Identification of hidden associations among eukaryotic genes through statistical analysis of coevolutionary transitions.

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes.

Isocitrate lyase promotes Puccinia striiformis f. sp. tritici susceptibility in wheat (Triticum aestivum) by suppressing accumulation of glyoxylate cycle intermediates.

Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-BY400*) was inhibited early during initial penetration, with the TaICL-BY400* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.

Metabolic Reprogramming of Klebsiella pneumoniae Exposed to Serum and Its Potential Implications in Host Immune System Evasion and Resistance.

The aim of this study was to identify, using proteomics, the molecular alterations caused by human serum exposure to Klebsiella pneumoniae ACH2. The analysis was performed under two different conditions, native serum from healthy donors and heat-inactivated serum (to inactivate the complement system), and at two different times, after 1 and 4 h of serum exposure. More than 1,000 bacterial proteins were identified at each time point. Enterobactin, a siderophore involved in iron uptake, and proteins involved in translation were upregulated at 1 h, while the chaperone ProQ and the glyoxylate cycle were identified after 4 h. Enzymes involved in the stress response were downregulated, and the SOD activity was validated using an enzymatic assay. In addition, an intricate metabolic adaptation was observed, with pyruvate and thiamine possibly involved in survival and virulence in the first hour of serum exposure. The addition of exogenous thiamine contributes to bacterial growth in human serum, corroborating this result. During 4 h of serum exposure, the glyoxylate cycle (GC) probably plays a central role, and the addition of exogenous succinate suppresses the GC, inducing a decrease in serum resistance. Therefore, serum exposure causes important changes in iron acquisition, the expression of virulence factors, and metabolic reprogramming, which could contribute to bacterial serum resistance.

Moderate high temperature is beneficial or detrimental depending on carbon availability in the green alga Chlamydomonas reinhardtii.

High temperatures impair plant growth and reduce agricultural yields, but the underlying mechanisms remain elusive. The unicellular green alga Chlamydomonas reinhardtii is an excellent model to study heat responses in photosynthetic cells due to its fast growth rate, many similarities in cellular processes to land plants, simple and sequenced genome, and ample genetic and genomics resources. Chlamydomonas grows in light by photosynthesis and with externally supplied acetate as an organic carbon source. Understanding how organic carbon sources affect heat responses is important for the algal industry but remains understudied. We cultivated wild-type Chlamydomonas under highly controlled conditions in photobioreactors at 25 °C (control), 35 °C (moderate high temperature), or 40 °C (acute high temperature) with or without constant acetate supply for 1 or 4 day. Treatment at 35 °C increased algal growth with constant acetate supply but reduced algal growth without sufficient acetate. The overlooked and dynamic effects of 35 °C could be explained by induced acetate uptake and metabolism. Heat treatment at 40 °C for more than 2 day was lethal to algal cultures with or without constant acetate supply. Our findings provide insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

Proteome reorganization and amino acid metabolism during germination and seedling establishment in Lupinus albus.

During germination plants rely entirely on their seed storage compounds to provide energy and precursors for the synthesis of macromolecular structures until the seedling has emerged from the soil and photosynthesis can be established. Lupin seeds use proteins as their major storage compounds, accounting for up to 40% of the seed dry weight. Lupins are therefore a valuable complement to soy as a source of plant protein for human and animal nutrition. The aim of this study was to elucidate how storage protein metabolism is coordinated with other metabolic processes to meet the requirements of the growing seedling. In a quantitative approach, we analysed seedling growth, as well as alterations in biomass composition, the proteome, and metabolite profiles during germination and seedling establishment in Lupinus albus. The reallocation of nitrogen resources from seed storage proteins to functional seed proteins was mapped based on a manually curated functional protein annotation database. Although classified as a protein crop, Lupinus albus does not use amino acids as a primary substrate for energy metabolism during germination. However, fatty acid and amino acid metabolism may be integrated at the level of malate synthase to combine stored carbon from lipids and proteins into gluconeogenesis.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

Transcriptome analysis reveals multiple targets of erythritol-related transcription factor EUF1 in unconventional yeast Yarrowia Lipolytica.

BACKGROUND: Erythritol is a four-carbon polyol with an unclear role in metabolism of some unconventional yeasts. Its production has been linked to the osmotic stress response, but the mechanism of stress protection remains unclear. Additionally, erythritol can be used as a carbon source. In the yeast Yarrowia lipolytica, its assimilation is activated by the transcription factor Euf1. The study investigates whether this factor can link erythritol to other processes in the cell. RESULTS: The research was performed on two closely related strains of Y. lipolytica: MK1 and K1, where strain K1 has no functional Euf1. Cultures were carried out in erythritol-containing and erythritol-free media. Transcriptome analysis revealed the effect of Euf1 on the regulation of more than 150 genes. Some of these could be easily connected with different aspects of erythritol assimilation, such as: utilization pathway, a new potential isoform of transketolase, or polyol transporters. However, many of the upregulated genes have never been linked to metabolism of erythritol. The most prominent examples are the degradation pathway of branched-chain amino acids and the glyoxylate cycle. The high transcription of genes affected by Euf1 is still dependent on the erythritol concentration in the medium. Moreover, almost all up-regulated genes have an ATGCA motif in the promoter sequence. CONCLUSIONS: These findings may be particularly relevant given the increasing use of erythritol-induced promoters in genetic engineering of Y. lipolytica. Moreover, use of this yeast in biotechnological processes often takes place under osmotic stress conditions. Erythritol might be produce as a by-product, thus better understanding of its influence on cell metabolism could facilitate processes optimization.

Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

The Transcription Factor StuA Regulates the Glyoxylate Cycle in the Dermatophyte Trichophyton rubrum under Carbon Starvation.

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

Evaluation of protein's interaction and the regulatory network of some drought-responsive genes in Canola under drought and re-watering conditions.

Drought stress is one of the most important environmental stresses that severely limits the growth and yield of Canola. The re-watering can compensate for the damage caused by drought stress. Investigation of protein's interaction of genes involved in important drought-responsive pathways and their regulatory network by microRNAs (miRNAs) under drought and re-watering conditions are helpful approaches to discovering drought-stress tolerance and recovery mechanisms. In this study, the protein's interaction and functional enrichment analyses of glycolysis, pentose phosphate, glyoxylate cycle, fatty acid biosynthesis, heat shock factor main genes, and the regulatory network of key genes by miRNAs were investigated by in silico analysis. Then, the relative expression of key genes and their related miRNAs were investigated in tolerant and susceptible genotypes of Canola under drought and re-watering conditions by Real-time PCR technique. The bna-miR156b/c/g, bna-miR395d/e/f, bna-miR396a, and all the studied key genes except HSFA1E and PK showed changes in expression levels in one or both genotypes after re-watering. The PPC1 and HSFB2B expression decreased, whereas the MLS and CAC3 expression increased in both genotypes under re-watering treatment after drought stress. It could cause the regulation of oxaloacetate production, the increase of the glyoxylate cycle, lipid biosynthesis, and the reduction of the negative regulation of HSFs under re-watering conditions. It seems that PPC1, G6PD2, MLS, CAC3, and HSFB2B were involved in the recovery mechanisms after drought stress of Canola. They were regulated by drought-responsive miRNAs to respond appropriately to drought stress. Therefore, regulating these genes could be important in plant recovery mechanisms. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01345-1.

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast.

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

Structural insights into the targeting specificity of ubiquitin ligase for S. cerevisiae isocitrate lyase but not C. albicans isocitrate lyase.

In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S.cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 Å, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S.cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

Transcriptome analysis provides insights into the root response of Chinese fir to phosphorus deficiency.

BACKGROUND: Phosphorus is one of the essential elements for plant growth and development, but available phosphorus (Pi) content in many soil types is low. As a fast-growing tree species for timber production, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir root responses to Pi deficiency are largely unexplored. In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured. The transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. RESULTS: We annotated 50,808 Unigenes from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and 17 genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term Pi stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30 days of Pi deficiency. CONCLUSION: Chinese fir roots showed increased expression of genes related with phosphorus starvation, citrate and malate synthesis genes, increased content of organic acids, and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of adaption to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.

Cappable-Seq Reveals Specific Patterns of Metabolism and Virulence for Salmonella Typhimurium Intracellular Survival within Acanthamoeba castellanii.

The bacterial pathogen Salmonella enterica, which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and multiplies inside their phagosomes. Although this relationship resembles that between Salmonella and mammalian phagocytes, the interaction mechanisms and bacterial genes involved are unclear. Here, we characterized global gene expression patterns of S. enterica serovar Typhimurium within Acanthamoeba castellanii at the early stage of infection by Cappable-Seq. Gene expression features of S. Typhimurium within A. castellanii were presented with downregulation of glycolysis-related, and upregulation of glyoxylate cycle-related genes. Expression of Salmonella Pathogenicity Island-1 (SPI-1), chemotaxis system, and flagellar apparatus genes was upregulated. Furthermore, expression of genes mediating oxidative stress response and iron uptake was upregulated within A. castellanii as well as within mammalian phagocytes. Hence, global S. Typhimurium gene expression patterns within A. castellanii help better understand the molecular mechanisms of Salmonella adaptation to an amoeba cell and intracellular persistence in protozoa inhabiting water and soil ecosystems.

What Constitutes a Gluconeogenic Precursor?

A gluconeogenic precursor is a biochemical compound acted on by a gluconeogenic pathway enabling the net synthesis of glucose. Recognized gluconeogenic precursors in fasting placental mammals include glycerol, lactate/pyruvate, certain amino acids, and odd-chain length fatty acids. Each of these precursors is capable of contributing net amounts of carbon to glucose synthesis via the tricarboxylic acid cycle (TCA cycle) because they are anaplerotic, that is, they are able to increase the pools of TCA cycle intermediates by the contribution of more carbon than is lost via carbon dioxide. The net synthesis of glucose from even-chain length fatty acids (ECFAs) in fasting placental mammals, via the TCA cycle alone, is not possible because equal amounts of carbon are lost via carbon dioxide as is contributed from fatty acid oxidation via acetyl-CoA. Therefore, ECFAs do not meet the criteria to be recognized as a gluconeogenic precursor via the TCA cycle alone. ECFAs are gluconeogenic precursors in organisms with a functioning glyoxylate cycle, which enables the net contribution of carbon to the intermediates of the TCA cycle from ECFAs and the net synthesis of glucose. The net conversion of ECFAs to glucose in fasting placental mammals via C3 metabolism of acetone may be a competent though inefficient metabolic path by which ECFA could be considered a gluconeogenic precursor. Defining a substrate as a gluconeogenic precursor requires careful articulation of the definition, organism, and physiologic conditions under consideration.

Anaplerotic reactions active during growth of Saccharomyces cerevisiae on glycerol.

Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h-1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.

Octyl gallate triggers dysfunctional mitochondria leading to ROS driven membrane damage and metabolic inflexibility along with attenuated virulence in Candida albicans.

Recently the high incidence of worldwide Candida infections has substantially increased. The growing problem about toxicity of antifungal drugs and multidrug resistance aggravates the need for the development of new effective strategies. Natural compounds in this context represent promising alternatives having potential to be exploited for improving human health. The present study was therefore designed to evaluate the antifungal effect of a naturally occurring phenolic, octyl gallate (OG), on Candida albicans and to investigate the underlying mechanisms involved. We demonstrated that OG at 25 µg/ml could effectively inhibit C. albicans. Mechanistic insights revealed that OG affects mitochondrial functioning as Candida cells exposed to OG did not grow on non-fermentable carbon sources. Dysfunctional mitochondria triggered generation of reactive oxygen species (ROS), which led to membrane damage mediated by lipid peroxidation. We explored that OG inhibited glucose-induced reduction in external pH and causes decrement in ergosterol levels by 45%. Furthermore, OG impedes the metabolic flexibility of C. albicans by inhibiting the glyoxylate enzyme isocitrate lyase, which was also confirmed by docking analysis. Additionally, OG affected virulence traits such as morphological transition and cell adherence. Furthermore, we depicted that OG not only prevented biofilm formation but eliminates the preformed biofilms. In vivo studies with Caenorhabditis elegans nematode model confirmed that OG could enhance the survival of C. elegans after infection with Candida. Toxicity assay using red blood cells showed only 27.5% haemolytic activity. Taken together, OG is a potent inhibitor of C. albicans that warrants further structural optimization and pharmacological investigations.

Repurposing of respiratory drug theophylline against Candida albicans: mechanistic insights unveil alterations in membrane properties and metabolic fitness.

AIMS: Drug repurposing is an attractive chemotherapeutic strategy that serves to make up for the inadequacy of current antifungal drugs. The present study aims to repurpose theophylline (THP) against Candida albicans. THP is a methylxanthine derived from cocoa beans and tea extracts, generally used as the first-line drug for asthma and other respiratory disorders. METHODS AND RESULTS: We investigated the antifungal activity of THP against C. albicans and non-albicans species. Mechanistic insights revealed that THP induces membrane damage. Enhanced ionic disturbances and depleted ergosterol levels with the concomitant rise in membrane fluidity due to elevated flippase activity confirmed the membrane damaging effect. THP impeded the metabolic adaptability of C. albicans by inhibiting malate synthase and isocitrate lyase enzymes of the glyoxylate cycle. In vivo efficacy of THP was depicted by increased survival of C. albicans infected Caenorhabditis elegans model. CONCLUSIONS: This study elucidates the antifungal potential of THP with mechanistic insights. SIGNIFICANCE AND IMPACT OF THE STUDY: This study unveils the antifungal potential of THP, a known respiratory drug that can be further utilized for a wider range of applications such as combating fungal infections. The effect of THP with the known antifungal drugs can be exploited in the combinatorial drug approach for treating candidiasis.