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In Drosophila melanogaster, this system operates through two biochemical pathways: Toll, also present in mammals; and immune deficiency (IMD) pathway, similar to the mammalian tumor necrosis factor (TNF)-* receptor pathway. Over the past decade, biologists have described, in some detail, the Toll system, which combats Gram-positive bacteria and fungi. The IMD pathway, which targets Gram-negative bacteria, has remained sketchier.
A discovery reported in recent F1000-recommended papers should open up the IMD pathway to greater scrutiny. Based on independently conducted studies, the three papers describe a putative transmembrane receptor that recognizes Gram-negative bacteria.1-3 A member, by homology, of the family of 13 Drosophila peptidoglycan-recognition proteins (PGRPs), the newly identified protein is called PGRP-LC.
Ezekowitz, senior author of one paper, was seeking genes that affect phagocytosis, when his lab chanced upon PGRP-LC.1 Applying the relatively new technique of RNA interference (RNAi), postdoc Mika Rämet exposed cultured macrophage-like Drosophila cells to RNA that encoded PGRP-LC. This treatment reduced phagocytosis of Escherichia coli, a Gram-negative bacterium, but not of Gram-positive Staphylococcus aureus.
The paper's approach impresses Lisa K. Denzin, an F1000 reviewer and immunologist at New York's Memorial Sloan-Kettering Cancer Center. She describes RNAi as a "very powerful technique" that allows researchers to create what are essentially gene-knockout cell lines. The main challenge is inserting double-stranded RNA into cells. "It apparently works really well for Drosophila," says Denzin, "but people are having problems in mammalian systems."
Another of Ezekowitz's postdocs, Pascal Manfruelli, generated mutant flies that produced little or no PGRP-LC. They were more susceptible to infection by E. coli than by Micrococcus luteus, a Gram-positive bacterium. PGRP-LC mutants yielded similar results in a study led by Jules A. Hoffmann at the Centre National de la Recherche Scientifique in Strasbourg, France. The Hoffmann group also found that PGRP-LC overexpression turns on the gene encoding diptericin, a peptide that acts against Gram-negative bacteria.2
Kathryn V. Anderson, a molecular biologist at Memorial Sloan-Kettering, identified PGRP-LC through a different approach. Her lab first isolated 56 fly mutants that responded defectively to bacteria. By combing through these mutants, graduate student Kwang-Min Choe learned that mutations of ird7, a chromosome 3 gene encoding PGRP-LC, strongly affected the antibacterial response. In infected ird7 mutants, various antibacterial peptide genes were not induced, and RNAi blocked such induction in a PGRP-LC wild-type cell line. Unlike wild-type flies, infected mutants also failed to process the IMD pathway's nuclear factor (NF)-*B-like transcription factor Relish.3
One unknown is PGRP-LC's location in vivo; an oft-mentioned candidate site is the fly's liver-like fat body, the source of most antimicrobial peptides. Another unknown: whether PGRP-LC is activated by the end-product of a proteolytic cascade, as occurs in the Toll pathway, or whether it interacts directly with bacteria. A possible ligand is lipopolysaccharide (LPS), contained in the cell walls of Gram-negative, but not Gram-positive, bacteria.
Yet another unsettled issue is whether PGRP-LC interacts exclusively with Gram-negative bacteria. According to Anderson's paper, ird7 mutation prevents induction of antibacterial peptide genes after infection by Gram-positive bacteria (including M. luteus) as well as Gram-negative bugs. (Both bacterial types, she notes, contain a possible PGRP-LC ligand, peptidoglycan, in their cell walls, though it is below the outer layer in Gram-negatives.) Ezekowitz acknowledges that although PGRP-LC "appears to favor binding to Gram-negatives and LPS, it has not been exhaustively shown that it does not bind [to] Gram-positives."
If further studies validate PGRP-LC's role as a receptor for the IMD pathway, the practical implications are intriguing. An agent that blocks the protein's interactions with bacteria might act as an insecticide by rendering insects more vulnerable to infection. Conversely, an agent that potentiates such interactions might allow insects' immune systems to better vanquish vectors that later cause such human diseases such as malaria. The challenge is figuring out how to introduce such agents into an insect's body. "I don't think just feeding [the agents] would work well," says Anderson.
Researchers also remain intrigued by the discovery of four PGRP genes in humans. Roman Dziarski, a microbiology and immunology professor at Indiana University School of Medicine in Gary, recently cloned three of those genes. Noting that mammals lack an IMD pathway, Dziarski hypothesizes that PGRPs' role in humans "is likely to be different than in insects."
1. M. Rëmet et al., "Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli," Nature, 416:644-8, April 11, 2002.
2. M. Gottar et al., "The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein," Nature, 416:640-4, April 11, 2002.
3. K.-M. Choe et al., "Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila," Science, 296:359-62, April 12, 2002.