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 Novel Isoflavone, ME-344, Enhances Venetoclax Antileukemic Activity Against AML Via Suppression of Oxidative Phosphorylation and Purine Biosynthesis

The 5-year survival rate for adult patients with acute myeloid leukemia (AML) treated with cytarabine-based chemotherapy remains less than 30%, due to drug resistance and disease relapse. Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was approved by the FDA in combination with low dose cytarabine or hypomethylating agents for treating newly diagnosed AML patients who are 75 years of age or older or for those who are unfit for standard chemotherapy, providing more treatment options for this group of patients. Although the response rate to these newly approved combination therapies is reported to be 70%, the median overall survival is only 10-18 months showing that the duration of response is limited. Therefore, novel therapeutic agents are in demand to enhance venetoclax activity against AML and to combat AML resistant to cytarabine-based chemotherapy. Cytarabine-resistant AML cells lead to relapse and rely on oxidative phosphorylation (OXPHOS) for survival. In addition, it has been reported that targeting OXPHOS can enhance venetoclax activity against preclinical models of AML. Thus, we hypothesize that OXPHOS suppressing agents would be good candidates to combine with and enhance venetoclax antileukemic activity against newly diagnosed AML and those with resistance to cytarabine. A novel isoflavone, ME-344, has been shown to suppress OXPHOS in cell lines derived from solid tumors by inhibiting Complex I of the electron transport chain. We hypothesized that combining ME-344 with venetoclax would result in synergistic antileukemic activity against AML. Consistent with our hypothesis, combining ME-344 with venetoclax resulted in synergistic induction of apoptosis in AML cell lines, including those with acquired cytarabine resistance. The combination of these two agents also resulted in synergistic antileukemic activity in one primary AML patient sample, as determined by MTT assay. The combination of ME-344 and venetoclax prolonged the median survival of MV4-11 leukemia- bearing NSGS mice by 37% (median survival of 48 days compared to 35 days for vehicle control treated mice, n=5 per arm, p<0.0001). This is in contrast to the venetoclax combination with cytarabine, which prolonged median survival of the same xenograft model by 7.5% (Luedtke et al., Signal Transduction and Targeted Therapy, 2020; 5:17). ME-344 alone (9-hour treatment) reduced basal mitochondrial respiration in AML cells by 10% prior to induction of apoptosis. When treated with ME-344 for 8-hours followed by combined ME-344 and venetoclax for an additional 1-hour, basal mitochondrial respiration was reduced by 18% (again prior to detection of apoptosis initiation). This sequential combination regimen also decreased the mitochondrial membrane potential (by JC-1 staining and flow cytometry analysis) when compared to untreated control and single treatment. Additionally, apoptosis induction by the combination of ME-344 and venetoclax or ME-344 alone was significantly enhanced when AML cells were forced to utilize OXPHOS by replacing glucose with galactose in the culture medium. Further investigation revealed that apoptosis induced by ME-344 was partially attenuated when Mcl-1 was overexpressed, Bak was knocked down, or caspase activation was inhibited. This suggests a mechanism that involves components of the intrinsic apoptosis pathway. Targeted metabolomics analyses of MV4-11 cells treated with ME-344 for 8 h revealed a significant reduction of essential metabolites involved in the de novo purine biosynthesis pathway, specifically AICAR (p=0.001) and IMP (p=0.004). Given the critical role of purine in cell proliferation and survival, suppression of purine biosynthesis by ME-344 may represent a novel mechanism underlying its enhancement on the antileukemic activity of venetoclax against AML. Interestingly, inhibition of this pathway by the purine biosynthesis inhibitor lometrexol, also synergistically enhanced apoptosis in AML cells induced by venetoclax. Taken together, these results suggest that ME-344 suppresses OXPHOS and the purine biosynthesis pathway to enhance the antileukemic activity of venetoclax against AML. Further in-depth mechanistic studies into the suppression of purine biosynthesis and OXPHOS, as well as studies of ME-344 and venetoclax against cytarabine-resistant AML in a mouse model are warranted. Disclosures Wiley: MEIPharma: Current Employment.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections

Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are “susceptible” in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints.

Aldh2 dependent formaldehyde metabolism fuels purine demands in melanocyte stem cells

ABSTRACTAldehyde-processing enzymes are viewed as essential clearing agents that rapidly deactivate harmful aldehydes. In the bone marrow, two specific enzymes, aldehyde dehydrogenase (ALDH) 2 and alcohol dehydrogenase (ADH) 5, were previously reported to protect hematopoietic stem cells from endogenous formaldehyde accumulation. Unexpectedly, we found that melanocyte stem cells (McSCs) in zebrafish depend on formate, an Aldh2-generated reaction product, to drive regeneration. Activated McSCs require Aldh2 (but not Adh5) to generate differentiated progeny, and by using scRNA-sequencing analysis, we identified a de novo purine biosynthesis program that is uniquely present in activated McSCs. Consistent with formate serving as one-carbon units for nucleotide biosynthesis, we found that purine supplementation (but not pyrimidine supplementation) was able to restore melanocyte regeneration in the absence of Aldh2. This work shows that Aldh2 enzymes generate reaction products that are needed to meet metabolic demands in regeneration.

From Bioorganic Models to Cells

I endeavor to share how various choices—some deliberate, some unconscious—and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Reduced purine biosynthesis in humans after their divergence from Neandertals

We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Inosine in Biology and Disease

The nucleoside inosine plays an important role in purine biosynthesis, gene translation, and modulation of the fate of RNAs. The editing of adenosine to inosine is a widespread post-transcriptional modification in transfer RNAs (tRNAs) and messenger RNAs (mRNAs). At the wobble position of tRNA anticodons, inosine profoundly modifies codon recognition, while in mRNA, inosines can modify the sequence of the translated polypeptide or modulate the stability, localization, and splicing of transcripts. Inosine is also found in non-coding and exogenous RNAs, where it plays key structural and functional roles. In addition, molecular inosine is an important secondary metabolite in purine metabolism that also acts as a molecular messenger in cell signaling pathways. Here, we review the functional roles of inosine in biology and their connections to human health.