In Vitro and In Vivo Activities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae.

Gram-negative bacteria are evolving to produce ß-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine ß-lactamase inhibitor which acts also as an antibiotic and as a ß-lactamase-independent ß-lactam "enhancer" against Enterobacteriaceae Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, in in vitro time-kill studies and an in vivo infection model against five strains of CTX-M-15-positive Escherichia coli and five strains of KPC-positive Klebsiella pneumoniae An OP0595 concentration of 4 µg/ml was found to be sufficient for an effective combination with all three ß-lactam agents. In both in vitro time-kill studies and an in vivo model of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all ß-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a ß-lactam agent is important to exert the antimicrobial functions of OP0595.

OP0595, a new diazabicyclooctane: mode of action as a serine ß-lactamase inhibitor, antibiotic and ß-lactam 'enhancer'.

OBJECTIVES: The production of a growing diversity of ß-lactamases by Gram-negative bacteria challenges antimicrobial chemotherapy. OP0595, discovered separately by each of Meiji Seika Pharma and Fedora Pharmaceuticals, is a new diazabicyclooctane serine ß-lactamase inhibitor that also acts as an antibiotic and as a ß-lactamase-independent ß-lactam 'enhancer'. METHODS: Inhibitory activity against serine ß-lactamases and affinity for PBPs were determined using nitrocefin and Bocillin FL, respectively. MICs alone and in combination with ß-lactam agents were measured according to CLSI recommendations. Morphological changes in Escherichia coli were examined by phase-contrast microscopy. RESULTS: IC50s of OP0595 for class A and C ß-lactamases were <1000 nM, with covalent binding demonstrated to the active-site serine of CTX-M-44 and AmpC enzymes. OP0595 also had direct antibiotic activity against many Enterobacteriaceae, associated with inhibition of PBP2 and conversion of the bacteria into spherical forms. Synergy between OP0595 and ß-lactam agents was seen against strains producing class A and C ß-lactamases vulnerable to inhibition. Lastly, OP0595 lowered the MICs of PBP3-targeted partner ß-lactam agents for a non-ß-lactamase-producing E. coli mutant that was resistant to OP0595 itself, indicating ß-lactamase-independent 'enhancer'-based synergy. CONCLUSIONS: OP0595 acts in three ways: (i) as an inhibitor of class A and C ß-lactamases, covalently binding at their active sites; (ii) as an antibacterial, by inhibiting PBP2 of several Enterobacteriaceae; and (iii) as an 'enhancer' of ß-lactam agents that bind to other PBPs besides PBP2 for several Enterobacteriaceae. OP0595 has considerable potential to overcome resistance when it is combined with various ß-lactam agents.

TLR2 - promiscuous or specific? A critical re-evaluation of a receptor expressing apparent broad specificity.

Of all pattern recognition receptors (PRR) in innate immunity, Toll-like receptor 2 (TLR2) recognizes the structurally broadest range of different bacterial compounds known as pathogen-associated molecular patterns (PAMPs). TLR2 agonists identified so far are lipopolysaccharides (LPSs) from different bacterial strains, lipoproteins, (synthetic) lipopeptides, lipoarabinomannans, lipomannans, glycosylphosphatidylinositol, lipoteichoic acids (LTA), various proteins including lipoproteins and glycoproteins, zymosan, and peptidoglycan (PG). Because these molecules are structurally diverse, it seems unlikely that TLR2 has the capability to react with all agonists to the same degree. The aim of this review is to identify and describe well-defined structure-function relationships for TLR2. Because of its biomedical importance and because its genetics and biochemistry are presently most completely known among all Gram-positive bacteria, we have chosen Staphylococcus aureus as a focus. Our data together with those reported by other groups reveal that only lipoproteins/lipopeptides are sensed at physiologically concentrations by TLR2 at picomolar levels. This finding implies that the activity of all other putative bacterial compounds so far reported as TLR2 agonists was most likely due to contaminating highly active natural lipoproteins and/or lipopeptides.

Thrombospondin-1 promotes cellular adherence of gram-positive pathogens via recognition of peptidoglycan.

Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. Here we report a novel role for the lectin TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that Streptococcus pneumoniae and other gram-positive pathogens including S. pyogenes, Staphylococcus aureus, and Listeria monocytogenes interact specifically with human TSP1. We also show for the first time that host cell-bound TSP1 promotes adherence of gram-positive pathogens to human epithelial and endothelial cell lines. Pretreatment of bacteria with sodium periodate but not Pronase E substantially reduced TSP1-mediated bacterial adherence to host cells, suggesting that a glycoconjugate on the bacterial cell surface functions as the receptor for TSP1. Lipoteichoic acids did not affect TSP1-mediated adherence of S. pneumoniae to host cells. In contrast, attachment of S. pneumoniae and other gram-positive pathogens to host cells via TSP1 was blocked by soluble peptidoglycan, indicating recognition of bacterial peptidoglycan by TSP1. In conclusion, our results demonstrate that recognition of gram-positive pathogens by TSP1 promotes bacterial colonization of host tissue cells. In this scenario, peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge linking gram-positive bacteria with receptors on the host cell.

Chemical synthesis of peptidoglycan fragments for elucidation of the immunostimulating mechanism.

Partial structures of peptidoglycan were chemically synthesized for elucidation of their precise biological activities. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of peptidoglycan were synthesized in good yields. The biological activity of synthetic fragments of peptidoglycan was evaluated by induction of TNF-alpha from human monocytes, and TLR2 and NOD2 dependencies by using transfected HEK293 cells, respectively. We revealed that TLR2 was not stimulated by the series of synthetic peptidoglycan partial structures, whereas NOD2 recognizes the partial structures containing the MDP moiety. We also synthesized potent NOD1 agonists, which showed several hundred-fold stronger activity than gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Interaction of PGRPs with synthetic peptidoglycan fragments is also described.

In vitro and in vivo activities of the diazabicyclooctane OP0595 against AmpC-derepressed Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common cause for healthcare-associated infections, which have been historically treated by antipseudomonal ß-lactam agents in the clinical setting. However, P. aeruginosa has evolved to overcome these ß-lactam agents via multiple endogenous resistance mechanisms, including derepression of the chromosomal cephalosporinase (AmpC). In this article, we investigated the effective concentration of OP0595 for combination with piperacillin, cefepime or meropenem in in vitro susceptibility tests, and the antibacterial activity of cefepime in combination with OP0595 in both in vitro time-kill studies and in vivo murine thigh infection model study with AmpC-derepressed P. aeruginosa. The sufficient combinational concentration of OP0595 was a 4 µg ml-1 with all these three ß-lactam agents. OP0595 increased the antibacterial activity of cefepime in both in vitro and in vivo studies against all strains tested. Taken together, OP0595 is the diazabicyclooctane serine ß-lactamase inhibitor with activity against AmpC-derepressed P. aeruginosa and its combinational use with a ß-lactam agent will provide a new approach for the treatment of P. aeruginosa infections.

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands.

UNLABELLED: B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. CONCLUSION: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.

Synthesis of peptidoglycan fragments and evaluation of their biological activity.

The peptidoglycan (PG) bacterial cell wall glycoconjugate has been well known as a strong immunopotentiator. Partial structures of PG were chemically synthesized for elucidation of precise biological activities. Effective construction of distinct repeating glycans of PG was accomplished by the coupling of a key disaccharide glucosaminyl-beta(1-4)-muramic acid unit. Stereoselective glycosylation of disaccharide units was achieved by neighboring group participation of the N-Troc (Troc = 2,2,2-trichloroethoxycarbonyl) group and appropriate reactivity of N-Troc-glucosaminyl trichloroacetimidate. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of PG were synthesized in high yields. The biological activity of synthetic fragments of PG was evaluated by induction of tumor necrosis factor-alpha (TNF-alpha) from human monocytes, and toll-like receptor 2 (TLR2) and Nod2 dependencies by using transfected HEK293 cells, respectively. Here we reveal that TLR2 was not stimulated by the series of synthetic PG partial structures, whereas Nod2 recognizes the partial structures containing the MDP moiety.

A synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade.

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.

Phagocytes containing a disease-promoting Toll-like receptor/Nod ligand are present in the brain during demyelinating disease in primates.

Recent studies claim a central role for Toll-like receptor (TLR) ligands in stimulating autoimmune disease by activation of antigen-presenting cells in the target organ, but it is unclear if and how TLR ligands reach target organs. Most evidence comes from rodent models, and it is uncertain whether this principle holds in primates. Here we identify which cells contain peptidoglycan (PGN) in multiple sclerosis brain and in two nonhuman primate experimental autoimmune encephalomyelitis (EAE) models with different disease courses: acute (rhesus monkey) versus chronic disease (marmoset). Because persistence of TLR ligands in the central nervous system might be consequential for disease progression, we also determined the expression of two major PGN-degrading enzymes, ie, lysozyme and N-acetylmuramyl-l-alanine amidase. Distinct phagocyte subsets, including granulocytes, macrophages, and dendritic cells, contained PGN in the brain and coexpressed the inflammatory cytokine interleukin-12. The number of phagocytes carrying PGN increased in acute and chronic EAE compared with control animals, with the highest number of PGN-containing cells in acute EAE brain. Lytic enzymes were scarcely expressed in monkey and multiple sclerosis brain, favoring PGN persistence. PGN stimulated interleukin-12p70 release by leukocytes from all three primate species. The presence of PGN in the inflamed brain may have major implications because TLR2/Nod ligation potentially promotes inflammation and disease progression.

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.