The role of antimicrobial peptides in animal defenses

Abstract

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.

Figure 1

Model outlining the major events in induction of sepsis by bacteria and the points at which cationic peptides are proposed to intervene.

Figure 2

Inhibition of LPS–LBP interaction and LPS-induced TNF-α production by structurally different cationic peptides. (A) Biotinylated LPS (45 ng/ml) was added to wells with immobilized LBP in the presence or absence of the indicated cationic peptides (10 μg/ml). The peptides were added to the wells at the same time as the LPS, and residual LPS binding was assessed by ELISA. (B) RAW 264.7 cells were incubated with E. coli O55:B5 LPS (100 ng/ml) in the presence or absence of the indicated peptides (20 μg/ml) for 6 h. TNF-α released into the culture supernatant was measured by ELISA. Data are from ref. .

Figure 3

Effect of CEMA on LPS-induced gene expression in RAW 264.7 cells. RAW 264.7 cells were stimulated for 4 h with media alone, S. typhimurium LPS (100 ng/ml), or S. typhimurium LPS (100 ng/ml) and CEMA (50 μg/ml). The RNA was isolated from the cells and used to make ³²P-labeled cDNA probes, which were hybridized to the CLONTECH Atlas arrays, and after a 3-day exposure, they were analyzed with a Phosphorimager and CLONTECH atlas software. The average percent inhibition of gene transcription by CEMA as measured by a change in fold intensity is shown in the graph. The following selected genes are shown: iNOS, inducible nitric oxide synthase; MIP-2 α, macrophage inflammatory protein (chemokine); MIP-1 β, macrophage inflammatory protein (chemokine); IL-15, interleukin-15; (cytokine), Stat3, acute-phase response factor; ICE, interleukin-converting enzyme; CD40L, CD40 ligand; TTP, tristetraprolin: (destabilizes TNF mRNA); p130 and p107, retinoblastoma proteins; Brn-3.2 POU, transcription factor 1; TF II D, transcription factor; A3R, adenosine A3 receptor; ATBF1, AT motif-binding factor (transcription factor); BKLF, CACCC Box-binding transcription factor. Data are from M.G.S., C. M. Rosenberger, M. R. Gold, B. B. Finlay & R.E.W.H. (unpublished results).

Figure 4

Influence of CEMA on gene expression in RAW 264.7 cells. Gene arrays were used to compare transcription in unstimulated cells and cells stimulated with CEMA (50 μg/ml) for 4 h. The average fold change is shown for the following genes: p21/Cip1, cyclin-dependent kinase (cdk)-inhibitor protein 1; Rab-3b, ras-related protein; p27kip1, G1 cyclin-cdk protein kinase inhibitor; jun-D, c-jun-related transcription factor; Egr-1, Zn-finger transcription factor; NFAT 1, transcription factor; H-ras, transforming G-protein. Data are from M.G.S., C. M. Rosenberger, M. R. Gold, B. B. Finlay & R.E.W.H. (unpublished results).