The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O 2˙؊ ) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.MccJ25 is active on gram-negative bacteria related to the producer strain, including some pathogenic strains (43,44,55). Four plasmid genes, mcjABCD, are involved in MccJ25 production: mcjA, mcjB, and mcjC code for an MccJ25 precursor and two processing enzymes required for the in vivo synthesis of the mature peptide, respectively, and mcjD encodes the McjD immunity protein (53). McjD, a homologous ABC exporter family protein, participates in MccJ25 secretion (52). Thus, immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level (53). Recently, it has been demonstrated that YojI, a chromosomal protein with ABC-type exporter homology (36), is also able to export MccJ25 from the cells (14). TolC, an E. coli outer membrane protein, is necessary for MccJ25 secretion mediated by either McjD or YojI (11,14). On the other hand, the uptake of MccJ25 by E. coli is dependent on the outer membrane receptor FhuA (15, 45) and the four inner membrane proteins TonB, ExbD, ExbB, and SbmA, the first three of which constitute the Ton complex (38), while the last one acts as a transporter (46).Convincing evidence showing that RNA polymerase (RNAP) is the target for MccJ25 action in E. coli was previously provided by our laboratory. The peptide inhibits the enzyme activity by obstructing the secondary channel and consequently preventing access of the substrates to its active sites (1,12,34,57). Later, it was demonstrated that MccJ25 can bind and penetrate into the phospholipid monolayer and disrupt the electric potential of liposomes composed of phospholipids from gram-negative bacteria (5, 40). These results encouraged the study of the effect of MccJ25 on the bacterial membrane. MccJ25 was found to disrupt the membrane potential inhibiting oxygen consumption in Salmonella enterica serovar Newport (41) and S. enterica serovar Typhimurium transformed with a plasmid carrying fhuA from E. coli (55), suggesting the presence of a se...

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