Peptidoglycan Stem Peptide Research Articles

Evolution of Transmembrane Protein Kinases Implicated in Coordinating Remodeling of Gram-Positive Peptidoglycan: Inside versus Outside

Members of a family of serine/threonine protein kinases (STPKs), unique to gram-positive bacteria, comprise an intracellular kinase domain and reiterated extracellular PASTA (for "penicillin-binding protein and serine/threonine kinase associated") domains. PASTA domains exhibit low affinity for beta-lactam antibiotics that are structurally similar to their likely normal ligands: stem peptides of unlinked peptidoglycan. The PASTA-domain STPKs are found in the actinobacteria and firmicutes and, as exemplified by PknB of Mycobacterium tuberculosis, they are functionally implicated in aspects of growth, cell division, and development. Whereas the kinase domains are well conserved, there is a wide divergence in the sequences of the multiple PASTA domains. Closer inspection reveals position-dependent evolution of individual PASTA domains: a domain at one position within a gene has a close phylogenetic relationship with a domain at a similar position in an orthologous gene, whereas neighboring domains have clearly diverged one from one another. A similar position-dependent relationship is demonstrated in the second family of proteins with multiple PASTA domains: the high-molecular-weight type II penicillin-binding protein (PBP2x) family. These transpeptidases are recruited to the division site by a localized pool of unlinked peptidoglycan. We infer that protein localization is guided by low-affinity interactions between structurally different unlinked peptidoglycan stem peptides and individual PASTA domains. The STPKs possess a greater multiplicity and diversity of PASTA domains, allowing interactions with a wider range of stem-peptide ligands. These interactions are believed to activate the intracellular kinase domain, allowing an STPK to coordinate peptidoglycan remodeling and reproduction of a complex cell wall structure.

Human peptidoglycan recognition protein S is an effector of neutrophil-mediated innate immunity

AbstractInnate immune responses to bacteria require cooperative interactions between host recognition molecules and phagocytes. The peptidoglycan recognition proteins (PGRPs) are a large group of proteins found in insects and mammals that bind to bacterial peptidoglycan (PGN). PGRP-S is located with other antimicrobial proteins, such as lysozyme, in the granules of human neutrophils. Whereas both PGRP-S and lysozyme recognize PGN, the exact binding specificity of human PGRP-S, its functional activity, and its potential synergy with other neutrophil-derived bactericidal proteins such as lysozyme have not been determined. Here we show that human PGRP-S binds to and inhibits the growth of Staphylococcus aureus (containing lysine-type PGN) and Escherichia coli (containing mesodiaminopimelic acid-type PGN). The binding affinity and thus antimicrobial activity of PGRP-S is determined by the third amino acid in the PGN stem peptide. Furthermore, the antimicrobial effect of PGRP-S against E coli is synergistic with lysozyme, and lysozyme and PGRP-S colocalize in neutrophil extracellular traps (NETs), suggesting that these granule-derived proteins act together to kill bacteria trapped in the NETs. Taken together, these results indicate that human PGRP-S plays a role in innate immunity in the context of neutrophils by contributing to the killing of intracellular and extracellular bacteria. (Blood. 2005;106:2551-2558)

Crystal Structure of the Actinomadura R39 DD-peptidase Reveals New Domains in Penicillin-binding Proteins

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.

Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 A resolution. PBP3 folds into an NH(2)-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated beta-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel beta-sheet surrounded by alpha-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k(cat)/K(m) = 50,500 M(-1) x s(-1)), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.

The carboxyl terminus of peptidoglycan stem peptides is a determinant for methicillin resistance in Staphylococcus aureus.

A mecA-containing Staphylococcus aureus strain was grown in the presence of high concentrations of D-serine, D-threonine, and D-phenylalanine. These growth conditions resulted in the replacement of the carboxyl-terminal (fifth) D-alanine residue of peptidoglycan stem peptides with the D-amino acid present in the growth medium and a reduced ability to grow in the presence of methicillin. The most dramatic effect was seen with D-serine. With 32 mM D-serine, strains that had been able to grow in the presence of 800 micro g of methicillin per ml were only able to grow in the presence of less than 50 micro g/ml. The results also suggest that in S. aureus vancomycin resistance mediated through the incorporation of precursors not terminating in D-alanyl-D-alanine would be mutually exclusive with expression of mecA-mediated methicillin resistance.

Experimental Chronic Arthritis and Granulomatous Inflammation Induced byBifidobacteriumCell Walls

Effects of cell walls (CWs) from two almost identical strains of Bifidobacterium adolescentis were studied in rats, using three different doses. A single i.p. injection of both CWs triggered a long-lasting arthritis with CW degradation products present in the joint tissue. Histologically, the arthritis was characterized by inflammatory cells, synovial hyperplasia, pannus formation and bone erosion, closely resembling human rheumatoid arthritis (RA). In addition, CWs of the other strain induced a remarkable granuloma formation in the spleen and liver. Both CWs have the same peptidoglycan (PG) type A4alpha/beta, but differ from each other in three aspects. CW of the granuloma inducing strain: firstly has more lysine and less ornithine in PG stem peptides; secondly is more resistant to lysozyme degradation, and thirdly is better retained in the spleen. All these in comparison to the other strain used. Such characteristics are associated with the capacity to induce chronic arthritis, but it remains open how crucial they are for the granuloma formation.

What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wall-induced arthritis.

To study what determines the arthritogenicity of the bacterial cell wall (CW) using Eubacterium CW-induced arthritis in the rat. Eubacterium aerofaciens, previously reported as arthritogenic, and E. limosum and E. alactolyticum, known as non-arthritogenic, were used. Gas chromatography-mass spectrometry (GC-MS) was applied to analyse the chemical composition of the bacterial cell wall. Cellular immune response was measured by concanavalin A (Con A) stimulation and FACScan analysis. Also, serum antibodies against the injected cell wall were determined. Unexpectedly, from the two strains of E. aerofaciens used only one proved to be arthritogenic (with a CW inducing chronic arthritis after a single intraperitoneal injection), even though these two strains were 100% identical by 16S rDNA analysis. CW of the other E. aerofaciens strain induced only transient acute arthritis; CW of E. limosum and E. alactolyticum induced weak signs of acute arthritis. Based on the GC-MS analysis and on the results published previously, putative structures of peptidoglycan (PG) in the four CW preparations are presented. It is apparent that the presence of lysine in position 3 of the PG stem peptide contributes to arthritogenicity but is alone not decisive. Both strains of E. aerofaciens were immunosuppressive, when tested by Con A response at 2 weeks after CW injection. Such an immunosuppression was not observed after injection of CW from E. limosum or E. alactolyticum. FACScan analysis for six T cell markers and studies on serum antibody responses did not reveal any differences in the effect of the four bacterial strains used. The results obtained suggest that the chemical structure of PG present in the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity. Therefore, from two bacterial strains belonging to normal human intestinal flora and 100% identical by 16S rDNA analysis, one proved to be arthritogenic and the other non-arthritogenic.

Digestion of Streptococcus pneumoniae Cell Walls with Its Major Peptidoglycan Hydrolase Releases Branched Stem Peptides Carrying Proinflammatory Activity

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr <750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr >1000) were >/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented </=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.

Glutamine synthetase and heteroresistance in methicillin-resistant Staphylococcus aureus.

Inactivation of femC in methicillin-resistant Staphylococcus aureus (MRSA) results in lowered methicillin resistance and a reduction in the amidation of the iso-D-glutamate of the peptidoglycan stem peptide. The femC phenotype is due to insertional inactivation of the glutamine synthetase repressor gene glnR by Tn551, which has a polar effect on glutamine synthetase (glnA) transcription. The complete glutamine synthetase operon (glnRA) of S. aureus was cloned and sequenced, and its transcriptional start was determined. The deduced amino acid sequence of the staphylococcal glutamine synthetase showed 76% identity and 87% similarity to the Bacillus subtilis glutamine synthetase. The staphylococcal glnRA operon was shown to complement an Escherichia coli glutamine synthetase-negative mutant and to restore methicillin resistance in femC mutants. femC mutants revert to resistance in the presence of high concentrations of methicillin. These revertants, which still carried the femC lesion, were shown to retain the lowered amidation of the iso-D-glutamate peptidoglycan stem peptide. A new chromosomal locus hmrC was postulated to have mutated to allow expression of high methicillin resistance in these femC revertants. Although the highly resistant hmrC revertant resembled phenotypically the highly methicillin-resistant subclones occurring in heterogeneously resistant MRSA, we could show by transduction that the locus hmrC was distinct from chr*, a chromosomal site postulated to confer high methicillin resistance in heterogeneous MRSA. This suggests that S. aureus can adopt multiple ways to achieve high methicillin resistance.

Altered peptidoglycan structure in a pneumococcal transformant resistant to penicillin.

A series of isogenic pneumococcal transformants differing in their levels of penicillin resistance and containing altered penicillin-binding proteins were compared for their cell wall structures by using a recently developed technique that can resolve the peptidoglycan stem peptides of Pneumococcus strains to over 40 components (J. F. Garcia-Bustos, B. T. Chait, and A. Tomasz, J. Biol. Chem. 32:15400-15405). The stem peptides from the highly resistant transformants differed strikingly from those of the susceptible recipient strain, and the peptide patterns were almost identical to that of the DNA donor. Four peptides representing the major components in the walls of susceptible cells were replaced by six new peptides that were only minor components of susceptible cell walls. A remarkable common feature of these new species was their high alanine content. Amino acid analysis, sequencing, and mass spectrometry allowed the assignment of the extra alanine residues to dialanine or alanylserine cross bridges in the six new stem peptides. The common feature of the four peptide species that were present as major components in the susceptible walls, but became minor species in the resistant cells, was the absence of a cross bridge in at least one of the stem peptide components. We suggest that the extensive remodelling of cell wall synthetic enzymes that accompanies acquisition of penicillin resistance eventually also alters the reactivity of these proteins towards their natural substrates in cell wall synthesis. As a result, highly penicillin-resistant pneumococci will shift from the use of wall precursors with linear stem peptides to a preferential use of precursors containing the more-hydrophobic peptides carrying dialanyl or alanylserine cross bridges.