Biology subjects

Shinde, D.

Publications and source records attributed to Shinde, D..

8 recordsLinked to original sources

bioRxiv2026

The β-lactam adjuvant guanosine potentiates anti-folate antibiotics and pyrimidine synthesis inhibitors by depleting thymidine in methicillin-resistant Staphylococcus aureus

Efforts to improve the effectiveness of existing interventions for antimicrobial-resistant (AMR) infections include identifying new ways to overcome resistance to licensed antibiotics using adjuvants or deploying antibiotics in novel combinations. Although antibiotics targeting the bacterial cell wall (e.g., {beta}-lactams) and folate metabolism (e.g., trimethoprim-sulfamethoxazole, TMP-SMX) remain cornerstones of modern healthcare, resistance to both classes poses an ongoing therapeutic challenge. We recently demonstrated that purine nucleosides can act as potent antibiotic adjuvants, restoring {beta}-lactam susceptibility in methicillin-resistant Staphylococcus aureus (MRSA). Here, we show that the {beta}-lactam adjuvant guanosine significantly reduces intracellular thymidine levels in MRSA and potentiates the activity of antifolate antibiotics (TMP-SMX) as well as the pyrimidine antimetabolites 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd). Incorporation of oxacillin into guanosine-antifolate or guanosine-pyrimidine analogue combinations further enhanced killing of both planktonic and biofilm-associated MRSA. Thymidine depletion was accompanied by elevated intracellular reactive oxygen species (ROS) and dissipation of membrane potential, providing mechanistic insight into the bactericidal effects of these combinations. Together, these findings demonstrate that guanosine expands MRSA susceptibility beyond {beta}-lactams to include additional clinically relevant antimicrobial drug classes commonly used to treat bacterial infections. Author Summary.Finding new ways to help antibiotics to work against resistant bacteria is an important part of efforts to address the antimicrobial resistance crisis. Adjuvants, which are boosters that help antibiotics to work against resistant bacteria, are important in this regard. We recently discovered that, guanosine, which is involved in the synthesis of DNA, makes methicillin-resistant Staphylococcus aureus (MRSA) more sensitive to penicillin-type antibiotics. Here we report that guanosine also improves the effectiveness of two other types of antimicrobial drugs (anti-folate antibiotics and pyrimidine antimetabolites) that are also used in clinical practice, expanding the potential of this adjuvant to improve the management of difficult-to-treat MRSA infections.

microbiology↗

bioRxiv2024

A thermodynamic bottleneck in the TCA cycle contributes to acetate overflow in Staphylococcus aureus

During aerobic growth, S. aureus relies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow in S. aureus have not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle, specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient for S. aureus to generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck provides S. aureus the flexibility to redirect carbon towards maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offers S. aureus distinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal-pathogen lifestyle.

systems biology↗

bioRxiv2023

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to {beta}-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased {beta}-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in {beta}-lactam resistance, which will support efforts to identify new drug targets and reintroduce {beta}-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other {beta}-lactam resistant pathogens. Author summaryHigh-level resistance to penicillin-type ({beta}-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of {beta}-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

microbiology↗

bioRxiv2022

Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae

The ubiquitous presence of toxic arsenate (AsV) in the environment has virtually raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that V. cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress- related systems to counter ROS production caused by intracellular AsV. Characterization of the var mutants suggested these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions.

microbiology↗

bioRxiv2022

PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

Phosphatidylinositol 3-phosphate (PI3P) and Phosphatidylinositol 5-phosphate (PI5P) are low abundant phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P, in vitro. In this study, we report a novel role for PIP4K in regulating PI3P levels in Drosophila tissues. Loss-of-function mutants of the only PIP4K gene in Drosophila (dPIP4K29) show reduced cell size in larval salivary glands. We find that PI3P levels are elevated in dPIP4K29 tissues and that reverting PI3P levels back towards wild type, without changes in PI5P levels, can also rescue the reduced cell size phenotype. dPIP4K29 mutants also show an upregulation in autophagy and the reduced cell size can be reverted by decreasing Atg8a, that is required for autophagosome maturation. Lastly, increasing PI3P levels in wild type salivary glands can phenocopy the reduction in cell size and associated upregulation of autophagy seen in dPIP4K29. Thus, our study reports for the first time, a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size regulation that may explain the reported role of PIP4K in regulating neurodegeneration and tumour growth.

cell biology↗

bioRxiv2022

PGC1β and ERRα promote glutamine metabolism and colorectal cancer survival via transcriptional regulation of PCK2

Previous studies have shown that Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1{beta}) and Estrogen-Related Receptor Alpha (ERR) are over-expressed in colorectal cancer and promote tumor survival. In this study, we show that amino acid motif LRELL on PGC-1{beta} is responsible for the physical interaction with ERR and promotes ERR mRNA and protein expression. We used RNAsequencing to determine the genes regulated by both PGC-1{beta} & ERR and found that mitochondrial Phosphoenolpyruvate Carboxykinase 2 (PCK2) was the gene that decreased most significantly after depletion of both genes. Depletion of PCK2 in colorectal cancer cells was sufficient to reduce anchorage-independent growth and inhibit glutamine utilization by the TCA cycle. Lastly, shRNA-mediated depletion of ERR decreased anchorage-independent growth and glutamine metabolism, which could not be rescued by plasmid derived expression of PCK2. These findings suggest that transcriptional control of PCK2 is one mechanism used by PGC-1{beta} and ERR to promote glutamine metabolism and colorectal cancer cell survival.

cancer biology↗

bioRxiv2019

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In this study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use 18O-ATP to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from both Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity levels from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

biochemistry↗

bioRxiv2018

Insulin sensitivity and PIP3 turnover in Drosophila are regulated by phosphatidylinositol 5 phosphate 4-kinase.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor and is critical for normal growth and metabolism. The lipid kinases and phosphatases regulating PIP3 levels are described but mechanisms controlling their activity remain unclear. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) function at the plasma membrane is required for normal PIP3 levels during insulin receptor activation. Depletion of PIP4K increases PIP3 levels and augments sensitivity to insulin through enhanced Class I phosphoinositide 3-kinase (PI3K) activity. Animals lacking PIP4K show enhanced insulin signalling dependent phenotypes in vivo and are resistant to the metabolic consequences of a high-sugar diet, highlighting the importance of PIP4K in normal metabolism and development. Thus, PIP4KS are key regulators of receptor tyrosine kinase signalling with implications for growth factor dependent processes including tumour growth, T-cell activation and metabolism.

cell biology↗