Biology subjects

Thomas, V. C.

Publications and source records attributed to Thomas, V. C..

9 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↗

bioRxiv2025

Activation of the Spx redox sensor counters cysteine-driven Fe(II) depletion under disulfide stress

In many low G+C Gram-positive bacteria, the global regulator Spx helps maintain thiol homeostasis during disulfide stress, when protein thiols form aberrant disulfide bonds that can lead to misfolding and oxidative damage. Spx-dependent gene expression is triggered when an intramolecular disulfide bond forms between two cysteines in its redox switch. Surprisingly, some Spx functions persist even in the absence of an active redox switch, highlighting the need to better understand the physiological significance of maintaining this regulatory feature. Here, we utilize a spxC10A mutant that encodes a redox-insensitive Spx variant to study the role of the Spx redox switch in Staphylococcus aureus. We show that the spxC10A mutant is hypersensitive to diamide-induced disulfide stress and exhibits widespread transcriptional dysregulation of genes that contribute to thiol maintenance and disulfide repair. Remarkably, the spxC10A mutant rapidly adapts to disulfide stress by increasing its intracellular pool of L-cysteine (L-Cys) through enhanced uptake, which helps restore a reduced intracellular environment. However, during this process increased L-Cys inadvertently depletes cytosolic Fe(II), leading to growth inhibition of the spxC10A mutant. Finally, we show that the Spx-dependent control of intracellular L-Cys is critical for S. aureus survival when it encounters human neutrophils. Overall, these findings suggest that staphylococcal adaptation to disulfide stress through intracellular L-Cys accumulation imposes significant fitness costs that S. aureus overcomes by rapid regulatory control of thiol homeostasis through a functional Spx redox switch. SignificanceAll cells have a pool of low molecular weight thiols, such as cysteine, glutathione, bacillithiol, and coenzyme A, to maintain redox balance under oxidative and disulfide stress. Among these, cysteine is a very effective thiol but is highly reactive, and its intracellular concentration must be tightly regulated. In S. aureus, we found that cysteine accumulates intracellularly during disulfide stress and if left unchecked, can inadvertently deplete cytosolic Fe(II), leading to growth inhibition. To prevent cysteine toxicity, S. aureus activates the global regulator Spx, which rapidly induces genes that restore thiol homeostasis and limits cysteine accumulation.

microbiology↗

bioRxiv2025

A D-alanine aminotransferase S180F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation

Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of D-alanine for cell wall synthesis, the target of {beta}-lactam antibiotics. Furthermore cycA-dependent alanine transport controls MRSA {beta}-lactam susceptibility in chemically defined medium (CDM) in a glucose-dependent manner. Here we report that S. aureus was auxotrophic for L-alanine in CDM, and that this growth defect was rescued by glucose (or compensatory mutations), but only when the alanine racemase (alr1) and D-alanine aminotransferase (dat) genes were functional. No role was observed for the alanine dehydrogenase 1 (ald1) and ald2 genes. As previously reported, alr1 and, to a lesser extent, cycA mutations increased susceptibility to D-cycloserine (DCS). In contrast, only alr1 mutation increased susceptibility to {beta}-chloro-D-alanine (BCDA), suggesting distinct targets for these alanine analogue antibiotics, which act synergistically against MRSA. Genome sequencing of a BCDA-resistant mutant identified a C539T mutation in dat, predicted to result in a S180F substitution. Expression of the datC539T operon in wild-type increased BCDA resistance. alr1/dat::Em and alr1/datC539T double mutants were auxotrophic for D-alanine, indicating that Dat-S180F transaminase activity is impaired, a conclusion supported by in vitro enzyme assays. Structural modeling revealed an active-site loop shift in Dat-S180F that altered PLP co-factor binding. Molecular docking showed that the S180F substitution promotes BCDA-PLP adduct dissociation by releasing inactivated BCDA, thereby conferring resistance. These data reveal essential roles for Alr1 and Dat during growth under nutrient-limiting conditions and the potential of combination therapy separately targeting both enzymes with DCS and BCDA to extend the treatment options for 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↗

bioRxiv2024

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin resistant Staphylococcus aureus

Infections caused by methicillin resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA have acquired resistance to next generation {beta}-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators which through poorly described mechanisms enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased {beta}-lactam MICs up to 16-folds and transformed MRSA cells with low level of resistance to being homogenously highly resistant to {beta}-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPRi to knock down spx transcription, we unambiguously linked hyper-resistance to accumulation of Spx. Spx was previously proposed to be essential, however, our data indicate that Spx is dispensable for growth at 37{degrees}C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in {beta}-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum {beta}-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

microbiology↗

bioRxiv2024

Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus. We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus. Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a substantial intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication. SignificanceUnder mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus. We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus.

microbiology↗

bioRxiv2023

Host autophagy is exploited by the intracellular parasite Toxoplasma gondii to enhance amino acids levels.

Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistance to available drugs. For its chronic persistence in the brain, the parasite relies on host cells nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustains its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in accumulation of unfolded protein with the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Remarkably, the persistence of latent forms in cell culture as well as behavioral changes in mice caused by the latent infection could be successfully reversed by restricting the availability of various amino acids during T. gondi infection. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis. ImportanceIntracellular parasites employ several mechanisms to manipulate the cellular environment, enabling them to persist in the host. Toxoplasma gondii, a single-celled parasite, possesses the ability to infect virtually any nucleated cell of warm-blooded vertebrates, including nearly 2 billion people worldwide. Unfortunately, existing treatments and immune responses are not entirely effective in eliminating the chronic persisting forms of the parasite. This study reveals that T. gondii induces the hosts autophagic pathway to boost amino acid levels in infected cells. The depletion of amino acids, in turn, influences the persistence of the parasites chronic forms, resulting in a reduction of neurological alterations caused by chronic infection in mice. Significantly, our investigation establishes the crucial role of host ER-phagy in the parasites persistence within the host during latent infection.

microbiology↗

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↗

bioRxiv2019

An integrated computational and experimental study to elucidate Staphylococcus aureus metabolism

Staphylococcus aureus is a metabolically versatile pathogen that colonizes nearly all organs of the human body. A detailed and comprehensive knowledge of staphylococcal metabolism is essential to understanding its pathogenesis. To this end, we have reconstructed and experimentally validated an updated and enhanced genome-scale metabolic model of S. aureus USA300_FPR3757. The model combined genome annotation data, reaction stoichiometry, and regulation information from biochemical databases and previous strain-specific models. Reactions in the model were checked and fixed to ensure chemical balance and thermodynamic consistency. To further refine the model, growth assessment of 1920 non-essential mutants from the Nebraska Transposon Mutant Library was performed and metabolite excretion profiles of important mutants in carbon and nitrogen metabolism were determined. The growth and no-growth inconsistencies between the model predictions and in vivo essentiality data were resolved using extensive manual curation based on optimization-based reconciliation algorithms. Upon intensive curation and refinements, the model contains 840 metabolic genes, 1442 metabolites, and 1566 reactions including transport and exchange reactions. To improve the accuracy and predictability of the model to environmental changes, condition-specific regulation information curated from the existing knowledgebase was incorporated. These critical additions improved the model performance significantly in capturing gene essentiality, substrate utilization, and metabolite production capabilities and increased the ability to generate model-based discoveries of therapeutic significance. Use of this highly curated model will enhance the functional utility of omics data and, therefore, serve as a resource to support future investigations of S. aureus and to augment staphylococcal research worldwide.

systems biology↗