On the control of the microflora in the gastro intestinal tract : functional examples of antibacterial peptides from Helicobacter pylori and mouse small intestine

The human microflora contains at least 400 different species mainly localized to the gastro-intestinal tract. This indigenous flora is controlled by host factors as well as by the interference within the microbial community through the action of antimicrobial components. These components are in the focus of this thesis.

A microprotocol was developed for biochemical analysis of antimicrobial peptides in tiny tissue material. The protocol was based on a one extraction step, followed by separation on HPLC and mass-spectrometry analysis. This method was sensitive enough for the detection of antimicrobial substances of the small intestine from a single mouse. Ribosomal proteins with antibacterial activity contributed substantially to the microbicidal activity, in addition to already known components such as defensins, lysozyme and phospholipase A. Using this protocol we could demonstrate that the processing of prodefensins to mature microbicidal defensin peptides involves at least two-steps. No major impact of the microflora on the production of antimicrobial components in the small intestine could be found except for the production of the peptide CRS4-4C.

We found that the gastro-intestinal bacteria Helicobacter pylori possessed antibacterial activity. The origin of this activity was traced to cecropin like N-terminal fragments of ribosomal protein L1 (RpL1). Synthetic peptides based on the H. pylori N-terminal sequence were antibacterial but lacked cytolytic as well as hemolytic properties. To H. pylori this antibacterial activity may confer survival advantage during the colonisation phase of a new host when the pH is raised and H. pylori may have to face other faster growing bacteria.

The antibacterial activity from ribosomal proteins was found to be a common theme for both the mouse small intestine and H. pylori. The impact of microbicidal substances on host microbe interactions is an important factor for the understanding of the dynamics of the normal microflora. By the use of the microprotocol described here it may become possible to biochemically analyse these substances from human biopsies.

List of scientific papers

I. Pütsep K, Brändén CI, Boman HG, Normark S (1999). "Antibacterial peptide from H. pylori" Nature 398(6729): 671-672
https://pubmed.ncbi.nlm.nih.gov/99241827

II. Pütsep K, Normark S, Boman HG (1999). "The origin of cecropins; implications from synthetic peptides derived from ribosomal protein L1" FEBS Lett 451(3): 249-252
https://pubmed.ncbi.nlm.nih.gov/99297609

III. Pütsep K, Axelsson LG, Boman A, Midvedt T, Normark S, Boman HG, Andersson M (2000). "Germ-free and colonized mice generate the same products from enteric prodefensins" (Submitted)

IV. Pütsep K, Andersson M, Axelsson LG, Boman A, Midvedt T, Normark S, Boman HG (2000). "Antimicrobal components in the mouse small intestine of germ-free, monoinfected and conventional mice" (Manuscript)