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1.
Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.  相似文献   

2.
Group 1B human pancreatic secretory phospholipase A2 (hp-sPLA2), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA2 constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH2; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA2, against E. coli. Full-length hp-sPLA2, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA2 possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.  相似文献   

3.
Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram-positive and Gram-negative, isolated from patients with sepsis, also bind kininogens, especially H-kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the entero-haemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curli, another fibrous bacterial surface protein, and the binding of HK to purified curli was specific, saturable, and of high affinity; Ka = 9 107M-1. Other contact phase proteins such as factor XI, factor XII, and prekallikrein bound to curliated E. coli, but not to an isogenic curli-deficient mutant strain, suggesting that contact phase activation may occur at the surface of curliated bacteria. Kininogens are also precursor molecules of the vasoactive kinins. When incubated with human plasma, curli-expressing bacteria absorbed HK. Addition of purified plasma kallikrein to the HK-loaded bacteria resulted in a rapid and efficient release of bradykinin from surface-bound HK. The assembly of contact phase factors at the surface of pathogenic bacteria and the release of the potent proinflammatory and vasoactive peptide bradykinin, should have a major impact on the host-microbe relationship and may contribute to bacterial pathogencity and virulence.  相似文献   

4.
一种新型贻贝抗菌肽的分离纯化及鉴定   总被引:1,自引:0,他引:1  
厚壳贻贝(Mytilus coruscus)广泛分布于我国东部海域,其体内富含各种抗菌肽分子,是研究软体动物免疫防御机制以及开发抗菌肽来源的新型生物抗生素的重要对象。采用多步反相高效液相色谱对厚壳贻贝血清进行分离纯化,获得一种分子量为6261.55 D的具有抗菌活性的多肽成分;经多肽N端测序和基因克隆,结果表明该抗菌肽由55个氨基酸残基构成,含6个半胱氨酸并形成三对二硫键。结构域分析表明该抗菌肽具有几丁质结合结构域(Chitin-biding domain),因此将该抗菌肽命名为mytichitin-A。Mytichitin-A对革兰氏阳性菌具有较强的抑制作用,同时对真菌及革兰氏阴性菌也具有抑制作用。荧光定量PCR检测表明,mytichitin-A主要在厚壳贻贝的性腺组织中表达且在细菌诱导后12h其表达量达到峰值。研究为深入了解厚壳贻贝抗菌肽的分子多样性及免疫机制奠定了基础。    相似文献   

5.
Mangoni ML  Fiocco D  Mignogna G  Barra D  Simmaco M 《Peptides》2003,24(11):1771-1777
Esculentin-1 is a 46-amino acid residue peptide isolated from skin secretions of Rana esculenta, displaying the most potent antimicrobial activity among the bioactive molecules found in the secretion, with negligible effects on eukaryotic cell membranes. From skin secretions, the 19-46 fragment of esculentin-1, devoid of antibacterial activity, was also isolated. We studied in detail the activity of the N-terminal fragment (1-18) of esculentin-1 using a synthetic amidated analogue. The results show that this fragment is highly active against most bacterial and fungal species, although at a lower extent than the full-length peptide, being four-fold more potent against Phytophthora nicotianae. It has a reduced activity against human erythrocytes with respect to the full-length peptide. The killing curves in liquid medium are similar for the two molecules and the shorter peptide is able to increase the bacterial outer and inner membrane permeability. Overall these data indicate that the antimicrobial properties of esculentin-1 are exerted by its N-terminal 1-18 region and that the positively charged residue distribution as well as peptide length represent important determinants for cell selectivity.  相似文献   

6.
The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.  相似文献   

7.
Two kininogens are found in mammalian sera: HK (high molecular weight kininogen) and LK (low molecular weight kininogen) with the exception of the rat which encompasses a third kininogen, T-Kininogen (TK). Kininogens are multifunctional glycosylated molecules related to cystatins (clan IH, family I25). They harbor three cystatin domains but only two of them are tight-binding inhibitors of cysteine cathepsins. HK and LK, but not TK, are precursors of potent peptide hormones, the kinins, which are released proteolytically by tissue and plasma kallikreins. Besides these classical features novel functions of kininogens have been recently discovered; they are described in the second part of this review. HKa, which corresponds to the kinin-free two-chain HK and its isolated domain D5 (kininostatin), possesses angiostatic and pro-apoptotic properties, inhibits the proliferation of endothelial cells and participates in the regulation of angiogenesis. Moreover, some HK-derived peptides display potent and broad-spectrum microbicidal properties against both Gram-positive and Gram-negative bacteria, and thus may offer a promising alternative to conventional antibiotic therapy. Of seminal interest, a kininogen-derived peptide inhibits activation of the contact phase system of coagulation and protects mice with invasive Streptococcus pyogenes infection from pulmonary lesions. On the other hand, TK is a biomarker of aging at the end of lifespan of elderly rats. However, although TK has been initially identified as an acute phase reactant, and earlier known as alpha-l-acute phase globulin, the increase of TK in liver and plasma is not known to relate to any inflammatory event during the senescence process.  相似文献   

8.
Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.  相似文献   

9.
A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.  相似文献   

10.
Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.  相似文献   

11.
Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.  相似文献   

12.
Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.  相似文献   

13.
Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.  相似文献   

14.
15.
The peptide hormone recently isolated from anglerfish endocrine pancreas (aPY) (Andrews, P. C., Hawke, D., Shively, J.E., and Dixon, J.E. (1985) Endocrinology 116, 2677-2681), is a member of a family of peptide hormones which includes pancreatic polypeptide, neuropeptide Y, and the gut peptide YY. A 30-residue carboxyl-terminal fragment of the precursor to aPY has been purified from anglerfish endocrine pancreas in two steps using both classical chromatographic methods and reversed-phase high pressure liquid chromatography. It was identified by sequence homology with the analogous peptide from human preproneuropeptide Y. The sequence was found by Edman degradation and fast atom bombardment mass spectrometry to be SSPEEAVAWLLFKADPSQDIEPRLDDDNAW. The high yield of this fragment (6.5 nmol . g-1) is similar to that previously reported for aPY (7.9 nmol . g-1) and suggests that it is a major product of pro-aPY processing. The data indicate that pro-aPY is proteolytically processed into two major products: the 37-residue aPY and the 30-residue carboxyl-terminal fragment.  相似文献   

16.
Conventional antibiotics might fail in the treatment of biofilm-associated infections causing infection recurrence and chronicity. The search for antimicrobial peptides has been performed with the aim to discover novel anti-infective agents active on pathogens in both planktonic and biofilm associated forms. The fragment 9–19 of human thymosin β4 was studied through 1 μs MD simulation. Two main conformations of the peptide were detected, both constituted by a central hydrophobic core and by the presence of peripheral charged residues suggesting a possible mechanism of interaction with two models of biological membranes, related to eukaryotic or bacterial membrane respectively. In addition, the peptide was chemically synthesized and its antimicrobial activity was tested in vitro against planktonic and biofilm form of a group of reference strains of Staphylococcus spp. and one P. aeruginosa strain. The human thymosin β4 fragment EIEKFDKSKLK showed antibacterial activity against staphylococcal strains and Pseudomonas aeruginosa ATCC 15442 at concentrations from 12.5 to 6.2 mg/ml and inhibited biofilm formation at sub-inhibitory concentrations (3.1–0.75 mg/ml). The activity of the fragment in inhibiting biofilm formation, could be due to the conformations highlighted by the MD simulations, suggesting its interaction with the bacterial membrane. Human thymosin β4 fragment can be considered a promising lead compound to develop novel synthetic or recombinant derivatives with improved pharmaceutical potential.  相似文献   

17.
Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.  相似文献   

18.
Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.  相似文献   

19.
IgA1 protease     
IgA1 proteases are proteolytic enzymes that cleave specific peptide bonds in the human immunoglobulin A1 (IgA1) hinge region sequence. Several species of pathogenic bacteria secrete IgA1 proteases at mucosal sites of infection to destroy the structure and function of human IgA1 thereby eliminating an important aspect of host defence. IgA1 proteases are known as autotransporter proteins as their gene structure encodes the information to direct their own secretion out of the bacterial cell. The iga gene structure is also thought to contribute to the antigenic heterogeneity demonstrated by the IgA1 proteases during infections and the cleavage specificity of the IgA1 proteases for human IgA1. The IgA1 proteases have therefore been implicated as important virulence factors that contribute to bacterial infection and colonisation. The development of strategies to inactivate these IgA1 proteases has become the subject of recent research, as this has the potential to reduce bacterial colonisation at mucosal surfaces.  相似文献   

20.
Mycobacterium avium complex is a causative organism for refractory diseases. In this study, we examined the effects of N-acetyl-cysteine on M. avium infection in vitro and in vivo. N-acetyl-cysteine treatment suppressed the growth of M. avium in A549 cells in a concentration-dependent manner. This effect was related to the induction of the antibacterial peptide human β-defensin-2. In a mouse model, N-acetyl-cysteine treatment significantly reduced the number of bacteria in the lungs and induced murine β-defensin-3. In interleukin-17-deficient mice, the effects of N-acetyl-cysteine disappeared, indicating that these mechanisms may be mediated by interleukin-17. Moreover, an additional reduction in bacterial load was observed in mice administered N-acetyl-cysteine in combination with clarithromycin. Our findings demonstrate the potent antimycobacterial effects of N-acetyl-cysteine against M. avium by inducing antimicrobial peptide, suggesting that N-acetyl-cysteine may have applications as an alternative to classical treatment regimens.  相似文献   

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