The de novo Purine Biosynthesis Pathway Is the Only Commonly Regulated Cellular Pathway during Biofilm Formation in TSB-Based Medium in Staphylococcus aureus and Enterococcus faecalis

Bacterial biofilms are involved in chronic infections and confer 10 to 1,000 times more resistance to antibiotics compared with planktonic growth, leading to complications and treatment failure. When transitioning from a planktonic lifestyle to biofilms, some Gram-positive bacteria are likely to modulate several cellular pathways, including central carbon metabolism, biosynthesis pathways, and production of secondary metabolites. These metabolic adaptations might play a crucial role in biofilm formation by Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. Here, we performed a transcriptomic approach to identify cellular pathways that might be similarly regulated during biofilm formation in these bacteria. Different strains and biofilm-inducing media were used to identify a set of regulated genes that are common and independent of the environment or accessory genomes analyzed. Our approach highlighted that the de novo purine biosynthesis pathway was upregulated in biofilms of both species when using a tryptone soy broth-based medium but not so when a brain heart infusion-based medium was used. We did not identify other pathways commonly regulated between both pathogens. Gene deletions and usage of a drug targeting a key enzyme showed the importance of this pathway in biofilm formation of S. aureus. The importance of the de novo purine biosynthesis pathway might reflect an important need for purine during biofilm establishment, and thus could constitute a promising drug target. IMPORTANCE Biofilms are often involved in nosocomial infections and can cause serious chronic infections if not treated properly. Current anti-biofilm strategies rely on antibiotic usage, but they have a limited impact because of the biofilm intrinsic tolerance to drugs. Metabolism remodeling likely plays a central role during biofilm formation. Using comparative transcriptomics of different strains of Staphylococcus aureus and Enterococcus faecalis, we determined that almost all cellular adaptations are not shared between strains and species. Interestingly, we observed that the de novo purine biosynthesis pathway was upregulated during biofilm formation by both species in a specific medium. The requirement for purine could constitute an interesting new anti-biofilm target with a wide spectrum that could also prevent resistance evolution. These results are also relevant to a better understanding of the physiology of biofilm formation.

A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify therapeutic targets for DMG, we conducted a genome-wide CRIPSR screen for DMG metabolic vulnerabilities, which revealed a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. The dependency is specific to pyrimidines as there is no selectivity for suppression of de novo purine biosynthesis. A clinical stage inhibitor of DHODH (a rate limiting enzyme in the de novo pathway) generates DNA damage and induces apoptosis through suppression of replication forks--an on target effect, as shown by uridine rescue. MALDI mass spectroscopy imaging demonstrates that BAY2402234 accumulates in brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts. Our results highlight BAY2402234, a brain-penetrant DHODH inhibitor, as a promising therapy against DMGs.

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities are coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells were genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggest SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systematically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states- glucose, galactose, OXPHOS inhibition, and absence of pyruvate-designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

DDRE-14. DE-NOVO PURINE BIOSYNTHESIS IS A MAJOR DRIVER OF CHEMORESISTANCE IN GLIOBLASTOMA

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumor is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is glioblastoma cells’ ability to adapt to complex changes within the tumor microenvironment. To elucidate this adaptation’s molecular mechanisms, specifically during chemotherapy temozolomide, we employed chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ALR13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with Liquid Chromatography-Mass Spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5’-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction function as a negative regulator for purine salvaging. Inhibition of ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using a Food and Drug Administration-approved drug, Mycophenolate Mofetil, that can block the IMPDH2 activity and enhance the therapeutic efficacy of TMZ. Our results suggest and support clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

EXTH-40. THERAPEUTICALLY TARGETING DE NOVO PURINE BIOSYNTHESIS IN DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse intrinsic pontine glioma (DIPG) is an incurable brainstem malignancy in children with median survival less than 1 year and 5-year overall survival only 2 percent. Little progress has been made in treating this deadly disease due to its inoperable location and treatments aimed at targets defined in adult gliomas. Despite recent advances in genetic characterization of DIPGs there are still no targeted therapies that significantly improve overall survival. We recently generated a metabolic profile for DIPG elucidating an upregulation in purine metabolism, specifically in de novo purine biosynthesis (DNPB). Normally nucleotide salvage maintains cellular purine levels by recycling degraded bases, however DNPB is needed when purine levels are depleted. Purine metabolism provides the basic components of nucleotides needed for tumor proliferation and thus considered a high-priority target in cancer treatment. DNPB is a sequential ten step enzymatic process resulting in the production of inosine monophosphate. The last step in DNPB is carried out by the bifunctional enzyme ATIC which is upregulated in DIPG cell lines, and in patient tumors. Our preliminary data demonstrates DIPG cell lines are sensitive to pharmacological inhibition and genetic ablation of multiple enzymes in the DNPB pathway. Strikingly, cell viability could be rescued by purine supplementation when inhibiting this pathway except when ATIC is inhibited indicating the mechanism of cell death for ATIC inhibition is independent of purine nucleotide levels. Furthermore, there is a therapeutic window for targeting ATIC in DIPG cell lines relative to normal neural stem cells and normal human astrocytes. Metabolic flux experiments have demonstrated DNPB is upregulated in DIPG cell lines and the reason these cells are more sensitive to ATIC inhibition is likely related to the rapid accumulation of a cytotoxic metabolite upstream of ATIC. In vivo studies are currently underway in pre-clinical mouse models for DIPG.

Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

De-novo purine biosynthesis is a major driver of chemoresistance in glioblastoma

This year nearly 20,000 lives will be lost to Glioblastoma (GBM), a treatment-resistant primary brain cancer. In this study, we identified a molecular circuit driven by epigenetic regulation that regulates the expression of ciliary protein ALR13B. We also demonstrated that ARL13B subsequently interacts with purine biosynthetic enzyme IMPDH2. Removal of ARL13B enhanced TMZ-induced DNA damage by reducing de-novo purine biosynthesis and forcing GBM cells to rely on the purine salvage pathway. Furthermore, targeting can be achieved by using an FDA-approved drug, Mycophenolate Moefitil. Our results suggest a clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

De Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus aureus and for the Hypervirulence Phenotype of a purR Mutant

ABSTRACT Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.