Innate Immune Responses in Peptidoglycan Recognition Protein L-Deficient Mice

The ability of the host to distinguish between self and nonself remains a central hallmark of innate immunity. Microbial organisms express distinct cell surface molecules, such as peptidoglycan (PGN) and lipoteichoic acid (LTA) on gram-positive bacteria and lipopolysaccharide (LPS) on gram-negative bacteria, that are structurally indispensable components of the cell wall. Recognition of these pathogen-associated molecular patterns, or PAMPs (14), is achieved predominantly by the vertebrate Toll-like receptor (TLR) family proteins, which collectively mediate induction of antimicrobial defensins, cytokines, chemokines, and dendritic cell maturation important to innate and adaptive immune responses. PGN recognition proteins (PGRPs) were first identified by their ability to bind PGN and complement the prophenoloxidase cascade that induces melanization around pathogens in insects (37). Thirteen PGRP family members are present in Drosophila (36), and each contains a conserved carboxyl-terminal region with homology to bacterial T7 lysozyme (11). Some family members, such as PGRP-SC1b, have enzymatic activity and can digest PGN by hydrolyzing the amide bond between N-acetylmuramic acid and l-alanine (20). The N-acetylmuramyl-l-alanine amidase activity may function to reduce the immunostimulatory effects of PGN (12). Definitive evidence indicating essential roles for PGRPs in Drosophila innate immunity was revealed in studies of mutants defective in Toll and Imd pathway-mediated antimicrobial responses. Infection of flies by gram-positive bacteria and fungi induces localized production of cleaved Spaetzle, the Toll ligand. Stimulation of Toll by ligand binding activates latent NF-κB-like transcription factors, resulting in expression of the antimicrobial peptide drosomycin. Infection by gram-negative bacteria results in activation of Imd, a Drosophila RIP-like adapter (5), and the Relish pathway, with expression of antibacterial peptides active against these bacteria, including diptericin, attacin, drosocin, and cecropins (11). Drosophila with a mutated PGRP-SA gene failed to activate drosomycin expression and was unable to survive challenge with gram-positive bacteria (21). In contrast, responses to gram-negative bacteria and fungi were not affected. A constitutively active Toll mutant could overcome the PGRP-SA mutation, suggesting that PGRP-SA functions upstream of Toll (21). The capacity of PGRP-SA to bind to PGN, likely in conjunction with gram-negative binding protein (6, 24), must therefore constitute a crucial and nonredundant recognition element upstream of Toll pathway activation in Drosophila. A similar screen identified PGRP-LC upstream of the Imd pathway (1, 7, 27). Like Imd mutants, PGRP-LC mutants poorly induced antimicrobial peptides, particularly diptericin, and demonstrated increased susceptibility to gram-negative bacteria; responses to gram-positive bacteria and fungi were unaffected. As in the Toll pathway, a constitutively active Imd mutation could rescue antimicrobial peptide induction attenuated by the PGRP-LC mutation (1, 7). Therefore, PGRP-LC plays a crucial and nonredundant role in detecting gram-negative bacteria and leading to activation of the Imd/Relish pathway. Although LPS is a major component of the gram-negative bacterial cell wall, PGN constitutes a shared cell wall element in both gram-positive and gram-negative bacteria (28). Structurally similar, these cell wall PGNs differ at the third amino acid of the peptide side chain, where lysine (Lys-PGN) and meso-diaminopimelic acid (Dap-PGN) are used in gram-positive and gram-negative organisms, respectively (28). Unlike LPS, Dap-PGN induced gram-negative-like Imd activation in flies, whereas Lys-PGN fully induced the gram-positive-like Toll pathway response (15, 18). Together, these studies suggest that different PGRP proteins in Drosophila mediate direct interactions with distinctive PGN moieties in bacterial cell walls as a proximal mechanism upstream of Toll and Imd activation. Indeed, PGRP-LE binds Dap-PGN but not Lys-PGN in vitro, consistent with this paradigm (31). PGRP is conserved in mice and humans, where four PGRP proteins are generated from three genes (19). The longest, PGRP-L, is a predicted membrane protein expressed mainly in liver. The shortest, PGRP-S, is a predicted soluble protein expressed in neutrophils. The intermediately sized family members, PGRP-Iα and -Iβ, are splice products from the same gene. PGRP-Iα/Iβ are predicted membrane proteins that are expressed in the esophagus (19). Like the Drosophila PGRPs, mammalian PGRPs have conserved carboxyl-terminal PGRP domains homologous to lysozyme (19). Only PGRP-L, however, has N-acetylmuramyl-l-alanine-amidase activity that digests PGN. The remaining family members have critical amino acid substitutions that abrogate enzymatic activity (4, 35), but all four mammalian proteins retain the ability to bind PGN in vitro (19). In contrast to the dramatic phenotype of PGRP-SA mutants in Drosophila, the PGRP-S null mouse displayed normal susceptibility to gram-negative and gram-positive challenge with Escherichia coli and Staphylococcus aureus. A nonpathogenic gram-positive Bacillus species was cleared less efficiently, however, and the killing and digestion of gram-positive bacteria by PGRP-SA-deficient neutrophils were compromised in vitro, although phagocytosis remained intact. Further, murine PGRP-S had no role in TLR-2-mediated cytokine induction (3). To define further the function of PGRPs in mammalian innate immunity, we disrupted the mouse PGRP-L gene and examined the role of this protein in host defense against bacterial and fungal challenges.