人源抗菌肽LL-37

LL-37是迄今在人体中发现的抗菌肽cathelicidin家族中的唯一成员,也是人体内唯一的双亲性α螺旋结构的抗菌肽. LL-37在人体的血液细胞和上皮细胞中广泛分布,具有广谱抗菌作用.抗菌活性依赖其螺旋构象的形成,通过“地毯样”机制杀灭细菌. LL-37有中和内毒素的作用,能与LPS和CD14结合,中和LPS的生物活性,还能利用formyl peptide receptor-like 1 (FPRL1) 作为受体介导趋化作用,招募免疫细胞到感染部位,清除病原物. 很多感染性疾病都与LL-37低表达或功能失活有关. LL-37是一种多功能的抗菌肽,有开发成为具有抗菌和增强免疫力双重作用药物的潜力.     

The Human Cathelicidin LL-37 Has Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.     

Antimicrobial Cathelicidin Peptide LL-37 Inhibits the LPS/ATP-Induced Pyroptosis of Macrophages by Dual Mechanism

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1β mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1β expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X₇-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X₇ to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X₇ activation.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Cathelicidin LL-37 bloodstream surveillance is down regulated during septic shock

Host defense peptides are ancient weapons of the innate immunity. The human cathelicidin LL-37 protects the epithelial barrier against infection and is constitutively secreted in the bloodstream by immune cells. Current knowledge claims that LL-37 is up regulated upon infection. LL-37 can protect against bacterial infections and possesses many immunomodulatory properties. Here, we show that the human host defense peptide LL-37 is down regulated during septic shock. Furthermore, we show that these effects are not related to vitamin D serum levels, a potent inducer of LL-37 gene expression, pointing out the complex regulation of cathelicidins during septic shock.     

Antiviral Activity of the Human Cathelicidin,LL-37, and Derived Peptides on Seasonal and Pandemic Influenza A Viruses

Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties.     

Cathelicidin LL-37 peptide regulates endothelial cell stiffness and endothelial barrier permeability

LL-37 peptide is a multifunctional host defense molecule essential for normal immune responses to infection or tissue injury. In this study we assess the impact of LL-37 on endothelial stiffness and barrier permeability. Fluorescence microscopy reveals membrane localization of LL-37 after its incubation with human umbilical vein endothelial cells (HUVECs). A concentration-dependent increase in stiffness was observed in HUVECs, bovine aortic endothelial cells (BAECs), human pulmonary microvascular endothelial cells, and mouse aorta upon LL-37 (0.5-5 μM) addition. Stiffening of BAECs by LL-37 was blocked by P2X7 receptor antagonists and by the intracellular Ca2(+) chelator BAPTA-AM. Increased cellular stiffness correlated with a decrease in permeability of HUVEC cell monolayers after LL-37 addition compared with nontreated cells, which was similar to the effect observed upon treatment with sphingosine 1-phosphate, and both treatments increased F-actin content in the cortical region of the cells. These results suggest that the antiinflammatory effect of LL-37 at the site of infection or injury involves an LL-37-mediated increase in cell stiffening that prevents increased pericellular permeability. Such a mechanism may help to maintain tissue fluid homeostasis.     

Endotoxin,Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 μM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.Neisseria meningitidis (meningococci) is a gram-negative, aerobic diplococci that is an obligate human pathogen. Infections caused by N. meningitidis are an important cause of morbidity and mortality worldwide. Meningococci colonize the nasopharyngeal mucosa of approximately 10% of healthy individuals but can cross epithelial and endothelial cell barriers and enter the bloodstream, causing septicemia, with mortality rates of 20 to 50% (4). Meningitis occurs when bacteria transverse the blood cerebrospinal fluid, causing a fatal outcome in 15 to 20% of infected patients. Bacterial adherence is initially mediated by type IV pili with host cell receptors. PilT is the molecular motor responsible for pili retraction, which mediates a tight interaction. An important virulence factor of N. meningitidis is the endotoxin lipooligosaccharide (LOS), which is located in the bacterial outer cell membrane. Meningococcal LOS is composed of a conserved inner core of membrane-associated lipid A (16) to which variable α- and β-chains attach (13).As one of many first lines of defense against invading pathogens like Neisseria bacteria, epithelial cells produce antimicrobial peptides (AMPs). These peptides are effector molecules for the innate immune response, with both direct antimicrobial activity and a broad spectrum of immunomodulatory functions (18, 22). LL-37 is the single known human cathelicidin and is expressed in various immune cells as well as in epithelial cells of inflamed skin, mouth, tongue, esophagus, and lungs. It has been shown that LL-37 interacts with bacterial membranes through both electrostatic and hydrophobic effects. It remains unknown whether LL-37 ultimately kills bacteria by formation of torroidal pores as described by Henzler Wildman et al. (11) or by detergent-like disintegration of the membrane via the carpet model as described by Shai (24), but increasing membrane permeability, osmotic swelling, and loss of the vital proton gradient are important characteristics of the killing process (21). Membrane interactions of LL-37 (and other AMPs) appear to be highly selective for the negative surface charge on prokaryotic membranes. However, it has been shown by Tzeng et al. (28) that meningococci regulate AMP attack via mechanisms that include lipid A modification and an efflux pump. LL-37 toxicity for eukaryotic cells remains low, probably because eukaryotic cell membranes do not have a negative net charge (31).In order to further investigate the bactericidal activity of LL-37, various Neisseria strains were examined for their susceptibility to LL-37. Our results show that LL-37 exhibits potent killing activity against N. meningitidis, whereas adhesion to host cells, LOS, and the capsule was found to contribute to resistance to LL-37. Neisseria bacteria can respond to sublethal doses of LL-37 to increase capsule production.     

Cathelicidin LL-37 increases lung epithelial cell stiffness, decreases transepithelial permeability, and prevents epithelial invasion by Pseudomonas aeruginosa

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 μM), sphingosine 1-phosphate (1 μM), or LPS (1 μg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.     

The Human Antimicrobial Peptide LL-37 Binds Directly to CsrS,a Sensor Histidine Kinase of Group A Streptococcus,to Activate Expression of Virulence Factors

Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.     

人源抗菌肽LL-37在毕赤酵母中的高效表达及其活性检测

根据GenBank CAA86115中的LL-37氨基酸序列, 选择毕赤酵母偏好密码子, 采用SOE方法合成了人源抗菌肽LL-37基因。所合成的LL-37基因全长为141 bp, 并在其N端引入kex2裂解位点, 以保证表达抗菌肽具有天然N端。基因克隆入pPICZa-A质粒, 构建分泌型重组酵母表达载体pPICZa-A-LL-37。pPICZa-A-LL-37经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。PCR鉴定为阳性的酵母转化子经甲醇诱导分泌LL-37于发酵上清液, 其表达量为206 mg/L。表达产物LL-37耐热性强, 在100℃条件下40 min内抗菌活性不变, 煮沸3 h以上仍具有活性。琼脂糖孔穴扩散法检测显示LL-37对多种革兰氏阴性菌和阳性菌均具有很好的抑制活性, 其对金黄色葡萄球菌 CowanⅠ(Staphylococcus aureus)、致病性大肠杆菌K99(Enteropathogenic E.coli)和鸡白痢沙门氏菌(Salmonella pullorum)的最小抑菌浓度(Minimal Inhibitory Concentration, MIC)分别为1.56 mg/mL、3.12 mg/mL和1.56 mg/mL。     

Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor.     

人源抗菌肽LL-37在毕赤酵母中的高效表达及其活性检测

根据GenBank CAA86115中的LL-37氨基酸序列,选择毕赤酵母偏好密码子,采用SOE 方法合成了人源抗菌肽LL-37基因.所合成的LL-37基因全长为141 bp,并在其N端引入kex2裂解位点,以保证表达抗茵肽具有天然N端.基因克隆入pPICZα-A质粒,构建分泌型重组酵母表达载体pPICZα-A-LL-37.pPICZα-A-LL-37经Sac Ⅰ酶切线性化后电转化导入毕赤酵母菌株X-33.PCR鉴定为阳性的酵母转化子经甲醇诱导分泌LL-37于发酵上清液,其表达量为206mg/L.表达产物LL-37耐热性强,在100℃条件下40 min内抗茵活性不变,煮沸3 h以上仍具有活性.琼脂糖孔穴扩散法检测显示LL-37对多种革兰氏阴性茵和阳性菌均具有很好的抑制活性,其对金黄色葡萄球菌Cowan I (Staphylococcus aureus)、致病性大肠杆菌K99(Enteropathogenic E.coli)和鸡白痢沙门氏菌(Salmonella pullorum)的最小抑菌浓度(Minimal Inhibitory Concentration,MIC)分别为1.56 μg/mL、3.12 μg/mL和1.56 μg/mL.     

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.     

LL-37 Peptide Enhancement of Signal Transduction by Toll-like Receptor 3 Is Regulated by pH: IDENTIFICATION OF A PEPTIDE ANTAGONIST OF LL-37

LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of ∼1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37·poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling.     

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog,mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.     

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized （sVP6）. The VP6 and sVp6 genes were transformed into tobacco （Nicotiana tabacum L.） plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein （TSP）. Then, BALB/ c female mice that had been gavaged weekly with 10 mg TSP containing 34 p.g VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.