ABSTRACT
An increase in the number of infections caused by resistant bacteria worldwide necessitates the development of alternatives to antibiotics. Human defensin (HD) 5 is an innate immune peptide with broad-spectrum antibacterial activity, but its complicated structure makes its preparation difficult. Herein, we truncated the HD5 structure by extracting the highly conserved γ-core motif. A structure-activity study showed that this motif was ineffective in killing bacteria in the absence of specific spatial conformation. Notably, after the introduction of two intramolecular disulfide bonds, its antibacterial activity was markedly improved. Glu and Ser residues were then replaced with Arg to create the derivative RC18, which exhibited stronger potency than HD5, particularly against methicillin-resistant S. aureus (MRSA). Mechanistically, RC18 bound to lipid A and lipoteichoic acid at higher affinities than HD5. Furthermore, RC18 was more efficient than HD5 in penetrating the bacterial membranes. Molecular dynamics simulation revealed that five Arg residues, Arg1, Arg7, Arg9, Arg15, and Arg18, mediated most of the polar interactions of RC18 with the phospholipid head groups during membrane penetration. In vivo experiments indicated that RC18 decreased MRSA colonization and dramatically improved the survival of infected mice, thus demonstrating that RC18 is a promising drug candidate to treat MRSA infections.
ABSTRACT
Recently, aging is considered a risk factor for various diseases. Although changes in the intestinal microbiota along with aging are thought to associate with the increased disease risk, mechanisms that cause age-related transition of the intestinal microbiota remain unknown. This study aims to clarify relationships between the amount of human defensin 5 (HD5), a Paneth cell α-defensin, which is known to regulate the intestinal microbiota, and age-related differences of the intestinal microbiota composition. Fecal samples from 196 healthy Japanese (35 to 81 years old) were collected and measured HD5 concentration. HD5 concentration in the elderly group (age > 70 years old) was significantly lower than the middle-aged group (age ≤ 70 years old). Furthermore, individual age was negatively correlated with HD5 concentration (r = - 0.307, p < 0.001). In ß-diversity, the intestinal microbiota of the elderly showed a significantly different composition compared to the middle-aged. At the genus level, relative abundance of Collinsella, Alistipes, Peptococcaceae; unassigned, Lactobacillus, Lactococcus, Weissella, Christensenellaceae R-7 group, Megasphaera, and [Eubacterium] eligens group was significantly higher, and Lachnospiraceae; unassigned, Blautia, Anaerostipes, Fusicatenibacter, Dorea, and Faecalibacterium was significantly lower in the elderly compared to the middle-aged. In addition, HD5 concentration was negatively correlated with Alistipes, Peptococcaceae; unassigned, and Christensenellaceae R-7 group and positively correlated with Lachnospiraceae; unassigned and Dorea. These results provide novel insights into the immunosenescence of enteric innate immunity, indicating low HD5 is suggested to contribute to the age-related differences in the intestinal microbiota and may relate to increased risk of diseases in elderly people.
Subject(s)
Gastrointestinal Microbiome , alpha-Defensins , Adult , Aged , Aged, 80 and over , Defensins , Feces , Humans , Japan , Middle Aged , alpha-Defensins/analysisABSTRACT
Human α-defensin 5 (HD5) is a host-defense peptide exhibiting broad-spectrum antimicrobial activity. The lipopolysaccharide (LPS) layer on the Gram-negative bacterial membrane acts as a barrier to HD5 insertion. Therefore, the pore formation and binding mechanism remain unclear. Here, the binding mechanisms at five positions along the bacterial membrane axis were investigated using Molecular Dynamics. (MD) simulations. We found that HD5 initially placed at positions 1 to 3 moved up to the surface, while HD5 positioned at 4 and 5 remained within the membrane interacting with the middle and inner leaflet of the membrane, respectively. The arginines were key components for tighter binding with 3-deoxy-d-manno-octulosonic acid (KDO), phosphates of the outer and inner leaflets. KDO appeared to retard the HD5 penetration.
Subject(s)
Anti-Infective Agents/metabolism , Cell Membrane/metabolism , Gram-Negative Bacteria/metabolism , Molecular Dynamics Simulation , alpha-Defensins/metabolism , Amino Acid Sequence , Anti-Infective Agents/chemistry , Arginine/metabolism , Humans , Hydrogen Bonding , Lipopolysaccharides/metabolism , Protein Binding , Protein Multimerization , Sugar Acids/metabolism , alpha-Defensins/chemistryABSTRACT
Human α-defensin 5 (HD5) is one of cationic antimicrobial peptides which plays a crucial role in an innate immune system in human body. HD5 shows the killing activity against a broad spectrum of pathogenic bacteria by making a pore in a bacterial membrane and penetrating into a cytosol. Nonetheless, its pore-forming mechanisms remain unclear. Thus, in this work, the constant-velocity steered molecular dynamics (SMD) simulation was used to simulate the permeation of a dimeric HD5 into a gram-negative lipopolysaccharide (LPS) membrane model. Arginine-rich HD5 is found to strongly interact with a LPS surface. Upon arrival, arginines on HD5 interact with lipid A head groups (a top part of LPS) and then drag these charged moieties down into a hydrophobic core resulting in the formation of water-filled pore. Although all arginines are found to interact with a membrane, Arg13 and Arg32 appear to play a dominant role in the HD5 adsorption on a gram-negative membrane. Furthermore, one chain of a dimeric HD5 is required for HD5 adhesion. The interactions of arginine-lipid A head groups play a major role in adhering a cationic HD5 on a membrane surface and retarding a HD5 passage in the meantime.
Subject(s)
Bacterial Outer Membrane/chemistry , alpha-Defensins/chemistry , Arginine/chemistry , Bacterial Outer Membrane/metabolism , Gram-Negative Bacteria/chemistry , Humans , Hydrogen Bonding , Lipopolysaccharides/chemistry , Molecular Dynamics Simulation , Protein Multimerization , alpha-Defensins/metabolismSubject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Enterocytes/virology , Paneth Cells/virology , Peptidyl-Dipeptidase A/physiology , Pneumonia, Viral/virology , alpha-Defensins/physiology , Angiotensin-Converting Enzyme 2 , COVID-19 , Cell Culture Techniques , Coronavirus Infections/metabolism , Coronavirus Infections/pathology , Enterocytes/metabolism , Humans , Microscopy, Interference , Pandemics , Paneth Cells/metabolism , Pneumonia, Viral/metabolism , Pneumonia, Viral/pathology , SARS-CoV-2 , Virus AttachmentABSTRACT
The escalating predicament of multidrug resistant cancer cells and associated side effects of conventional chemotherapy necessitates the exploration of alternative anticancer therapies. The present study evaluated anticancer therapeutic potential of human defensin 5 (HD-5) against colon cancer. The in vivo anticancer efficacy of HD-5 against 1,2-dimethylhydrazine (DMH) induced colon cancer was elucidated in terms of tumor biostatistics, number of aberrant crypt foci (ACF), in situ apoptosis assay,changes in morphological as well as histological architecture of colon(s). The direct interaction of peptide was investigated by incubating peptide with normal and/or cancerous colonocytes followed by phase contrast, Hoechst 3342 and AO/PI staining as well as confocal microscopy. Changes in membrane dynamics were evaluated by MC 540 and N-NBD-PE staining. In vivo decrease(s) in tumor parameters, number of aberrant crypt foci along with marked increase in the rate of apoptosis was observed.H&E staining revealed neutrophils infiltration and restoration of normal architecture in treated colon(s) which was consistent with scanning electron microscopic observations. Furthermore, non-membranolytic mechanism was found to be acquired by peptide as it could traverse cell membrane gaining access to nucleus and cytoplasm thereby disintegrating cellular architecture. MC 540 and NBD-PE staining revealed that peptide could bind to cancerous cells by taking advantage of altered fluidity levels. Our results indicated that HD-5 exhibited strong cancer cell killing and does not affect normal host cells. The peptide can be exploited as promising option to combat developing menace of colon cancer and/or can at least be used as an adjunct to present day chemotherapies.
Subject(s)
Colonic Neoplasms/prevention & control , Peptide Fragments/pharmacology , alpha-Defensins/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Carcinogens/toxicity , Colon/drug effects , Colon/pathology , Colonic Neoplasms/chemically induced , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dimethylhydrazines/toxicity , Female , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
Human α -defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and plays an essential role in innate immunity in the human gut and other organ systems. Although its antimicrobial mechanism of action remains unclear, the high salt concentration seems to attenuate the antimicrobial function of HD5 via an unknown mechanism. In this work, we employ Molecular Dynamics (MD) simulations to analyse the oligomerization behaviour of HD5 when exposed to different salt concentration. We demonstrate that the presence of salt, such as sodium chloride (NaCl), promotes HD5 to form higher-order oligomers (up to heptamers) in our simulations. In addition, we also analyse the electrostatic interactions between the two Glu residues (E14 and E21) and their neighbouring residues. Our data confirm that the E14 residue is essential for the structural integrity, whereas the E21 residue contributes to the dimerization of HD5, suggesting that these Glu residues are important for the antimicrobial function of this peptide.
Subject(s)
Molecular Dynamics Simulation , alpha-Defensins/chemistry , Humans , Sodium Chloride/chemistry , Solutions , Static Electricity , alpha-Defensins/chemical synthesisABSTRACT
Human α-defensin 5 (HD5) is one of the important antimicrobial peptides (AMPs) used against a broad-spectrum of pathogens, especially Gram-negative bacteria. HD5 kills by disrupting and making a pore in the bacterial membrane. The presence of lipopolysaccharide (LPS), located on a membrane surface, is found to have an impact on HD5's activity, where such binding mechanism in microscopic detail remains unclear. In this work, we therefore employed molecular dynamics (MD) simulations to investigate the binding mechanisms of HD5 on LPS in comparison to a bare DMPC lipid membrane. Two oligomers, dimer and tetramer, are studied here. Apparently, the membrane structure influences the protein binding affinity. HD5 binds tighter to a lipid membrane than LPS. Both dimeric and tetrameric HD5 can penetrate deeply into a phosphate layer in a lipid membrane, whereas only facial contacts are observed for LPS systems. The proteins appear to stay in the polar area instead of diving into a hydrophobic region. Furthermore, it happens in all cases that residues in the active region (A1, T2, R6, R13, R32) contribute to the membrane adsorption. The breakdown of tetramer into two dimers is also found. This implies that the dimer is more favorable for membrane binding. Moreover, both dimeric and tetrameric HD5 can significantly disrupt a LPS layer, whilst no serious distortion of lipid membrane is obtained. This emphasizes the importance of LPS on HD5 activity.
Subject(s)
Cell Membrane/chemistry , Gram-Negative Bacteria/cytology , Lipopolysaccharides/chemistry , Molecular Dynamics Simulation , alpha-Defensins/chemistry , Adsorption , Cell Membrane/metabolism , Humans , Lipopolysaccharides/metabolism , Protein Binding , Protein Conformation , alpha-Defensins/metabolismABSTRACT
The gut epithelium is critically involved in maintaining intestinal immune homeostasis. Acting as a physical barrier, it separates the intestinal microflora from cells of the immune system. In addition to its barrier function, the intestinal epithelium expresses defensins, natural, endogenous antimicrobial peptides. In humans, specialized epithelial cells, termed Paneth cells, located primarily in the small intestine express two defensins, Human Defensin-5 (HD-5) and Human Defensin-6 (HD-6). Previously, we have shown that HD-5 potently kills bacteria and induces secretion of interleukin-8 by intestinal epithelial cells. We show that HD-6 specifically and synergistically enhances the HD-5-induced IL-8 secretion, but does not alter its anti-bacterial activity. Further, we find that HD-5 decreases the trans-epithelial electrical resistance of intestinal epithelial cells and that HD-6 negates this effect of HD-5.
Subject(s)
Defensins/physiology , Humans , Interleukin-8/biosynthesis , Intestinal Mucosa/physiologyABSTRACT
Defensins are cationic antimicrobial peptides that contribute to regulation of host cell function also. Here, we report on the regulation of cell death by Human Defensin 5, the major antimicrobial peptide of ileal Paneth cells. We find that Human Defensin 5-mediated cellular effects depend on functional expression of Tumor Necrosis Factor receptors and downstream mediators of TNF signaling. Our data indicate the involvement of interactions between Human Defensin 5 and the extra-cellular domain of Tumor Necrosis Factor receptor 1. Human Defensin-5 also induces apoptosis intrinsically by targeting the mitochondrial membrane.
Subject(s)
Defensins/metabolism , Macrophages/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , alpha-Defensins/metabolism , Animals , Apoptosis , Caspase 8/metabolism , Cell Survival , Cells, Cultured , Defensins/genetics , Flow Cytometry , Humans , Interleukin-8/metabolism , Jurkat Cells , Macrophages/cytology , Mice , Mice, Knockout , Microscopy, Confocal , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/genetics , alpha-Defensins/geneticsABSTRACT
Defensins are cationic antimicrobial peptides that play an important role in innate immunity by primarily acting against microbes. Their antimicrobial properties have been widely studied and are well understood. Defensins contribute to regulation of host immunity also. Their effects on cells of the host however are less well understood. Here, we report on the pro-inflammatory and apoptotic properties of Human Defensin 5, the major antimicrobial peptide of ileal Paneth cells. We find that HD-5 up-regulates expression of genes involved in cell survival and inflammation in a NF-kB-dependent fashion in epithelial cells. Further, we find that HD-5 has pro-apoptotic effects on intestinal epithelial cells as well as primary CD4+ T cells.