Differential gene expression in rat colon by dietary heme and calcium.

Dietary heme and calcium are alleged modulators of colon cancer risk. Little is known about the molecular and cellular changes in the colon epithelium that are induced by consumption of these unabsorbed nutrients. In this nutrigenomics study, we fed rats high- and low-calcium diets with or without heme. In agreement with previous studies, we found that dietary heme increased the cytotoxicity of fecal water in the colon and elevated epithelial proliferation, a risk factor in colon carcinogenesis. Calcium reduced cytotoxicity and inhibits heme-induced effects. Among 365 colon-expressed genes, we could identify 10 diet-modulated genes that show >2-fold altered expression, of which several are related to colon cell turnover and disease. Mucosal pentraxin (Mptx) was the strongest differentially expressed gene, approximately 10-fold down-regulated by dietary heme and 3-fold up-regulated by calcium. cDNA microarray and quantitative PCR analysis show that calcium significantly inhibits the effects of heme, which correlates with the physiological effects. Our results indicate that Mptx expression is related to colonic cell turnover, and that Mptx might be a marker for diet-modulated mucosal integrity. We also show that Mptx expression is restricted to the intestine, and occurs predominantly in the colon.

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity.

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.

Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic.

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

Pore formation by nisin involves translocation of its C-terminal part across the membrane.

Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.

The orientation of nisin in membranes.

Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.

Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity.

Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

The C-terminal region of nisin is responsible for the initial interaction of nisin with the target membrane.

The interaction of nisin Z and a nisin Z mutant carrying a negative charge in the C-terminus ([Glu-32]-nisin Z) with anionic lipids was characterized in model membrane systems, and bacterial membrane systems. We focused on three possible steps in the mode of action of nisin, i.e., binding, insertion, and pore formation of nisin Z. Increasing amounts of anionic lipids in both model and natural membranes were found to strongly enhance the interaction of nisin Z with the membranes at all stages. The results reveal a good correlation between the anionic lipid dependency of the three stages of interaction, of which the increased binding is probably the major determinant for antimicrobial activity. Maximal nisin Z activity could be observed for negatively charged lipid concentrations exceeding 50-60%, both in model membrane systems as well as in bacterial membrane systems. We propose that the amount of negatively charged lipids of the bacterial target membrane is a major determinant for the sensitivity of the organism for nisin. Nisin Z induced leakage of the anionic carboxyfluorescein was more efficient as compared to the leakage of the potassium cation. This lead to the conclusion that an anion-selective pore is formed. In contrast to the results obtained for nisin Z, the binding of [Glu-32]-nisin Z to vesicles remained low even in the presence of high amounts of negatively charged lipids. The insertion and pore-forming ability of [Glu-32]-nisin Z were also decreased. These results demonstrate that the C-terminus of nisin is responsible for the initial interaction of nisin, i.e., binding to the target membrane.