The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

Introduction. Plasmid-mediated resistance to the glycopeptide antibiotics vancomycin and teicoplanin was first detected in 1986 (35, 53). It was rapidly shown that glycopeptide-resistant enterococci had a broad geographical distribution and were phenotypically and genotypically heterogeneous (Table 1). Strains displaying the VanA resistance phenotype have been studied extensively, leading to the first elucidation of a mechanism of resistance to glycopeptides and to an insight into the regulation and mode of dissemination of the corresponding genes. We review the experimental approaches used to characterize the molecular basis of VanA resistance and briefly discuss the similarities and differences between the VanA phenotype and other phenotypes. The clinical implications (19, 31) and the mechanism (59) of glycopeptide resistance have recently been reviewed. The VanA phenotpe. Inducible resistance to high levels of vancomycin and teicoplanin defines the VanA phenotype (18). This type of resistance reduces the activities of all glycopeptides to various extents, but there is no crossresistance with other cell wall inhibitors (35, 40). Vancomycin induces the synthesis of two proteins that are readily detectable in enterococcal membrane fractions: a 39-kDa protein (5, 41, 50), which was identified as the VanA ligase necessary for cell wall synthesis in the presence of glycopeptides (21), and a D,D-carboxypeptidase (4) that is not required for resistance (7). High-level resistance to glycopeptides is generally transferable to susceptible enterococci by conjugation and was shown, at least in certain strains, to be mediated by self-transferable plasmids (20, 28, 36, 41, 43, 50, 53). It has recently been shown (9) that the genes necessary and sufficient for expression of the VanA phenotype are carried by a transposon designated TnI546. Dissemination of this transposon appears to be responsible for the spread of high-level glycopeptide resistance among clinical isolates of enterococci (9). Overview of Tn1546. Originally detected on plasmid pIP816 from Enterococcus faecium BM4147 (9, 35), TnJS46 consists of 10,851 bp and encodes nine polypeptides (Fig. 1) that can be assigned to four functional groups (7-9): transposition functions (open reading frames [ORFs] ORF1 and ORF2), regulation of vancomycin resistance genes (VanR and VanS), resistance to glycopeptides by production of depsipeptides (VanH, VanA, and VanX), and accessory proteins that may be involved in peptidoglycan synthesis,

Genetics and mechanisms of glycopeptide resistance in enterococci.

Vancomycin resistance in Enterococcus faecium BM4 147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alaninemalanine ligase of altered substrate specificity (Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-20211, while the sequence of VanH is related to those of a-keto acid dehydrogenases (Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)). We report purification of VanH to homogeneity, characterization as a D-specific a-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2- hydroxybutyrate (K, = 0.60 mM). The VanA product ~-alanyl-~-2-hydroxybutyrate could then be in- corporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D- Ala-D- Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-~-alanyl-~-2-hydroxybutyrate (& > 73 mM) was > 1 000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (& = 54 pM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.

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Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Many surface proteins are thought to be anchored to the cell wall of Gram-positive bacteria via their C-terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C-terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N-terminal fragment, while the C-terminal fragment remains within the cytoplasm. N-terminal sequencing of the C-terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram-positive bacteria.

Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram-positive bacteria

It has been hypothesized that penicillin acts as a structural analog of the acyl-D-alanyl-D-alanine terminus of nascent bacterial cell wall and that it consequently binds to and acylates the active site of the enzyme(s) that crosslinks the cell wall to form an inactive penicilloyl enzyme [Tipper, D.J. & Strominger, J.L. (1965) Proc. Natl. Acad. Sci. USA 64, 1133-1138]. This study directly proves that penicillin acylates the active site of two penicillin-sensitive enzymes, D-alanine carboxypeptidases from Bacillus stearothermophilus and Bacillus subtilis. Active site peptides were generated by chemical or enzymatic cleavage of these carboxypeptidases after covalently labeling with [14C]penicillin G or after trapping an acyl-enzyme intermediate derived from the depsipeptide substrate. [14C]diacetyl-L-lysyl-D-alanyl-D-lactate. The amino acid sequences of the penicillin- and substrate-labeled peptides were identical. Both penicillin and substrate were covalently bound via an ester linkage to the same active site residue, a serine at position 36 of the B. stearothermophilus carboxypeptidase and the corresponding serine in the B. subtilis carboxypeptidase. The two D-alanine carboxypeptidases showed significant homology around the active site. Moreover, homology between these two enzymes and four beta-lactamases of known sequence suggests that these two groups of enzymes are evolutionally related.

https://www.pnas.org/content/pnas/76/6/2730.full.pdf

Mechanism of penicillin action: penicillin and substrate bind covalently to the same active site serine in two bacterial D-alanine carboxypeptidases

The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k2), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k3) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (Vmax/Km) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [14C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [14C]depsipeptide and precipitated with acetone.

http://www.pnas.org/content/75/1/84.full.pdf

Utilization of a depsipeptide substrate for trapping acyl—enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases

The present invention provides methods and compositions for treating autoimmune diseases and other unwanted immune reactions comprising administering a copolymer that binds to one or more HLA-DQ molecules and modulates DQ-restricted T cell responses. The copolymers are random copolymers of amino acids and copolymers comprising anchor residues to facilitate binding to the DQ binding pockets.

Methods and compositions for treatment of autoimmune diseases

The invention relates to novel methods and kits for treating or preventing disease through the administration of random copolymers. The invention also relates to the treatment of autoimmune diseases, such as multiple sclerosis, and to the administration of random copolymers in treatment regimen comprising formulations that are administered at intervals greater than 24 hours, or to sustained release formulations which administer the copolymer over a period greater than 24 hours. The invention further relates to methods for conducting a pharmaceutical business comprising manufacturing, licensing, or distributing kits containing or relating to the formulations or dosing regimens of random copolymer described herein.

James Rasmussen

Papers

Mechanism of penicillin action: penicillin and substrate bind covalently to the same active site serine in two bacterial D-alanine carboxypeptidases

Utilization of a depsipeptide substrate for trapping acyl—enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases

Methods and compositions for treatment of autoimmune diseases

Random copolymer compositions for treating unwanted immune response

Methods of treating unwanted immune response with random copolymers