Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion.     

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.     

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.     

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.     

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 mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid origin

Cryptdin is a Paneth cell corticostatin/defensin in the mouse small bowel. To help define the intestinal role of cryptdin, cryptdin-related sequence (CRS) mRNAs have been characterized with respect to developmental regulation, sequence homology, putative coding function, and occurrence in myeloid cells. Cryptdin, CRS1C, and CRS4C mRNAs are transcribed from separate genes, occur at equivalent abundance in small intestine, and appear in the small bowel in concert during the 2nd and 3rd weeks postpartum. Cryptdin and CRS1C mRNAs are not detectable in adult mouse bone marrow, but probes specific for the 5'- or the 3'-untranslated regions of CRS4C mRNA hybridize to a moderately abundant 1.05-kilobase bone marrow mRNA in contrast to a highly abundant 0.75-kilobase mRNA in small intestine. Nucleotide sequences corresponding to the deduced prepro-coding regions of cryptdin, CRS1C, and CRS4C mRNAs contain a highly conserved 200-base pair region of 92% sequence similarity (CSE.2), but the mRNAs are not homologous otherwise. The deduced CRS1C and CRS4C polypeptides are apparent precursors of secreted, cationic, proline- and cysteine-rich peptides that contain Cys-Pro-X repeats. Unlike cryptdin, however, the proposed CRS1C and CRS4C mature peptide regions lack the structural motif characteristic of defensins. Attempts to find homologies between the putative CRS peptides and existing protein sequences have been unsuccessful, leading us to speculate that CRS1C and CRS4C represent a new family of nondefensin antimicrobial peptides in the mouse small bowel.     

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.     

Paneth cells of the human small intestine express an antimicrobial peptide gene.

Mucosal surfaces of several organ systems are important interfaces for host defense against microbes. Recent evidence suggests that antimicrobial peptides contribute to the defense of these surfaces. Defensins are one family of antimicrobial peptide, but their known distribution in humans has been limited to four members found in cells of myeloid origin. We sought to determine if the human defensin family was more complex. We found that the family of human defensins is diverse and is not restricted to expression in leukocytes. Southern blot and genomic clone analyses reveal that numerous defensin-related sequences are present in the human genome. A gene for a new human defensin family member was characterized. This gene, designated human defensin-5, is highly expressed in Paneth cells of the small intestine. This is the first example of an antimicrobial peptide gene expressed in an epithelial cell in humans. The data support the hypotheses that epithelial defensins equip the human small bowel with a previously unrecognized defensive capability which would augment other antimicrobial defenses.     

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.     

Cryptdin 3 forms anion selective channels in cytoplasmic membranes of human embryonic kidney cells

Cryptdins are antimicrobial peptides secreted by Paneth cells located at the base of intestinal crypts. In addition to their antimicrobial function, cryptdins may also regulate salt and water secretion by intestinal epithelial cells. Recent work with short-circuit current measurements indicated that at least one cryptdin peptide, cryptdin 3, induces apical conductance(s) in Cl(-) secretory, including cystic fibrosis, epithelia. In the present study, we characterized the cryptdin 3-induced anion channel activity in human embryonic kidney (HEK) cells with single-channel patch-clamp techniques. The patch pipette was filled with solution containing different concentrations of cryptdin 3, and, after gigaseal formation, the channel activity was recorded with either cell-attached or inside-out patch modes. We found an anion selective channel with a conductance of 15 pS and open probability of 0.19, regardless of cryptdin 3 concentration. The mean open and closed times varied with the cryptdin 3 concentration. For cryptdin 3 concentrations of 10, 4, 1, and 0.5 microg/ml in the pipette, the corresponding mean open times were 1.2, 7.0, 9.0, and 17.4 ms and the corresponding mean closed times were 1.1, 1.6, 4.2, and 12.5 ms. These results suggest that cryptdin 3 forms anion-selective channels on the cytoplasmic membrane of HEK cells and that the kinetics of one such channel are affected by its interaction with other such channels.     

Functional Role of Metaplastic Paneth Cell Defensins in Helicobacter pylori-Infected Stomach

Background and Aims:  Chronic gastritis is caused by Helicobacter pylori infection, and gastritis is classified as inflammation, atrophy, and intestinal metaplasia. Detailed pathologic studies have shown that H. pylori settles on the surface of gastric mucosa, and that it is eliminated from metaplastic mucosa. However, its mechanism of natural protection is not well known.
Methods:  Antimicrobial human enteric defensin expression was determined in the RNA and protein levels. Recombinant enteric defensins were produced with a bacterial expression system and their anti- H. pylori activities were assessed by bactericidal assay.
Results:  Human enteric defensin (HD)-5 and HD-6 were detected in Paneth cells, which are observed in the gastric metaplastic mucosa as well as small intestinal epithelia. HD-5 protein was coexpressed with trypsin, which is considered to be an activating enzyme of HD-5. Less H. pylori was observed in the intestinal metaplasia with HD-5 expressing Paneth cells. The recombinant defensins showed killing activity against H. pylori at a low concentration in vitro.
Conclusions:  The human defensins that are expressed in the metaplastic Paneth cells eliminate H. pylori . Metaplastic change may be a purposive development of the human stomach.     

Structure-dependent functional properties of human defensin 5

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.     

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.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.     

Defensins in granules of phagocytic and non-phagocytic cells

Antimicrobial proteins stored in lysosome-like granules of neutrophils and macrophages probably play an important role in killing phagocytosed microbes after delivery to the phagolysosome. Among the granules' antimicrobial armamentarium are defensins, peptides that kill a broad spectrum of microorganisms in vitro. Antimicrobial defensins were recently also isolated from non-phagocytic granulocytes of the mouse small intestinal epithelium, from where they are secreted into the lumen to function extracellularly. Clarification of the antimicrobial mechanisms of defensins in intracellular and extracellular environments will provide a key to understanding peptide-mediated host defence.     

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.     

The defensin-related murine CRS1C gene: expression in Paneth cells and linkage to Defcr, the cryptdin locus.

The site of defensin-related CRS1C gene expression in mouse small bowel and the chromosomal location of the CRS1C locus, Defcr-rs1, have been determined. CRS1C (cryptdin-related sequence 1C) mRNA is an abundant small intestinal sequence that exhibits extensive similarity to the prepro-coding regions of defensin mRNAs yet does not encode a defensin (A. J. Ouellette and J. C. Lualdi, 1990, J. Biol. Chem. 265: 9831-9837). Using sequence-specific probes, CRS1C mRNA was detected in Paneth cells at the base of intestinal crypts by in situ hybridization. Southern blot analysis of genomic DNAs from inbred and recombinant inbred (RI) mouse strains, also conducted with probes specific for CRS1C, showed that the CRS1C locus maps to the proximal region of Chromosome 8. In 62 RI strains, no discordancies were found between Defcr-rs1 and Defcr, the cryptdin gene. Thus, both the Defcr-rs1 and the Defcr genes are expressed in Paneth cells and both are genetically inseparable within 1.58 cM on Chromosome 8. These studies identify a second defensin-related Paneth cell gene in mice.     

Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3

Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell derived α-defensin HD5 and the major colonic β-defensins HBD-1–3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD-1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteremia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species- but also strain-specific and may be clinically relevant in the host–bacteria interaction influencing mucosal translocation and systemic infection.