVID-19 patients and controls. Logistic regression analysis (adjusted for age, gender and body mass index) confirmed the significance of hBD2 (odds ratio [OR] = 0.996; corrected p = 0.004) and hBD4 (OR = 4.948; corrected p < 0.001) in susceptibility to COVID-19. In conclusion, the study indicated that hBD2 showed low levels in serum of patients infected with severe COVID-19, while hBD4 showed elevated levels. These differences in HBDs were not influenced by age, gender, body mass index, or chronic disease.
Project description: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: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.
Project description:Chronic wounds remain a large problem in the field of medicine and are often associated with risk of infection and amputation. Recently, a commercially available human cryopreserved viable amniotic membrane (hCVAM) has been shown to effectively promote wound closure and reduce wound-related infections. A sprevious study indicates that hCVAM can inhibit the growth of bacteria associated with chronic wounds. In the present study, we investigated the mechanism of hCVAM antimicrobial activity. Our data demonstrate that antimicrobial activities against common pathogens in chronic wounds such as P.aeruginosa, S.aureus and Methicillin-resistant S.aureus (MRSA) are mediated via the secretion of soluble factors by viable cells in hCVAM and that these factors are proteins in nature. Further, we show that genes for antimicrobial peptides (AMPs) including human beta-defensins (HBDs) are expressed by hCVAM and that expression levels positively correlate with antimicrobial activity of hCVAM. At the protein level, our data indicate that HBD2 and HBD3 are secreted by hCVAM and directly contribute to its activity against P. aeruginosa. These data provide evidence that soluble factors including AMPs are hCVAM antimicrobial agents and are consistent with a role for AMPs in mediating antimicrobial properties of the membrane.
Project description:Background:Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide variety of serious infections worldwide. There are few studies on the prevalence, antimicrobial susceptibility, and molecular characteristics of MRSA contamination in the environment of airports. Materials and methods:A cross-sectional survey was conducted in Guangzhou Baiyun Airport. Environmental surface sampling was conducted in frequently touched locations for S. aureus analysis. All isolates were characterized by multilocus sequence typing (MLST) and tested for antimicrobial susceptibility, resistance genes, and virulence genes. Data were analyzed by chi-squared test and correspondence analysis. Results:Of the 1,054 surface samples, the contamination rate was 7.2% (76/1,054) for S. aureus and 2.2% (23/1,054) for MRSA. There were 62.9% (56/89) S. aureus isolates classified as multidrug resistant (MDR), with six linezolid-resistant isolates and two cfr-carrying isolates. The most prevalent S. aureus genotypes were CC6 (ST6), CC59 (ST59), and CC188 (ST188), with ST59-MRSA-IV (pvl-) as the predominant MRSA. There were significant differences between methicillin-resistant and methicillin-sensitive isolates in rates of resistance to tetracycline (P<0.001) and sek carriage (P=0.029). The correspondence analyses revealed significant corresponding relationships between genotypes and phenotype-genotype characteristics of S. aureus isolates. Conclusion:Our findings revealed a potential risk of cross-transmission of MRSA between human beings and environments, suggesting more stringent contamination control measures. In addition, this study revealed significant corresponding relationships between genotypes and phenotype-genotype characteristics of S. aureus isolates, which may provide new ideas for monitoring the latest epidemiological trends.