Germ-free and colonized mice generate the same products from enteric prodefensins.
The use of germ-free mice offers the possibility to study antibacterial components in a gut uncolonized by bacteria. We have developed a method to extract and high pressure liquid chromatography-fractionate the antibacterial factors present in the small intestine of a single mouse. By mass spectrometry and sequence analyses of fractions exhibiting antimicrobial activity, we identified and characterized the defensin region in germ-free mice as well as in colonized mice. Defensins made up around 15% of the total antibacterial activity both in germ-free and colonized mice. The intestine of germ-free mice exhibited the same set of mature enteric defensins (defensins 1, 2, 3, 4, and 6) as mice colonized by a normal microflora. Mature defensins are generated through processing of larger precursors by enzymatic removal of a signal peptide and a propiece. We found that all prodefensins were cleaved at a Ser/Ala-Leu bond, giving 34-residue propiece peptides and only trace amounts of the predicted 39-residue peptide. This first step must be followed by the removal of a residual peptide to render the mature defensins, indicating that the processing is more complex than previously anticipated. The same propieces were found in both germ-free and colonized mice, suggesting that the same processing operates independent of bacterial presence in the intestine.
Putsep
K
K
Microbiology and Tumor Biology Center, Medical Biochemistry and Biophysics, Chemistry I, and Cell and Molecular Biology, Laboratory for Medical Microbial Ecology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Axelsson
L G
LG
Boman
A
A
Midtvedt
T
T
Normark
S
S
Boman
H G
HG
Andersson
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D023082:Defensins / Q000737:chemistry / Q000378:metabolism*; D005856:Germ-Free Life; D007421:Intestine, Small / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D023082:Defensins; D010446:Peptide Fragments; D011498:Protein Precursors