Biology subjects

Cross, J. R.

Publications and source records attributed to Cross, J. R..

10 recordsLinked to original sources

bioRxiv2026

Loss of riboflavin biosynthesis leads to accumulation of select aromatic amino acids and loss of infectivity in Mycobacterium tuberculosis

Riboflavin biosynthesis is required for in vitro survival of the human pathogen Mycobacterium tuberculosis (Mtb). However, despite the lack of a known transporter, growth can be rescued by exogenous riboflavin. The riboflavin biosynthesis pathway is also predicted essential in vivo, but whether riboflavin levels available in the host can support survival has not been directly tested. Here we constructed a set of inducible CRISPR interference (CRISPRi) knockdown and targeted gene deletion strains for known riboflavin biosynthesis genes (ribA2, ribG, ribH, ribC) as tools to characterize riboflavin requirements, uptake, and metabolite changes and to assess in vivo essentiality. We found that riboflavin, but not flavin adenine dinucleotide or flavin mononucleotide, rescued auxotrophy for all strains tested. Further, riboflavin uptake did not show strong evidence of being dependent on active or facilitated transport, supporting the mechanism of passive diffusion. Targeted metabolite profiling after removal of riboflavin from growth medium confirmed reduced riboflavin levels. While other riboflavin intermediates were not detected, significant accumulation of aromatic amino acids (Phe, Tyr) was observed across all assayed strains, as well as alteration in a vitamin B9 metabolite. Selecting the ribC knockout as a representative strain, we found that riboflavin depletion had a bacteriostatic effect as late as 3 weeks after removal. Unexpectedly, {Delta}ribC lacked infectivity in an aerosol mouse infection, suggesting that the potential to scavenge riboflavin from the host is not sufficient to survive in vivo. Overall, our results show that altered metabolism upon loss of riboflavin biosynthesis leads to compromised Mtb infectivity. IMPORTANCETuberculosis remains one of the worlds most deadly infectious diseases, underscoring the need to explore new drug targets. Riboflavin (vitamin B2) biosynthesis has emerged as a promising target because Mycobacterium tuberculosis (Mtb) depends on this pathway for survival. The riboflavin pathway also produces metabolites that modulate host mucosal-associated invariant T (MAIT) cell activity, towards understanding potential strategies for host-directed therapies. Here we found that disrupting riboflavin biosynthesis led to not only compromised survival, but also widespread changes to metabolism and loss of the ability to establish infection in an animal model. These findings improve our understanding of how Mtb adapts to metabolic stress, with implications for developing drugs that target riboflavin biosynthesis and for alterations in host immunity to be explored in future studies.

microbiology↗

bioRxiv2025

Macropinocytosis enables metabolic recycling of extracellular DNA in cancer cells

Avid nutrient consumption is a metabolic hallmark of cancer and leads to regional depletion of key metabolites within the tumor microenvironment (TME). Cancer cells consequently employ diverse strategies to acquire the fuels needed for growth, including bulk uptake of the extracellular medium by macropinocytosis. Here, we show that breast and pancreatic cancer cells macropinocytically internalize extracellular DNA (exDNA), an abundant component of the TME, and deliver it to lysosomes for degradation. This provides a supply of nucleotides that sustains growth when de novo biosynthesis is impaired by glutamine restriction or pharmacological blockade. Mechanistically, this process is dependent on the non-redundant lysosomal equilibrative nucleoside transporter SLC29A3 (ENT3), which mediates the export of nucleosides from the lysosomal lumen into the cytosol. Accordingly, genetic ablation of SLC29A3 or pharmacological disruption of lysosomal function prevents exDNA scavenging and potently sensitizes breast tumors to antimetabolite chemotherapy in vivo. These findings reveal a previously unrecognized nutrient acquisition pathway through which cancer cells recycle exDNA into metabolic building blocks and highlight SLC29A3 as a mediator of metabolic flexibility and a potential target to improve chemotherapy response.

cancer biology↗

bioRxiv2025

Intracellular acidification by bacteria-derived valeric acid is a mechanism of trans-kingdom ecology against Candida parapsilosis colonization

In hematopoietic cell transplant patients, intestinal Candida parapsilosis expansion and translocation cause life-threatening candidemia, yet how commensal intestinal bacteria prevent Candida expansion remains incompletely defined. Here, we trained a machine learning model on supernatant metabolomic profiles of commensal bacteria to identify bacteria-derived inhibitors of fungal growth, with valeric and butyric acid as top hits. Experimental validation confirmed in silico predictions in three systems. First, in patient fecal samples, valeric and butyric acid levels inversely correlated with C. parapsilosis growth. Second, in vitro, valeric acid potently inhibited C. parapsilosis growth by causing intracellular acidification. Third, administration of glycerol valerate, and free or microencapsulated valeric acid blunted C. parapsilosis growth at murine intestinal sites where valeric acid could be detected. Thus, machine learning could identify a mechanistic driver of trans-kingdom ecology limiting C. parapsilosis intestinal expansion and may inform strategies to reduce patient risk of developing candidiasis during high-risk periods.

microbiology↗

bioRxiv2025

A genetically encoded bifunctional enzyme mitigates redox imbalance and lipotoxicity via engineered Gro3P-Glycerol shunt

Dihydroxyacetone phosphate (DHAP), glycerol-3-phosphate (Gro3P) and reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+) are key metabolites of the Gro3P shuttle system that forms a redox circuit, allowing transfer of reducing equivalents between cytosol and mitochondria. Targeted activation of Gro3P biosynthesis was recently identified as a promising strategy to alleviate reductive stress by promoting NAD+ recycling, including in cells with an impaired mitochondrial complex I. However, because Gro3P constitutes the backbone of triglycerides under some circumstances, its accumulation can lead to excessive fat deposition. Here, we present the development of a novel genetically encoded tool based on a di-domain glycerol-3-phosphate dehydrogenase from algae Chlamydomonas reinhardtii (CrGPDH), which is a bifunctional enzyme that can recycle NAD+ while converting DHAP to Gro3P. In addition, this enzyme possesses an N-terminal domain which cleaves Gro3P into glycerol and inorganic phosphate (Pi) (in humans and other organisms, this reaction is catalyzed by a separate glycerol-3-phosphate phosphatase, a reaction also known as "glycerol shunt"). When expressed in mammalian cells, CrGPDH diminished Gro3P levels and boosted the TCA cycle and fatty acid {beta}-oxidation in mitochondria. CrGPDH expression alone supported proliferation of HeLa cells under conditions of either inhibited activity of the mitochondrial electron transport chain or hypoxia. Moreover, human kidney cancer cells, which exhibit abnormal lipid accumulation, had decreased triglycerides levels when expressing CrGPDH. Our findings suggest that the coordinated boosting of both Gro3P biosynthesis and glycerol shunt may be a viable strategy to alleviate consequences of redox imbalance and associated impaired lipogenesis in a wide repertoire of conditions, ranging from primary mitochondrial diseases to obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD).

bioengineering↗

bioRxiv2024

Hexa-acylated lipopolysaccharides from the gut microbiota enhance cancer immunotherapy responses

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment, but they are only effective for a minority of patients. The gut microbiome plays a crucial role in modulating immunotherapy treatment responses, and previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis. However, LPS from diverse bacterial species have activities ranging from immunostimulatory to inhibitory. By functionally analyzing fecal metagenomes from 112 melanoma patients prior to anti-PD-1 therapy, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in the microbiomes of clinical responders. We confirmed robust activation of the NF-kB pathway by hexa-acylated LPS in vitro, and this activation was significantly inhibited by penta-acylated LPS in a dose-dependent manner. Importantly, oral administration of hexa-acylated LPS augmented anti-PD-1-mediated anti-tumor immunity in an in vivo mouse model of cancer immunotherapy. Microbiome hexa-acylated LPS may therefore represent an accessible predictor and potential enhancer of clinical anti- PD-1 immunotherapy responses. Statement of significanceFunctional rather than taxonomic profiling of patient gut microbiomes reveals hexa-acylated LPS as a novel biomarker of responsiveness and a targetable pathway for enhancing responses to anti-PD-1, informing future studies and current patient treatment.

microbiology↗

bioRxiv2024

Characterization, structure and inhibition of the human succinyl-CoA:glutarate-CoA transferase, a genetic modifier of glutaric aciduria type 1

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or non-toxic metabolites. Here, we report a novel target, SUGCT, which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA. We report the structure of SUGCT, the first eukaryotic structure of a type III CoA transferase, develop a high-throughput enzyme assay and a cell-based assay, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme validating the screening approach. These results may form the basis for future development of new pharmacological intervention to treat GA1.

biochemistry↗

bioRxiv2022

A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells

Impaired reduction/oxidation (redox) metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration, and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies which can perturb cellular redox metabolism and model pathology in various cellular, tissue, or organismal backgrounds without creating additional and potentially confounding metabolic perturbations. To date, most studies involving impaired redox metabolism have focused on oxidative stress and reactive oxygen species (ROS) production; consequently, less is known about the settings where there is an overabundance of reducing equivalents, termed reductive stress. NADH reductive stress has been modeled using pharmacologic inhibition of the electron transport chain (ETC) and ethanol supplementation. Still, both these methods have significant drawbacks. Here, we introduce a soluble transhydrogenase from E. coli (EcSTH) as a novel genetically encoded tool to promote NADH overproduction in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially-targeted version (mitoEcSTH), can elevate the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determine the metabolic and transcriptomic signatures of NADH reductive stress in mammalian cells. We also find that cellular responses to NADH reductive stress, including blunted proliferation, are dependent on cellular background and identify the metabolic reactions that sense changes in the cellular NADH/NAD+ balance. Collectively, our novel genetically encoded tool represents an orthogonal strategy to perturb redox metabolism and characterize the impact on normal physiology and disease states.

bioengineering↗

bioRxiv2022

Cytosolic peptide accumulation activates the NLRP1 and CARD8 inflammasomes

NLRP1 and CARD8 are related sensors that form inflammasomes, but the danger signals that they detect are not fully established. These proteins undergo autoproteolysis, generating repressive N-terminal (NT) and inflammatory C-terminal (CT) fragments. The proteasome- mediated degradation of the NT releases the CT from autoinhibition, but the CT is then sequestered in a complex with the full-length sensor and DPP9. Here, we show that cytosolic peptide accumulation activates these inflammasomes. We found that a diverse array of peptides accelerates NT degradation, and those with N-terminal XP sequences also destabilize the ternary complexes. Peptides interfere with many biological processes, including protein folding. We show that unrelated agents that disrupt protein folding also induce NT degradation, but do not cause inflammasome activation because DPP9 sequesters the CT fragments in the absence of XP peptides. Overall, these results indicate that NLRP1 and CARD8 detect protein misfolding that is associated with peptide accumulation.

immunology↗

bioRxiv2022

Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels

Female Aedes aegypti mosquitoes feed on human blood, which they use to develop their eggs. It has been widely noted that some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. Here we tested mosquito attraction to skin odor collected from human subjects and identified people who are exceptionally attractive or unattractive to mosquitoes. Notably, these preferences were stable over several years, indicating consistent longitudinal differences in skin odor between subjects. We carried out gas chromatography/quadrupole time of flight-mass spectrometry to analyze the chemical composition of human skin odor in these subjects and discovered that highly attractive people produce significantly increased levels of carboxylic acids. Mosquitoes could reliably distinguish a highly attractive human from their weakly attractive counterparts unless we substantially diluted the odor of the "mosquito magnet." This is consistent with the hypothesis that odor concentration is a major driver of differential attraction, rather than the less-favored skin odor blend containing repellent odors, although these are not mutually- exclusive. Mosquitoes detect carboxylic acids with a large family of odor-gated ion channels encoded by the Ionotropic Receptor gene superfamily. Mutant mosquitoes lacking any of the Ionotropic Receptor (IR) co-receptors Ir8a, Ir25a, and Ir76b, were severely impaired in attraction to human scent but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in "mosquito-magnet" human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.

neuroscience↗

bioRxiv2019

Mapping interactions of microbial metabolites and human receptors

Despite evidence linking the human microbiome to health and disease, the mechanistic details of how the microbiota affects human physiology remain largely unknown. Metabolites encoded by bacteria are expected to play an integral role in the microbiotas effect on its human host. Assigning function to these metabolites is therefore critical to determining the molecular underpinnings of the host-microbe relationship and ultimately developing microbiota inspired therapies. Here we use large-scale functional screening of small molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein coupled receptors (GPCR). This analysis revealed a complex network of metabolite host receptor interactions and guided our identification of multiple microbiota derived agonists of GPCRs associated with diverse biological functions within the nervous and immune systems, among others. Collectively, the metabolite-receptor pairs we uncovered indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.\n\nStatement of SignificanceBacteria residing within the human body have been shown to influence human health. It is likely that physiological responses to the human microbiota are mediated by the collection of small molecules encoded within these bacteria. In this study we use direct functional screening of small molecules produced by individual members of a simplified human microbiota to identify new G protein coupled receptor-metabolite interactions that seek to explain the molecular underpinnings of the microbiotas influence on its human host.

microbiology↗