Person:

Kahne, Suzanne

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNALeuUAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive, but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-GlcNAc transferase, an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.

Gram-negative bacteria contain an unusual outer membrane that prevents the entry of most currently available antibiotics. This membrane contains a complex glycolipid, LPS, on the exterior. It is not understood how such a large molecule, which can contain hundreds of sugars and six fatty acyl chains, is transported across the cell envelope from its site of synthesis in the cytoplasmic membrane to the cell surface. Using a combination of genetics, biochemistry, and structural biology, we characterized residues in the protein that powers LPS transport to gain mechanistic insight into how ATP hydrolysis is coupled to the biological function of the transporter. These tools help us understand how to design antibiotics targeting this essential pathway.

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.

Purpose: Wall teichoic acids (WTAs) are major polyanionic polymer components of the cell wall of Staphylococcus aureus. However, little is known about their role at the host–pathogen interface, especially in endophthalmitis. This study was designed to investigate the extent to which WTAs contribute to the pathogenicity of S. aureus in models of endophthalmitis and to determine whether there would be value in targeting their biosynthesis as a new therapeutic approach. Methods: S. aureus RN6390 and its isogenic WTA-null mutant (RN6390ΔtarO) were used to evaluate the role of WTAs in endophthalmitis. RN6390 and RN6390ΔtarO were cultured in bovine vitreous humor (VH) in vitro or inoculated into the vitreous chamber of C57B6 mice. Changes in the number of bacteria, organ function as determined by electroretinography (ERG), and histopathologic changes were assessed throughout the course of infection. In addition, the efficacy of WTA biosynthesis inhibitors in VH in vitro was examined. Results: It was observed that a component of VH synergized with WTA biosynthesis inhibitors in vitro and killed the S. aureus. This effect was also seen when mutants incapable of expressing WTA were exposed to VH. The killing activity of VH was lost on treatment with a protease inhibitor. RN6390ΔtarO could not survive in mouse eyes and did not affect organ function, nor was it able to establish endophthalmitis. Conclusions: WTAs are essential cellular constituents for the manifestation of virulence by S. aureus in endophthalmitis, and appears to be a viable target for treating the endophthalmitis caused by S. aureus strains.

Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The MIC of an antibiotic against S. aureus, as well as other microbes, is determined by the affinity of the antibiotic for its target in addition to a complex interplay of many other cellular factors. Identifying nontarget factors impacting resistance to multiple antibiotics could inform the design of new compounds and lead to more-effective antimicrobial strategies. We examined large collections of transposon insertion mutants in S. aureus using transposon sequencing (Tn-Seq) to detect transposon mutants with reduced fitness in the presence of six clinically important antibiotics-ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin, and vancomycin. This approach allowed us to assess the relative fitness of many mutants simultaneously within these libraries. We identified pathways/genes previously known to be involved in resistance to individual antibiotics, including graRS and vraFG (graRS/vraFG), mprF, and fmtA, validating the approach, and found several to be important across multiple classes of antibiotics. We also identified two new, previously uncharacterized genes, SAOUHSC_01025 and SAOUHSC_01050, encoding polytopic membrane proteins, as important in limiting the effectiveness of multiple antibiotics. Machine learning identified similarities in the fitness profiles of graXRS/vraFG, SAOUHSC_01025, and SAOUHSC_01050 mutants upon antibiotic treatment, connecting these genes of unknown function to modulation of crucial cell envelope properties. Therapeutic strategies that combine a known antibiotic with a compound that targets these or other intrinsic resistance factors may be of value for enhancing the activity of existing antibiotics for treating otherwise-resistant S. aureus strains.

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria. For decades it was thought that only class A penicillin-binding proteins (aPBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the unrelated SEDS protein family, also acts as a peptidoglycan polymerase. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly. FtsW was previously proposed to translocate the peptidoglycan precursor Lipid II across the cytoplasmic membrane. We report here that purified FtsW polymerizes Lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP (bPBP). We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

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.