Combining Signal Peptidase and Lipoprotein Processing Inhibitors Overcomes Ayr Resistance in Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens is an ongoing public health concern. The arylomycins are a class of natural product antibiotics that target the type I signal peptidase, which carries out the terminal step in protein secretion. Here, we used transposon sequencing (Tn-Seq) to profile the effects of the optimized arylomycin derivative G0775 in Staphylococcus aureus. Our transposon libraries include both upregulation and inactivation mutants, allowing us to identify resistance mechanisms and targets for synergism. We identified several cell envelope pathways that, when inactivated, sensitize S. aureus to the arylomycin G0775. These pathways include the lipoprotein processing pathway, and we have shown that inhibitors of this pathway synergize with G0775 even though lipoprotein processing is nonessential in S. aureus. Moreover, we found that blocking this pathway completely reverses Ayr resistance, which is a major resistance mechanism to arylomycins, including G0775. Our Tn-Seq data also showed that upregulation of mprF and several other genes is protective against G0775. Because a subset of these genes was previously found in a Tn-Seq profile of the clinically important antibiotic daptomycin, we tested a set of daptomycin-nonsusceptible clinical isolates with gain-of-function mutations in mprF for susceptibility to arylomycin G0775. Despite structural and mechanistic differences between these antibiotics, we observed similar decreases in susceptibility. Taken together, our results highlight how Tn-Seq profiles that include both gene inactivation and upregulation can identify targets, antibiotic resistance mechanisms, and strategies to overcome resistance.

Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus.

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.

Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic.

Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Human CHD1 is required for early DNA-damage signaling and is uniquely regulated by its N terminus.

CHD1 is a conserved chromatin remodeling enzyme required for development and linked to prostate cancer in adults, yet its role in human cells is poorly understood. Here, we show that targeted disruption of the CHD1 gene in human cells leads to a defect in early double-strand break (DSB) repair via homologous recombination (HR), resulting in hypersensitivity to ionizing radiation as well as PARP and PTEN inhibition. CHD1 knockout cells show reduced H2AX phosphorylation (γH2AX) and foci formation as well as impairments in CtIP recruitment to the damaged sites. Chromatin immunoprecipitation following a single DSB shows that the reduced levels of γH2AX accumulation at DSBs in CHD1-KO cells are due to both a global reduction in H2AX incorporation and poor retention of H2AX at the DSBs. We also identified a unique N-terminal region of CHD1 that inhibits the DNA binding, ATPase, and chromatin assembly and remodeling activities of CHD1. CHD1 lacking the N terminus was more active in rescuing the defects in γH2AX formation and CtIP recruitment in CHD1-KO cells than full-length CHD1, suggesting the N terminus is a negative regulator in cells. Our data point to a role for CHD1 in the DSB repair process and identify a novel regulatory region of the protein.

Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan.

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton1. In Staphylococcus aureus, the peptidoglycan assembly enzymes relocate during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells2-4. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood5,6. Here, we used a transposon screen to identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes stem peptides from uncrosslinked peptidoglycan and a partner protein that controls its activity. Amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so that they can separate7. However, this amidase controls cell growth. In its absence, peptidoglycan synthesis becomes spatially dysregulated, which causes cells to grow so large that cell division is defective. We show that the cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given subcellular location. Removal of stem peptides from peptidoglycan at the cell periphery promotes peptidoglycan synthase relocation to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.