Enteric defensins: antibiotic peptide components of intestinal host defense

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.     

Activation of Paneth cell alpha-defensins in mouse small intestine.

Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.     

Quantitative interactions between cryptdin-4 amino terminal variants and membranes

Paneth cells secrete alpha-defensins into the lumen from the base of small intestinal crypts, and cryptdin-4 (Crp4) is the most potent mouse alpha-defensin in vitro. Purified recombinant Crp4 and Crp4 variants with (des-Gly)-, (Gly1Val)-, (Gly1Asp)-, and (Gly1Arg)-substitutions were all bactericidal with Crp4 and (Gly1Arg)-Crp4 being slightly more active than other variants. Bactericidal activities correlated directly with permeabilization of live Escherichia coli, with equilibrium binding to E. coli membrane phospholipid bilayers and vesicles, and with induced graded fluorophore leakage from phospholipid vesicles. The Crp4 peptide N-terminus affects bactericidal activity modestly, apparently by influencing peptide binding to phospholipid bilayers and subsequent permeabilization of target cell membranes.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.     

Paneth cell cryptdins act in vitro as apical paracrine regulators of the innate inflammatory response

Intestinal-specific antimicrobial alpha-defensins, termed cryptdins, are secreted into the intestinal lumen by mouse Paneth cells in response to microbial pathogens. Cryptdins kill microbes by forming pores in their limiting membranes. The cryptdin isoforms 2 and 3 also can form anion-conductive pores in eukaryotic cell membranes, thus affecting cell physiology. Here, we find that when applied to apical membranes of the human intestinal cell line T84, cryptdin 3 (Cr3) induces secretion of the proinflammatory cytokine interleukin 8 (IL-8) in a dose-dependent manner. The induction of IL-8 secretion is specific to the cryptdins that form channels in mammalian cell membranes because cryptdin 4, which does not form pores in T84 cells, does not induce IL-8 secretion. Cr3 induces inflammatory cytokine secretion by activating NF-kappaB and p38 mitogen-activated protein kinase in a Ca2+-dependent manner, but influx by extra-cellular Ca2+ is not involved. Unlike other known inflammatory agonists, signal transduction by Cr3 occurs slowly, suggesting a novel mechanism of action. These results show that selective cryptdins may amplify their roles in innate immunity by acting as novel paracrine agonists to coordinate an inflammatory response with the antimicrobial secretions of Paneth cells.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

Matrix metalloproteinase-7 activation of mouse paneth cell pro-alpha-defensins: SER43 down arrow ILE44 proteolysis enables membrane-disruptive activity

Small intestinal Paneth cells secrete alpha-defensin microbicidal peptides as mediators of innate enteric immunity. In mice, production of mature Paneth cell alpha-defensins, termed cryptdins (Crps), requires proteolytic activation of inactive precursors (pro-Crps) by the convertase matrix metalloproteinase-7. Proteolysis of mouse (pro-Crp4)(20-92) produces the specific cleavage intermediates pro-Crp4(44-92), pro-Crp4(54-92), and pro-Crp4(59-92). To identify which cleavage event enables bactericidal activity, recombinant pro-Crp4-processing intermediates were purified to homogeneity and assayed for bactericidal peptide activity. The in vitro bactericidal activities of pro-Crp4-processing intermediates were very similar to fully processed Crp4, contrasting the lack of bactericidal and membrane-disruptive activity shown by pro-Crp4(20-92). Thus, cleavage of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) is sufficient to activate bactericidal activity, and amino acids in the pro-Crp4(20-43) of the proregion maintain the precursor in an inactive state. Because cationic Arg residues are determinants of Crp4 bactericidal peptide activity, we hypothesized that Asp and Glu residues in pro-Crp4(20-43) neutralize Crp4 Arg side chains in pro-Crp4(20-92). Therefore, a pro-Crp4(20-92) variant with Gly substitutions at all pro-Crp4(20-43) Asp and Glu positions ((DE/G)-pro-Crp4) was prepared, and it was bactericidal and lysed phospholipid vesicles under conditions where native pro-Crp4(20-92) lacks activity. These findings show that MMP-7 proteolysis of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) converts inactive precursors to bactericidal forms by removal of covalently associated, inhibitory acidic amino acids from proximity with the Crp4 component of the molecule.     

Structure-activity determinants in paneth cell alpha-defensins: loss-of-function in mouse cryptdin-4 by charge-reversal at arginine residue positions

Paneth cells secrete microbicidal enteric alpha-defensins into the small intestinal lumen, and cryptdin-4 (Crp4) is the most bactericidal of the mouse alpha-defensin peptides in vitro. Here, site-directed Arg to Asp mutations in Crp4 have been shown to attenuate or eliminate microbicidal activity against all of the bacterial species tested regardless of the Arg residue position. R31D/R32D charge-reversal mutagenesis at the C terminus and mutations at R16D/R18D, R16D/R24D, and R18D/R24D in the Crp4 polypeptide chain eliminated in vitro bactericidal activity, blocked peptide-membrane interactions, as well as Crp4-mediated membrane vesicle disruption. Lys for Arg charge-neutral substitutions in (R16K/R18K)-Crp4 did not alter the bactericidal activity relative to Crp4, showing that bactericidal activity appears not to require the guanidinium side chain of Arg at those two positions. Partial restoration of (R31D/R32D)-Crp4 bactericidal activity occurred when an electropositive Arg for Gly substitution was introduced at the peptide N terminus and the (G1R/R31D/R32D)-Crp4 peptide exhibited intermediate membrane binding capability. Also, the loss of peptide bactericidal activity in (G1D/R31D/R32D)-Crp4 and (R16D/R24D)-Crp4 mutants corresponded with diminished phospholipid vesicle disruptive activity. Fluorophore leakage from anionic phospholipid vesicles induced by the charge-reversal variants was negligible relative to Crp4 and lower than that induced by pro-Crp4, the inactive Crp4 precursor. Thus, Arg residues function as determinants of Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. The role of the Arg residues, however, was surprisingly independent of their position in the polypeptide chain.     

Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.     

Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine.     

Events at the host-microbial interface of the gastrointestinal tract. V. Paneth cell alpha-defensins in intestinal host defense

Host defense of the small intestine is mediated, in part, by antimicrobial peptides, including alpha-defensins. In the small intestine, Paneth cells, specialized secretory epithelial cells located at the base of the crypt invaginations lining the intestinal wall, produce alpha-defensins. The alpha-defensins are cysteine-rich cationic peptides with antibiotic activity against a wide range of bacteria and other microbes. Studies of transgenic and knockout mice have supported a pivotal role of Paneth cell alpha-defensins in protection from bacterial pathogens. New data suggest that deficient expression of Paneth cell alpha-defensins may contribute to the pathophysiology of Crohn's disease, a chronic inflammatory bowel disease.     

α-Defensins from blood leukocytes of the monkey Papio hamadryas

Three antimicrobial peptides named PHD1-3 (Papio hamadryas defensin) have been isolated from hamadryas baboon blood leukocytes using preparative electrophoresis and reverse-phase HPLC. The primary structures of these peptides have been determined by automated Edman degradation and mass-spectrometry. The results suggest that the peptides belong to the alpha-defensin family. Structural homology analysis reveals that among alpha-defensins from other animal species, PHD3 is the most closely related to RMAD5 (rhesus macaque alpha-defensin) (90% homology) from rhesus macaque leukocytes and also highly similar to human alpha-defensin HD5 (60% homology), which is produced by intestinal Paneth cells. The homology of PHD3 with human neutrophil alpha-defensin HNP1 (human natural peptide) was 30%. The primary structures of PHD1 and PHD2 are most similar to RED1 (rhesus enteral defensin), one of six enteral alpha-defensins of rhesus monkeys. PHD1-3 have been shown to be active against the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, and the fungus Candida albicans, similarly to the human HNP1 defensin.     

Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes

The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC, MMP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.     

防御素研究进展

防御素是广泛分布于自然界中的广谱性抗菌多肽,在生物体内由特殊的细胞合成,经过一系列的加工后分泌表达出功能分子,由于其抗菌活性高,且作用范围广,引起了人们极大的兴趣,目前通过对其天然表达细胞的培养、合成肽和基因表达等手段制备,并进一步对其作用机理进行了探讨,但临床应用仍未见报道。     

Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.     

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin.

We have purified and determined the amino acid sequence of cryptdin-1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid-extracted intestinal protein was fractionated by cation-exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti-Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid-urea PAGE. The peptide was purified to homogeneity by RP-HPLC and sequenced. These studies demonstrate defensin expression in non-myeloid tissue. The N-terminal extension of cryptdin-1 is a unique structural feature of this novel epithelial defensin.     

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 ?) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.     

A novel murine beta -defensin expressed in tongue, esophagus, and trachea

beta-Defensins are broad spectrum antimicrobial peptides expressed at epithelial surfaces. Two human beta-defensins, HBD-1 and HBD-2, have been identified. In the lung, HBD-2 is an inducible product of airway epithelia and may play a role in innate mucosal defenses. We recently characterized rat homologs (RBD-1, RBD-2) of the human genes and used these sequences to identify novel mouse genes. Mouse beta-defensin-4 (MBD-4) was amplified from lung cDNA using polymerase chain reaction primers designed from conserved sequences of RBD-2 and HBD-2. A full-length cDNA was cloned which encodes a putative peptide with the sequence MRIHYLLFTFLLVLLSPLAAFTQIINNPITCMTNGAICWGPCPTAFRQIGNCGHFKVRCCKIR. The peptide shares approximately 40% identity with HBD-2. MBD-4 mRNA was expressed in the esophagus, tongue, and trachea but not in any of 20 other tissues surveyed. Cloning of the genomic sequence of MBD-4 revealed two nearly (>99%) identical sequences encoding MBD-4 and the presence of numerous additional highly similar genomic sequences. Radiation hybrid mapping localized this gene to a region of chromosome 8 near several other defensins, MBD-2, MBD-3, and alpha-defensins (cryptdins)-3 and -17, consistent with a gene cluster. Our genomic cloning and mapping data suggest that there is a large beta-defensin gene family in mice. Identification of murine beta-defensins provides an opportunity to understand further the role of these peptides in host defense through animal model studies and the generation of beta-defensin-deficient animals by gene targeting.