Dataset Information

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA.

ABSTRACT: Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

SUBMITTER: Wang L

PROVIDER: S-EPMC1618108 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA.

Wang Lihui L Weber Alexander N R AN Atilano Magda L ML Filipe Sergio R SR Gay Nicholas J NJ Ligoxygakis Petros P

The EMBO journal 20061005 20

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to h ...[more]

PMID: 17024181

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Project description:Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. The peptidoglycan of the bacterial cell wall is turned over steadily during growth. As peptidoglycan fragments were found in large amounts in spent medium of exponentially growing Gram-positive bacteria, their ability to recycle these fragments has been questioned. We conclusively showed recycling of the peptidoglycan component MurNAc in different Gram-positive model organisms and revealed that a MurNAc-6P etherase (MurQ or MurQ ortholog) enzyme is required in this process. We further demonstrated that recycling occurs predominantly during the transition to stationary phase in S. aureus and B. subtilis, explaining why peptidoglycan fragments are found in the medium during exponential growth. We quantified the intracellular accumulation of recycling products in MurNAc-6P etherase gene mutants, revealing that about 5% and 10% of the MurNAc of the cell wall per generation is recycled in S. aureus and B. subtilis, respectively. Importantly, we showed that MurNAc recycling and salvaging does not sustain growth in these bacteria but is used to enhance survival during late stationary phase.

Project description:BACKGROUND: The metabolism of bacterial peptidoglycan is a dynamic process, synthases and cleavage enzymes are functionally coordinated. Lytic Transglycosylase enzymes (LT) are part of multienzyme complexes which regulate bacterial division and elongation. LTs are also involved in peptidoglycan turnover and in macromolecular transport systems. Despite their central importance, no LTs have been identified in the human pathogen Streptococcus pneumoniae. We report the identification of the first putative LT enzyme in S. pneumoniae and discuss its role in pneumococcal peptidoglycan metabolism. RESULTS: Homology searches of the pneumococcal genome allowed the identification of a new domain putatively involved in peptidoglycan cleavage (PECACE, PEptidoglycan CArbohydrate Cleavage Enzyme). This sequence has been found exclusively in Gram-positive bacteria and gene clusters containing pecace are conserved among Streptococcal species. The PECACE domain is, in some instances, found in association with other domains known to catalyze peptidoglycan hydrolysis. CONCLUSIONS: A new domain, PECACE, putatively involved in peptidoglycan hydrolysis has been identified in S. pneumoniae. The probable enzymatic activity deduced from the detailed analysis of the amino acid sequence suggests that the PECACE domain may proceed through a LT-type or goose lyzosyme-type cleavage mechanism. The PECACE function may differ largely from the other hydrolases already identified in the pneumococcus: LytA, LytB, LytC, CBPD and PcsB. The multimodular architecture of proteins containing the PECACE domain is another example of the many activities harbored by peptidoglycan hydrolases, which is probably required for the regulation of peptidoglycan metabolism. The release of new bacterial genomes sequences will probably add new members to the five groups identified so far in this work, and new groups could also emerge. Conversely, the functional characterization of the unknown domains mentioned in this work can now become easier, since bacterial peptidoglycan is proposed to be the substrate.

Dataset Information

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA.

Publications

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA.

Similar Datasets

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