Nitrosative stress: Metabolic pathway involving the flavohemoglobin

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms l-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)—compounds identified with antimicrobial activities of NO synthase—elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.

PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.

Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

The role of antimicrobial peptides in animal defenses

It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.