The PGRPs

Peptidoglycan, which is mainly found in the Grampositive bacteria cell wall, is a highly potent target for recognition by eukaryotic cells. The term peptidoglycan-recognition protein was first introduced by Ashida's group (Yoshida et al, 1996); they isolated a 19 kDa protein from the haemolymph of the silkworm B. mori that showed a high affinity for Gram-positive bacteria and peptidoglycan and activated the downstream prophenoloxidase cascade that leads to melanization of microbial substances (Ochiai and Ashida, 1999). Subsequently, genes encoding PGRP-related structures were identified in other organisms, from Drosophila to Anopheles to humans (Kang et al, 1998; Liu et al, 2001; Christophides et al, 2002; Waterhouse et al, 2007), all having a peptidoglycan-recognition domain of approximately 165 amino acids with structural similarity to the peptidoglycan-binding region of lysozyme (Kim et al., 2003).

Based on the length of their gene products, insect PGRPs have been grouped into two classes: short PGRPs (PGRP-S), which are small extracellular proteins (19-20 kDa) similar to the originally described PGRP, and long PGRPs (PGRP-L), which are either intracellular or membrane-spanning proteins (Christophides et al, 2002). The C-terminal region of the PGRP-Ls, which is the most highly conserved and is a homologue of PGRP-Ss, has three domains (I, II, and III) (Werner et al, 2000). Drosophila has 13 PGRP genes that are transcribed into at least 17 PGRP proteins (Werner et al, 2000; Christophides et al, 2002). Of these 17, seven are short DPGRPs (SA, SB1, SB2, SC1a, SC1b, SC2, and SD), and 10 are long DPGRPs (LAa, LAb, LAc, LB, LCa, LCx, LCy, LD, LE, and LF) that either have a signal peptide and a predicted transmembrane domain or are intracellular proteins that are secreted by unknown mechanisms. Separate reduplication of two adjacent PGRP-LC domains in Drosophila has generated a novel gene, PGRP-LF, which is absent from mosquitoes (Christophides et al, 2002).

Anopheles has seven PGRP genes (Christophides et al, 2002; Waterhouse et al, 2007), three encoding short AgPGRPs (S1, S2, and S3) and the remaining four encoding six long AgPGRPs (LA1, LA2, LB, LC1, LC2 and LC3), some of which are splice variants (see Figure 5.2 for PRGP domain organization). Sequencing of the human genome has led to the identification of two additional PGRP homologues, HPGRP-Ia and HPGRP-Iß (Liu et al, 2001). The crystal structures of Drosophila PGRP-LB and -SA and human PGRP-Ia have been determined in separate studies (Kim et al, 2003; Guan et al, 2004; Reiser et al, 2004) and found to be very similar to bacteriophage T7 lysozyme.

Most PGRP genes are expressed in all post-embryonic stages, and insect PGRP-S and other short PGRPs are present in the haemolymph, cuticle, fat body, epidermal cells, gut, and, to a lesser extent, haemocytes. Long insect PGRPs are mainly expressed in haemocytes (Ochiai and Ashida, 1999; Werner et al, 2000; Christophides et al, 2002; Dimopoulos et al, 2002). The expression of several short and long PGRPs has been shown to be upregulated in D. melanogaster and in A. gambiae by exposure to various bacteria, to bacterial pep-tidoglycan, and by P. berghei challenge (Kang et al, 1998; Dimopoulos et al, 2002).

In D. melanogaster DPGRP-SA mutants, activation of the Toll pathway by Gram-positive bacteria has been shown to be blocked, and resistance to

ITZ^H tflyiwmm AgPGRP-S1, -S2, -S3 (extracellular) amrD— AgPGRP-LA2 (intracellular)

. Ag-PGRP-LA1, -LB, -LC1, -LC2, -LC3 (membrane spanning)

23 Signal peptide HHPGRP domain III

HTH PGRP domain I d Transmembrane domain

100 amino acids

Figure 5.2 The domain organization (signal peptide and PGRP domains) of the PGRP protein family of A. gambiae is shown; the thin horizontal black line indicates the length of each protein and boxes indicate specific domains The short PGRPs (S1, S2, and S3) are extracellular and among the long ones PGRP-LA2 is intracellular while the other five (PGRPLA1, -LB, -LC1, -LC2, and -LC3) are membrane-spanning.

Gram-positive infection has been found to be decreased (Michel et al., 2001; Gobert et al., 2003). In another report, a mutation in the gene PGRP-LC was shown to reduce survival in Gram-negative sepsis but to have no effect on the response to Gram-positive bacteria or natural fungal infections (Gottar et al., 2002). Experiments using RNA interference (RNAi) in Drosophila mbn-2 cells have shown that PGRP-LCx is the only isoform that is required to mediate signals from Gram-positive bacteria and purified bacterial peptidoglycan. In contrast, the recognition of Gram-negative bacteria and bacterial lipopolysaccharide requires both PGRP-LCa and -LCx. The third isoform, LCy, is expressed at lower levels than the other two and may be partially redundant (Werner et al, 2003). In Drosophila larvae, the Imd-mediated antibacterial defence has been shown to be activated by PGRP-LE. The product of this gene binds to the diaminopimelic acid (DAP)-type peptidoglycan, a cell-wall component of the bacteria that is capable of activating the Imd pathway, but not to the lysine-type peptidoglycan (Takehana et al, 2002). In a later study, PGRP-SC1/-2-depleted flies demonstrated a specific over-activation of the Imd signalling pathway after bacterial challenge (Bischoff et al., 2006).

In summary, the Drosophila PGRP-SA is required to activate the Toll receptor in response to the cleavage of the Toll-ligand Spätzle in the protease cascade that occurs as a result of infection with a Grampositive bacterium (Michel et al., 2001). PGRP-LC

is required for the activation of the Imd receptor in response to fungal and Gram-negative bacterial infection (Choe et al, 2002). It is also presumably involved in the phagocytosis of Gram-negative bacteria, since inhibition of PGRP-LC expression in Drosophila cell lines results in a decreased phagocytosis of Escherichia coli (Ramet et al, 2002). PGRP-SC1b has been suggested to possess an amidase activity that can degrade peptidoglycan (Mellroth et al, 2003). Thus, the PGRPs play a multi-faceted, pivotal role in D. melanogaster innate immunity and in the mosquito's defences against Gram-positive bacteria.

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