Non-Proteinogenic Amino Acids in Lacticin 481 Analogues Result in More Potent Inhibition of Peptidoglycan Transglycosylation

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.Chemistry and Chemical Biolog

Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

This work is licensed under a Creative Commons Attribution 4.0 International License.As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.NIH U19AI111143Milstein Program in Chemical Biology and Translational MedicineWilliam Randolph Hearst TrustWelch Foundation (A-0015

Reconstitution of Peptidoglycan Cross-Linking Leads to Improved Fluorescent Probes of Cell Wall Synthesis

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

Substrate mimics of bacterial Hsp70s inhibit chaperone function through distinct allosteric mechanisms

Here, we set up high-throughput multiprotein assays to screen a large collection (>25K) of structurally diverse small molecules against the Mtb DnaK chaperone system including a J-protein and GrpE. We measure the level of ATP hydrolysis by this system as a proxy to detect small molecule-mediated modulation. Like others, we aimed to identify allosteric inhibitors that might affect DnaK function through distal site or cofactor binding, instead of ATP competitive inhibitors that might exhibit promiscuity against the plethora of cellular nucleotide binding proteins. In doing so, we identify several small molecules that affect the activity of the Mtb DnaK chaperone complex, and focus on two unique compounds that have been used as clinical drugs. Both of these molecules represent mimics of substrates that bind to the NBD or SBD of DnaK, but they lead to proposed conformational changes that inactive the protein. We show that one of these molecules has activity against mycobacterial cells that have compromised folding function. Our work contributes to a small but growing list of chemical scaffolds that have activity against protein chaperones, and strengthens our understanding of how to achieve specificity in bacterial DnaK targeting

Lipoprotein Activators Stimulate Escherichia coli Penicillin-Binding Proteins by Different Mechanisms

In Escherichia coli, the bifunctional penicillin-binding proteins (PBPs), PBP1A and PBP1B, play critical roles in the final stage of peptidoglycan (PG) biosynthesis. These synthetic enzymes each possess a PG glycosyltransferase (PGT) domain and a transpeptidase (TP) domain. Recent genetic experiments have shown that PBP1A and PBP1B each require an outer membrane lipoprotein, LpoA and LpoB, respectively, to function properly in vivo. Here, we use complementary assays to show that LpoA and LpoB each increase the PGT and TP activities of their cognate PBPs, albeit by different mechanisms. LpoA directly increases the rate of the PBP1A TP reaction, which also results in enhanced PGT activity; in contrast, LpoB directly affects PGT domain activity, resulting in enhanced TP activity. These studies demonstrate bidirectional coupling of PGT and TP domain function. Additionally, the transpeptidation assay described here can be applied to study other activators or inhibitors of the TP domain of PBPs, which are validated drug targets.Chemistry and Chemical BiologyOther Research Uni

Preference of bacterial rhamnosyltransferases for 6‑deoxysugars reveals a strategy to deplete O‑antigens

Bacteria synthesize hundreds of bacteria-specific or “rare” sugars that are absent in mammalian cells and enriched in 6-deoxy monosaccharides such as L-rhamnose (L-Rha). Across bacteria, L-Rha is incorporated into glycans by rhamnosyltransferases (RTs) that couple nucleotide sugar substrates (donors) to target biomolecules (acceptors). Since L-Rha is required for the biosynthesis of bacterial glycans involved in survival or host infection, RTs represent potential antibiotic or antivirulence targets. However, purified RTs and their unique bacterial sugar substrates have been difficult to obtain. Here, we use synthetic nucleotide rare sugar and glycolipid analogs to examine substrate recognition by three RTs that produce cell envelope components in diverse species, including a known pathogen. We find that bacterial RTs prefer pyrimidine nucleotide-linked 6-deoxysugars, not those containing a C6-hydroxyl, as donors. While glycolipid acceptors must contain a lipid, isoprenoid chain length, and stereochemistry can vary. Based on these observations, we demonstrate that a 6-deoxysugar transition state analog inhibits an RT in vitro and reduces levels of RT-dependent O-antigen polysaccharides in Gram-negative cells. As O-antigens are virulence factors, bacteria-specific sugar transferase inhibition represents a novel strategy to prevent bacterial infections