Formylmethionyl-leucylphenylalanine and the SOS operon in Escherichia coli: a model of host-bacterial interactions.

To determine the biological significance of the existence of highly specific receptors for the bacterial chemotactic peptide formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) on neutrophil leucocytes, we investigated the role of this peptide in bacterial metabolism. The UmuD protein of the Escherichia coli SOS operon was identified as having an N-terminal fMet-Leu-Phe sequence and a recombinant E. coli with the umuD gene on plasmid pSB13 was shown to be an over-producer of both UmuD and fMet-Leu-Phe. Activation of SOS genes in conventional wild-type E. coli (K12) by u.v. light or hydrogen peroxide increased fMet-Leu-Phe production up to 4-fold. A RecA- strain, incapable of SOS activation, was a low basal producer of fMet-Leu-Phe and showed no increased production with u.v. light or oxidant stress. We propose that host phagocytes respond to fMet-Leu-Phe and closely related peptides because they are generated by bacteria under oxidant stress. Increased fMet-Leu-Phe production may signal to the host a change in the organism's biological status from commensal to pathogen because of the invasion into tissues exposing bacteria to high pO2 levels and oxidant stress.

Isolation and purification of N-formylmethionine aminopeptidase from rat intestine.

The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.

Identification of formyl Met-Leu-Phe in culture filtrates of Helicobacter pylori.

Helicobacter pylori synthesizes and secretes a substance which co-chromatographs and is antigenically cross-reactive with the bacterial chemotactic peptide fMet-Leu-Phe. Using reverse phase and affinity chromatography this substance has now been purified. Carboxypeptidase Y microsequencing has verified that this material is fMet-Leu-Phe. The infiltration of polymorphonuclear leucocytes to sites of H. pylori infection may be a response to mucosal permeation of soluble, diffusable bioreactive substances such as fMet-Leu-Phe.

Radio-immunoassay for formyl methionyl leucyl phenylalanine. I. Development and application to assessment of chemotactic peptide production by enteric bacteria.

Bacterial chemotactic peptides are low molecular weight peptides which stimulate a wide range of neutrophil functions following binding to specific leucocyte receptors. Formyl methionyl leucyl phenylalanine (FMLP) is the major chemotactic peptide in Escherichia coli culture supernatants. This paper reports the development and validation of a radio-immunoassay (RIA) for FMLP and its application to the analysis of formyl peptide production by enteric bacteria in vitro. The assay was moderately sensitive (10 nmol/L FMLP) and highly specific showing cross reactivity with F-met-leu-tyr, F-nle-leu-phe and F-met-met-met sequences (ID50 = 200, 100 and 250 nmol/L, respectively) but no significant cross reactivity with non-formylated or other formylated di- and tri-peptides (ID50 = 10(5) nmol/L. Culture supernatants from five species of enteric bacteria were filtered, concentrated and fractionated by reverse phase high performance liquid chromatography before RIA. All five organisms produced immunoreactive F-met peptides. A major peak of immunoreactivity co-chromatographing with authentic FMLP was found in all supernatants, but additional peaks representing more hydrophobic peptides were found in Streptococcus faecalis and Bacteroides fragilis cultures. In E. coli culture supernatants, concentration of immunoreactive FMLP increased in a linear fashion during 3 h of log phase growth reaching 31.2 nmol/L(s.e.m. = 10) with final bacterial concentrations of 3 +/- 0.73 x 10(8)/mL (n = 6). These findings extend earlier work showing production of bioactive formyl oligopeptides by different species of enteric bacteria and suggest that a RIA for FMLP will be a useful tool for investigating the production and metabolic fate of such peptides in man.

Partial purification and characterization of a formylmethionine deformylase from rat small intestine.

A formylmethionine deformylase from rat small-intestinal mucosa has been isolated, characterized and partially purified. The enzyme catalyses the release of equimolar amounts of formate and the free amino acid. The deformylase was active against formylmethionine (Km 7.1 mM) and formylnorleucine, but showed reduced activity against formyl-leucine. It was inactive against a range of other polar and nonpolar formyl-amino acids and against formyl di- and tri-peptides. The Mr of the native enzyme was between 45,000 and 66,000, as determined by h.p.l.c. gel permeation. Further purification of the enzyme either by h.p.l.c. ion-exchange chromatography and concanavalin A-Sepharose or by isoelectric focusing yielded a preparation with one predominant band of Mr 50,000 on SDS/polyacrylamide-gel electrophoresis. Bacteria in the intestine present the host with substantial amounts of formylmethionine (fMet) from proteinase and carboxypeptidase digestion of bacterial formyl-peptides in the intestinal lumen. fMet (0.01-1.0 mM) inhibited translation of a test RNA from brome mosaic virus in vitro, indicating that it could have adverse effects on cellular metabolism. Gut epithelial fMet deformylase may be required for deformylation of this exogenous (bacterial) and also endogenous (mitochondrial) fMet.