Identification of CRAMP, a Cathelin-related Antimicrobial Peptide Expressed in the Embryonic and Adult Mouse (1997)

Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, namedCramp for cathelin-relatedantimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic α-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5–8.0 μm) and by permeabilizing the inner membrane of Escherichia colidirectly at 1 μm. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair. Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, namedCramp for cathelin-relatedantimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic α-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5–8.0 μm) and by permeabilizing the inner membrane of Escherichia colidirectly at 1 μm. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair. Endogenous antimicrobial peptides play an important role in innate immunity (1Martin E. Ganz T. Lehrer R.I. J. Leukocyte Biol. 1995; 58: 128-136Crossref PubMed Scopus (297) Google Scholar, 2White S.H. Wimley W.C. Selsted M.E. Curr. Opin. Struct. Biol. 1995; 5: 521-527Crossref PubMed Scopus (378) Google Scholar, 3Boman H.G. Cell. 1991; 65: 205-207Abstract Full Text PDF PubMed Scopus (539) Google Scholar). More than 100 microbicidal peptides have been isolated from plants and animals (4Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1520) Google Scholar). The role of these defense peptides in mammals has been inferred from their expression in neutrophil granules and at sites exposed to multiple microbes such as the skin and gastrointestinal tract. To exert their antimicrobial effect these peptides adhere to and permeabilize the surface membranes of potential pathogens. This activity is a consequence of several common features such as a high content of basic residues and the tendency of some to adopt an amphipathic conformation. However, antimicrobial peptides show marked diversity in structure and antimicrobial spectrum. One class of antimicrobial peptides, the cathelicidin-derived peptides, contains a highly conserved prepro region that is homologous to cathelin, a putative cysteine-proteinase inhibitor originally isolated from pig leukocytes (5Ritonja A. Kopitar M. Jerala R. Turk V. FEBS Lett. 1989; 255: 211-214Crossref PubMed Scopus (129) Google Scholar). Cathelicidins have been identified in several species including pig, cow, sheep, rabbit, and man (6Agerberth B. Lee J.Y. Bergman T. Carlquist M. Boman H.G. Mutt V. Jörnvall H. Eur. J. Biochem. 1991; 202: 849-854Crossref PubMed Scopus (296) Google Scholar, 7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar, 8Storici P. Zanetti M. Biochem. Biophys. Res. Commun. 1993; 196: 1058-1065Crossref PubMed Scopus (65) Google Scholar, 9Gennaro R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar, 10Bagella L. Scocchi M. Zanetti M. FEBS Lett. 1995; 376: 225-228Crossref PubMed Scopus (102) Google Scholar, 11Mahoney M.M. Lee A.Y. Brezinski-Caliguri D.J. Huttner K.M. FEBS Lett. 1995; 377: 519-522Crossref PubMed Scopus (76) Google Scholar, 12Tossi A. Scocchi M. Skerlavaj B. Gennaro R. FEBS Lett. 1994; 339: 108-112Crossref PubMed Scopus (84) Google Scholar, 13Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar). The high degree of conservation of their cathelin domain suggests that the members of this family evolved from a common ancestor gene through duplication and modification (7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar). In general, the precursors of these peptides are stored in neutrophil granules. Upon stimulation, the cathelin domain is cleaved proteolytically to allow the mature COOH-terminal antimicrobial peptide to be released. The antimicrobial portion of the cathelicidin-derived gene family is highly diverse in terms of structure and function. In the pig, the cathelicidin PR-39 has been found to have potent activity against Gram-negative bacteria and also to function as a stimulator of syndecan-1 and -4 expression on fibroblasts and endothelia (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar). Observations of functions beyond antimicrobial activity have also been made for other host defense peptides (15Murphy C.J. Foster B.A. Mannis M.J. Selsted M.E. Reid T.W. J. Cell. Physiol. 1993; 155: 408-413Crossref PubMed Scopus (187) Google Scholar) and suggest the need for further study of the role these peptides play in vivo. The mouse is a highly useful animal model to study the function of the immune system in events such as wound repair and cutaneous inflammation. Surprisingly, despite the well characterized immune system in the mouse, antimicrobial peptides have only been identified in the mouse intestine (16Yount N.Y. Wang M.-S.C. Yuan J. Banaiee N. Ouellette A.J. Selsted M.E. J. Immunol. 1995; 155: 4476-4484PubMed Google Scholar, 17Ouellette A.J. Hsieh M.W. Cano-Gauci D.F. Nosek M.T. Huttner K.M. Buick R.N. Selsted M.E. Infect. Immun. 1994; 62: 5040-5047Crossref PubMed Google Scholar) and appear to be absent from neutrophils (18Eisenhauer P.B. Lehrer R.I. Infect. Immun. 1992; 60: 3446-3447Crossref PubMed Google Scholar). Thus, in this investigation we sought to identify a mouse member of the cathelicidin gene family and describe its expression and antimicrobial function. We report a full-length cDNA sequence derived from mouse marrow which is a member of the cathelicidin gene family. This gene, named Cramp, forcathelin-relatedantimicrobial peptide, mapped to a single region on murine chromosome 9, homologous to the map locations of cathelicidins in man and pig. Transcripts for Cramp were expressed in multiple mature tissues and during embryogenesis. Finally, CRAMP protein was identified by immunostaining in murine bone marrow cells and neutrophils and behaved structurally and functionally as a potent antimicrobial agent. Amino acids and coupling reagents for peptide synthesis were from PerSeptive Biosystems (Framingham, MA) and Novabiochem (Laufelfingen, Switzerland). HPLC-grade acetonitrile, 1The abbreviations used are: HPLC, high performance liquid chromatography; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; Fmoc,N-(9-fluorenyl)methoxycarbonyl; MIC, minimum inhibitory concentration(s); PBS, phosphate-buffered saline. N-methyl-2-pyrrolidone, dichloromethane, andN,N-dimethylformamide were from Lab-Scan (Dublin, Ireland). Trifluoroacetic acid, N-methylmorpholine, and trifluoroethanol were from Janssen Chimica (Beerse, Belgium).o-Nitrophenyl-β-d-galactopyranoside was from Sigma. Mueller-Hinton broth, Bacto-agar, dextrose, mycological peptone, and yeast extract powder were from Unipath Ltd (Basingstoke, U. K.). All other chemicals were of analytical grade. Total RNA was extracted from C57BL/6 mouse femoral marrow cells with guanidinium thiocyanate (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). To isolate potential murine cDNA homologs to the cathelin-related antimicrobial peptides, a 5′ and 3′ RACE strategy was applied (Life Technologies, Inc.; 3′ and 5′ RACE systems, Gaithersburg, MD). For 3′ RACE, cDNA synthesis was carried out using adapter primer 5′-GGCCACGCGTCGACTAGTAC(T)17-3′. Amplification toward the 3′ end was done first with the universal amplification primer 5′-(CUA)4GGCCACGCGTCGACTAGTAC-3′ and with a cathelin-specific primer-1, 5′-TCGGAAGCTAATCTCTAC-3–3′, which was designed based on a base pairs 165–182 sequence of porcine prepro-PR-39. A second nested amplification was then done with a cathelin-specific primer-2, 5′-(CAU)4CTGGACCAGCCGCCCAAG-3′ designed based on base pairs 195–212 of prepro-PR-39 and the universal amplification primer. For amplification toward the 5′ end, the gene-specific primer-1, 5′-TTTGCGGAGAAGTCCAGC-3′, based on a sequence derived from 3′ RACE, was used for cDNA synthesis. Amplification was done with an anchor primer, provided by Life Technologies, Inc., and with a gene-specific primer-2, 5′-(CAU)4GAAATTTTCTTGAACCG-3′. Products of 3′ and 5′ RACE were for by in using against and were in Northern blot analysis of RNA was as (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar). of RNA was extracted from C57BL/6 mouse at and by through a RNA was to a membrane was carried out at in and using corresponding to base pairs of murine CRAMP were for in 1 at and then in at Total RNA from testis, stomach, liver, skeletal brain, heart, spleen, and intestine of C57BL/6 was with guanidinium thiocyanate (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). of RNA from was at with in a and The were then by at a of of and 1 of reverse (Life Technologies, at for The cDNA was then with the and based on base pairs and of the CRAMP cDNA These were used with the for of for 1 for 1 and for and a of for A second of was with nested and based on base pairs and of the CRAMP cDNA Amplification were as but a of was Products were on and using peptide synthesis of was done on a The synthesis was using andN,N-dimethylformamide as were carried out with a of an of acid, and in the of from the was with andN,N-dimethylformamide in the of To the was to the was and coupling and and and for the coupling of residues and was obtained by of Amino were with and and from the were carried out using a of for at peptides were extracted with and by reverse on a using a in the of The peptide was by the of at and using a of C.J. Anal. Biochem. 1989; PubMed Scopus Google Scholar). were with an were at on a μm) were in in the or of and The content was by the of 13: PubMed Scopus Google Scholar) from the using and as the for and of with the of and minimum inhibitory concentrations were by a R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar). The bacterial strains were Escherichia and and the gene by L. of of and The were at for The activity was using of and The were similar to used for that the species were and in liquid and the was at for The activity of and against of E. P. and S. was in were in the or in the of of peptides in a of a at were with PBS, in on Mueller-Hinton and for to allow R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar). inner membrane was with as B. Romeo D. Gennaro R. Infect. Immun. 58: PubMed Google Scholar). to were in by an of of in by of of CRAMP in at was mouse bone marrow cells in 100 of PBS, or blood cells in were to by at for in a in then for 1 with a of in PBS, were then for 1 with a of or To of were with CRAMP that been for 1 at with were for in and then for 1 at with a of was for with saline. were with and cells with on a Cramp was mapped by analysis of the of or M. M. M. S. B.A. PubMed Scopus Google Scholar) and M. or J. 1991; PubMed Scopus Google Scholar). of these have been for including the 9 and as R. 1995; PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar). were stored an using the by C. E. MD). and were to and in Google were by the of To isolate potential murine homologs to the family of antimicrobial peptides by a were designed using sequence from the conserved domain of the porcine gene A nested RACE strategy was on the highly prepro domain from species to derived were in to the cDNA sequence in other were identified by this approach despite than for by the 5′ cathelin domain but in the 3′ The murine cathelin-related cDNA contains an of base pairs and a predicted of the prepro of the predicted murine pig and cathelicidins identify sequence the murine protein with PR-39 and in the domain and with This sequence the conservation of at and potential sites at the of the cathelin portion of the peptide. The cDNA of and porcine PR-39 peptides in the 3′ the predicted of CRAMP is in this To evaluate the expression of Cramp in the mouse, RNA was extracted from murine isolated at and from adult mouse Northern blot of RNA from abundant 1-kilobase A and was as early as and and to RNA during development. In adult this transcript was by Northern blot analysis only in marrow RNA not To the for in adult a reverse strategy was Total RNA from brain, heart, spleen, testis, liver, stomach, and skeletal was and reverse was using nested to a for reverse for were and were RNA or reverse Cramp were in spleen, testis, stomach, and intestine corresponding to Cramp were also in heart, and skeletal but were not in brain, and All were with and in the of RNA or reverse the base pairs of Cramp, we of in M. in and and in M. of the was in the of of and with of than and mapped in these CRAMP showed to on 9 and was to This region of mouse 9 is homologous to with the of the of this The of the cathelicidins are to peptides with antimicrobial To the of the peptide predicted by the CRAMP peptides were synthesized. The first peptide, was based on the predicted at and is a peptide. The was based on potential at the at and is a peptide. of and that these peptides will form an amphipathic similar to that for B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar) and antimicrobial peptides such as the and To evaluate this spectral analysis of and was done The of in suggest that the peptide is in a and that will a structure in the of The CD of were similar to The of and to function as antimicrobial was on a variety of microbes peptides potent antimicrobial activity against Gram-negative bacteria with in the of 0.5–8.0 μm. CRAMP peptides were against strains μm) and not at the concentrations against P. C. and strains of S. The CRAMP peptides were also found to have potent activity in these the was at concentrations as as 1 activity of and and was as the of peptide growth with bacteria for or with for All strains were in Mueller-Hinton broth, B. which was in and C. and C. which were in liquid were using and and are the of at with a of not than with to in a activity of and microbicidal activity of and was by bacteria with the of peptide in the of of a at were with PBS, in on Mueller-Hinton and for to allow are expressed as of bacteria with to in the of peptide. in a was as the of peptide growth with bacteria for or with for All strains were in Mueller-Hinton broth, B. which was in and C. and C. which were in liquid were using and and are the of at with a of not than with to The microbicidal activity of and was by bacteria with the of peptide in the of of a at were with PBS, in on Mueller-Hinton and for to allow are expressed as of bacteria with to in the of peptide. The of E. inner membrane was for peptides. The reaction for with was than for a of has the to permeabilize bacteria and activity at a that is of that by bacterial was at the with by the potent to bacterial inner the CRAMP peptides μm) not or membranes not The expression of CRAMP protein in was with a against immunostaining was in myeloid cells mouse femoral marrow This was for CRAMP of peptide In CRAMP was detected in in neutrophil granules but was not in or Thus, CRAMP protein is expressed in in a similar to that for cathelin-related antimicrobial peptides in other species. In this study we and a antimicrobial peptide in the mouse. This is a member of a family of antimicrobial peptide precursors found in B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar, G.H. B. Odeberg J. Bergman T. B. R. Eur. J. Biochem. PubMed Scopus Google pig (6Agerberth B. Lee J.Y. Bergman T. Carlquist M. Boman H.G. Mutt V. Jörnvall H. Eur. J. Biochem. 1991; 202: 849-854Crossref PubMed Scopus (296) Google Scholar, 8Storici P. Zanetti M. Biochem. Biophys. Res. Commun. 1993; 196: 1058-1065Crossref PubMed Scopus (65) Google Scholar, R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar, P. Zanetti M. Biochem. Biophys. Res. Commun. 1993; 196: PubMed Scopus Google Scholar, C. L. Lehrer R.I. FEBS Lett. 1994; PubMed Scopus Google R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar, Gennaro R. M. Romeo D. J. Biol. Full Text PDF PubMed Google A. Scocchi M. Skerlavaj B. Gennaro R. FEBS Lett. 1994; 339: 108-112Crossref PubMed Scopus (84) Google and L. Scocchi M. Zanetti M. FEBS Lett. 1995; 376: 225-228Crossref PubMed Scopus (102) Google Scholar, 11Mahoney M.M. Lee A.Y. Brezinski-Caliguri D.J. Huttner K.M. FEBS Lett. 1995; 377: 519-522Crossref PubMed Scopus (76) Google Scholar). The the precursors of these antimicrobial peptides in a common NH2-terminal region of the as cathelin and has to use of the to describe the gene family. The mouse gene a high degree of with the cathelin domain members of this to a region of mouse chromosome 9 homologous to the of cathelicidins in pig and is and is expressed in neutrophil granules. this gene, a antimicrobial peptide and a host defense in mouse for further study of of innate The cathelicidins exert their antimicrobial effect based on the structurally diverse COOH-terminal domain of these peptides (7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar). To identify cathelicidins in mouse, were designed against the highly conserved NH2-terminal cathelin domain of these gene This approach has been in multiple cathelin-related genes in several species (7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar). a single cathelicidin was identified by this approach in mouse despite the use of multiple and for RNA and of than cathelin-related myeloid gene has been in mouse which contains a domain is not to be antimicrobial B. J. Cell. Biochem. 1995; PubMed Scopus Google Scholar). The to this transcript in a reverse approach such as in in Thus, antimicrobial cathelicidin has been in the work out the of multiple cathelin-related antimicrobial peptides such as found in other species. of a murine cathelin-related gene to use the mouse for study of and We found that the expression of Cramp in the mouse is in a and In the transcript expression was abundant in The was with that the protein was found in and in marrow cells of myeloid lineage and with the sites of expression of cathelicidins in man and pig (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar, G.H. B. Odeberg J. Bergman T. B. R. Eur. J. Biochem. PubMed Scopus Google Scholar, J. M.M. J. Leukocyte Biol. 1994; PubMed Scopus Google Scholar). However, CRAMP was also at in mouse testis, the gastrointestinal spleen, heart, and skeletal muscle. is that the of CRAMP in a variety of tissues is to the of of myeloid precursors in However, a of CRAMP in liver, and brain, tissues that are to of blood against pig cathelicidins have been found in the gastrointestinal and the cathelicidin has been found in B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar). are by the expression of cathelicidins in other species. of RNA during that CRAMP are also expressed during embryogenesis. was detected as early as in the mouse and The of CRAMP expression with the earliest of the murine with cells but the of D. J. PubMed Scopus Google Scholar). Thus, as in adult CRAMP RNA be expressed by The of these is expression of in a variety of tissues suggests that these tissues have an innate to microbial or an inflammatory antimicrobial peptides such as PR-39 are to activity including the to function as of surface expression (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar). of CRAMP in a variety of tissues and during embryogenesis is with functions in to antimicrobial work to evaluate expression and function of CRAMP is to these The 3′ region of CRAMP a that is an antimicrobial peptide. of the of the cathelicidins suggested that CRAMP be cleaved at multiple sites G.H. B. Odeberg J. Bergman T. B. R. Eur. J. Biochem. PubMed Scopus Google Scholar, M. Skerlavaj B. Romeo D. Gennaro R. Eur. J. Biochem. 1992; PubMed Scopus Google Scholar). of potential predicted that the mature peptides an with an amphipathic This is similar to a of cathelicidins which the and pig but not the cathelicidins characterized by and or mature peptides. analysis of CRAMP peptides this and the antimicrobial function of CD spectral analysis that these peptides can an done against a variety of microbes potent activity against Gram-negative bacteria and rapid of the inner membrane of E. These that CRAMP is a peptide. a similar cDNA from the mouse was FEBS Lett. PubMed Scopus Google Scholar). The corresponding peptide NH2-terminal acids used in and was not This is to be for the of antimicrobial activity analysis done in other cathelicidin-derived peptides has that the NH2-terminal domain of the mature peptide is for activity M. M. H. J. 1993; PubMed Scopus Google Scholar). of the peptide from the mouse will be to its In of CRAMP and of its expression and antimicrobial activity that the murine host defense system members of the cathelicidin family. This in the mouse to be applied to the study of these antimicrobial peptides. work to the function of these peptides in from and We and for for and Huttner for