RESUMO
Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of ß-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3â3 cross-links compared to 4â3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4â3 (MepM) and 3â3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4â3 and 3â3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4â3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of ß-lactams.
Assuntos
Endopeptidases/metabolismo , Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Antibacterianos/farmacologia , Catálise , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Hidrólise , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Resistência beta-LactâmicaRESUMO
(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum ß-lactam resistance as mediated by the ß-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent ß-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the ß or ß' RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although ß-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by ß-lactams.
Assuntos
Guanosina Pentafosfato , Peptidoglicano , Andinocilina , Parede Celular , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genéticaRESUMO
The D,D-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of ß-lactam antibiotics that block peptidoglycan polymerization. ß-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a ß-lactam-insensitive L,D-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by ß-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond L,D-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of ß-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of ß-lactams involves self-promoted penetration through the outer membrane.
Assuntos
Peptidil Transferases , beta-Lactamas , beta-Lactamas/farmacologia , beta-Lactamas/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli, the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance.