Synthesis and Application of Methyl N,O-Hydroxylamine Muramyl Peptides.

The innate immune system's interaction with bacterial cells plays a pivotal role in a variety of human diseases. Carbohydrate units derived from a component of bacterial cell wall, peptidoglycan (PG), are known to stimulate an immune response. Nonetheless, access to modified late-stage peptidoglycan intermediates is limited due to their synthetic complexity. A method to rapidly functionalize PG fragments is needed to better understand the natural host-PG interactions. Here methyl N,O-hydroxylamine linkers are incorporated onto a synthetic PG derivative, muramyl dipeptide (MDP). The modification of MDP maintained the ability to stimulate a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) immune response dependent on the expression of nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Intrigued by this modification's maintenance of biological activity, several applications were explored. Methyl N,O-hydroxylamine MDP was amendable to N-hydroxylsuccinimide (NHS) chemistry for bioconjugation to fluorophores as well as a self-assembled monolayer for Nod2 surface plasmon resonance analysis. Finally, linker incorporation was applicable to larger PG fragments, both enzymatically generated from Escherichia coli or chemically synthesized. This methodology provides rapid access to PG probes in one step and allows for the installation of a variety of chemical handles to advance the molecular understanding of PG and the innate immune system.

Postsynthetic Modification of Bacterial Peptidoglycan Using Bioorthogonal N-Acetylcysteamine Analogs and Peptidoglycan O-Acetyltransferase B.

Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG)3, a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.

Concurrent zero-dimensional and one-dimensional biomineralization of gold from a solution of Au3+ and bovine serum albumin.

A technique was developed for preparing a novel material that consists of gold nanoparticles trapped within a fiber of unfolded proteins. These fibers are made in an aqueous solution that contains HAuCl4 and the protein, bovine serum albumin (BSA). By changing the ratio of gold to BSA in solution, two different types of outcomes are observed. At lower gold to BSA ratios (30-120), a purple solution results after heating the mixture at 80 °C for 4 h. At higher gold to BSA ratios (130-170), a clear solution containing purple fibers results after heating the mixture at 80 °C for 4 h. UV-Vis spectroscopy and light scattering techniques show growth in nanocolloid size as gold to BSA ratio rises above 100. Data indicate that, for the higher gold to BSA ratios, the gold is sequestered within the solid material. The material mass, visible by eye, appears to be an aggregation of smaller individual fibers. Scanning electron microscopy and transmission electron microscopy indicate that these fibers are primarily one-dimensional aggregates, which can display some branching, and can be as narrow as 400 nm in size. The likely mechanism for the synthesis of the novel material is discussed.

Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2.

The human gut must regulate its immune response to resident and pathogenic bacteria, numbering in the trillions. The peptidoglycan component of the bacterial cell wall is a dense and rigid structure that consists of polymeric carbohydrates and highly cross-linked peptides which offers protection from the host and surrounding environment. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a human membrane-associated innate immune receptor found in the gut epithelium and mutated in an estimated 30% of Crohn's disease patients, binds to peptidoglycan fragments and initiates an immune response. Using a combination of chemical synthesis, advanced analytical assays, and protein biochemistry, we tested the binding of a variety of synthetic peptidoglycan fragments to wild-type (WT)-NOD2. Only when the protein was presented in the native membrane did binding measurements correlate with a NOD2-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) response, supporting the hypothesis that the native-membrane environment confers ligand specificity to the NOD2 receptor for NF-κB signaling. While N-acetyl-muramyl dipeptide (MDP) has been thought to be the minimal peptidoglycan fragment necessary to activate a NOD2-dependent immune response, we found that fragments with and without the dipeptide moiety are capable of binding and activating a NOD2-dependent NF-κB response, suggesting that the carbohydrate moiety of the peptidoglycan fragments is the minimal functional epitope. This work highlights the necessity of studying NOD2-ligand binding in systems that resemble the receptor's natural environment, as the cellular membrane and/or NOD2 interacting partners appear to play a crucial role in ligand binding and in triggering an innate immune response.