In Pseudomonas aeruginosa the LasR protein is required for activation of lasB and several other virulence genes. A diffusible signal molecule, the P. aeruginosa autoinducer (PAI), produced by the bacterial cel and released into the growth medium, is required for activity ofLasR. By cloning a lasB::lacZ fusion and a lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI. We have used this assay to foUlow the purification of PAI from cel-free culture supernatant fluids in which P. aeruginosa or E. coli containing the P. aeruginosa gene required for autoinducer synthesis, lasI, had been grown. Chemical analyses indicated the purified material was 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)dodecanamide. To confirm this assignment, the compound was synthesized and the synthetic compound was shown to have chemical and biological properties identical to those of PAI purified from culture supernatant fluids. The elucidation of the PAI structure suggests therapeutic approaches toward control of P. aeruginosa infections.

Previous studies have demonstrated that beta-defensins exhibit chemotactic activity by sharing the chemokine receptor CCR6 with the CC chemokine ligand CCL20/macrophage-inflammatory protein-3alpha (MIP-3alpha). Structural analysis of CCL20/MIP-3alpha revealed that most of the positively charged residues are concentrated at one area of its topological surface, a characteristic considered to be important for the antimicrobial activity of defensins. Here, we report that similar to defensins, CCL20/MIP-3alpha has antimicrobial effects on Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Streptococcus pyogenes, Enterococcus faecium, Staphylococcus aureus, and Candida albicans. Additionally, by screening a total of 30 human chemokines, we have identified an additional 17 human chemokines, which exhibit antimicrobial activity in vitro. Collectively, about two-thirds of the chemokines investigated so far has the capacity to kill microorganisms in vitro, suggesting that antimicrobial activity may be another host-defense function for certain chemokines. Comparison of the structural characteristics between antimicrobial and nonantimicrobial chemokines suggests that topological formation of a large, positively charged electrostatic patch on the surface of the molecule is likely to be a common structural feature of antimicrobial chemokines.

Six a-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human a-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known a-and b-defensins. Like HNP3 but in contrast to murine or rabbit a-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human a-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human a-defensins.

Human ␤-defensin 3 (hBD3) is a highly basic 45-amino-acid protein that acts both as an antimicrobial agent and as a chemoattractant molecule. Although the nature of its antimicrobial activity is largely electrostatic, the importance of the molecular structure on this activity is poorly understood. Two isoforms of hBD3 were synthesized: the first with native disulfide linkages and the second with nonnative linkages. In a third synthetic peptide, all cysteine residues were replaced with ␣-aminobutyric acid, creating a completely linear peptide. A series of six small, linear peptides corresponding to regions of hBD3 with net charges ranging from ؉4 to ؉8 (at pH 7) and lengths ranging from 9 to 20 amino acids were also synthesized. The linear full-length peptide showed the highest microbicidal activity against Escherichia coli and Staphylococcus aureus, while all three full-length forms showed equal activity against Candida albicans. The linear peptide also showed high activity against Enterococcus faecium and Pseudomonas aeruginosa. Peptides corresponding to the C terminus showed higher activities when tested against E. coli, with the most active peptides being the most basic. However, only the peptide corresponding to the N terminus of hBD3 showed any activity against S. aureus and C. albicans. Further, N-terminal deletion mutants of native hBD3 showed diminished activities against S. aureus. Thus, the antimicrobial properties of hBD3 derivatives are determined by both charge and structure.