In-silico evaluation of Bismurrayaquinone-A phytochemical as a potential multifunctional inhibitor targeting dihydrofolate reductase: implications for anticancer and antibacterial drug development.

Dihydrofolate reductase (DHFR) has gained significant attention in drug development, primarily due to marked distinctions in its active site among different species. DHFR plays a crucial role in both DNA and amino acid metabolism by facilitating the transfer of monocarbon residues through tetrahydrofolate, which is vital for nucleotide and amino acid synthesis. This considers its potential as a promising target for therapeutic interventions. In this study, our focus was on conducting a virtual screening of phytoconstituents from the IMPPAT2.0 database to identify potential inhibitors of DHFR. The initial criterion involved assessing the binding energy of molecules against DHFR and we screened top 20 compounds ranging energy -13.5 to -11.4 (kcal/Mol) while Pemetrexed disodium bound with less energy -10.2 (kcal/Mol), followed by an analysis of their interactions to identify more effective hits. We prioritized IMPHY007679 (Bismurrayaquinone-A), which displayed a high binding affinity and crucial interaction with DHFR. We also evaluated the drug-like properties and biological activity of IMPHY007679. Furthermore, MD simulation was done, RMSD, RMSF, Rg, SASA, PCA and FEL explore the time-evolution impact of IMPHY007679 comparing it with a reference drug, Pemetrexed disodium. Collectively, our findings suggest that IMPHY007679 recommend further investigation in both in vitro and in vivo settings for its potential in developing anticancer and antibacterial therapies. This compound holds promise as a valuable candidate for advancing drug research and treatment strategies.Communicated by Ramaswamy H. Sarma.

Toxicity and detoxification of T-2 toxin in poultry.

This review summarizes the updated knowledge on the toxicity of T-2 on poultry, followed by potential strategies for detoxification of T-2 in poultry diet. The toxic effects of T-2 on poultry include cytotoxicity, genotoxicity, metabolism modulation, immunotoxicity, hepatotoxicity, gastrointestinal toxicity, skeletal toxicity, nephrotoxicity, reproductive toxicity, neurotoxicity, etc. Cytotoxicity is the primary toxicity of T-2, characterized by inhibiting protein and nucleic acid synthesis, altering the cell cycle, inducing oxidative stress, apoptosis and necrosis, which lead to damages of immune organs, liver, digestive tract, bone, kidney, etc., resulting in pathological changes and impaired physiological functions of these organs. Glutathione redox system, superoxide dismutase, catalase and autophagy are protective mechanisms against oxidative stress and apoptosis, and can compensate the pathological changes and physiological functions impaired by T-2 to some degree. T-2 detoxifying agents for poultry feeds include adsorbing agents (e.g., aluminosilicate-based clays and microbial cell wall), biotransforming agents (e.g., Eubacterium sp. BBSH 797 strain), and indirect detoxifying agents (e.g., plant-derived antioxidants). These T-2 detoxifying agents could alleviate different pathological changes to different degrees, and multi-component T-2 detoxifying agents can likely provide more comprehensive protection against the toxicity of T-2.

Identification of Anti-tuberculosis Compounds From Aurone Analogs.

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains has made tuberculosis (TB) control more difficult. Aurone derivatives have demonstrated promising anti-bacterial activities, but their effects against Mtb have not been thoroughly determined. In this study, we aimed to develop anti-TB compounds from aurone analogs. We used a fluorescent protein tdTomato labeled Mtb CDC1551 strain to screen 146 synthesized aurone derivatives for effective anti-TB compounds. The 9504, 9505, 9501, 9510, AA2A, and AA8 aurones inhibited the growth of Mtb with minimal inhibitory concentrations of 6.25, 12.5, 25, 25, 25, and 50 µM, respectively. We also examined cytotoxicities of the six leads against the human liver cell line HepG2, the primate kidney cell line Vero and human monocyte THP-1 derived macrophages. Three of the aurone leads (9504, 9501, and 9510) showed low cytotoxic effects on all three cell lines and high Mtb inhibitory efficacy (selectivity index > 10). Aurone 9504, 9501, AA2A, or AA8 significantly reduced the Mtb load in the lungs of infected mice after a 12-days treatment. We determined that the aurone leads inhibit Mtb chorismate synthase, an essential enzyme for aromatic acid synthesis. Our studies demonstrate the promise of synthetic aurones as novel anti-TB therapeutics.

Mycobacterium tuberculosis LipE Has a Lipase/Esterase Activity and Is Important for Intracellular Growth and In Vivo Infection.

Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.

Rv1075c of Mycobacterium tuberculosis is a GDSL-Like Esterase and Is Important for Intracellular Survival.

Mycobacterium tuberculosis lipid metabolism pathways facilitate access to carbon and energy sources during infection. M. tuberculosis gene Rv1075c was annotated as a conserved hypothetical protein. We identified that Rv1075c amino acid sequence shares similarities with other bacterial lipase/esterases and we demonstrated that it has esterase activity, with preference for short-chain fatty acids, particularly acetate, with highest activity at 45°C, pH 9. Site-direct mutagenesis revealed its activity triad as Ser80, Asp244, and His247. We further determined that rRv1075c hydrolyzed triacetin and tributyrin, and it was mainly distributed in cell wall and membrane. Its expression was induced at pH 4.5, mimicking the acidic phagosome of macrophages. Mutation of Rv1075c led to reduced bacterial growth in THP-1 cells and human peripheral blood mononuclear cell-derived macrophages, and attenuated M. tuberculosis infection in mice. Our data suggest that Rv1075c is involved in ester and fatty acid metabolism inside host cells.

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

The impact of Fusarium mycotoxins on human and animal host susceptibility to infectious diseases.

Contamination of food and feed with mycotoxins is a worldwide problem. At present, acute mycotoxicosis caused by high doses is rare in humans and animals. Ingestion of low to moderate amounts of Fusarium mycotoxins is common and generally does not result in obvious intoxication. However, these low amounts may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host pathogen interactions and thus a different outcome of infection. This review summarizes the current state of knowledge about the impact of Fusarium mycotoxin exposure on human and animal host susceptibility to infectious diseases. On the one hand, exposure to deoxynivalenol and other Fusarium mycotoxins generally exacerbates infections with parasites, bacteria and viruses across a wide range of animal host species. Well-known examples include coccidiosis in poultry, salmonellosis in pigs and mice, colibacillosis in pigs, necrotic enteritis in poultry, enteric septicemia of catfish, swine respiratory disease, aspergillosis in poultry and rabbits, reovirus infection in mice and Porcine Reproductive and Respiratory Syndrome Virus infection in pigs. However, on the other hand, T-2 toxin has been shown to markedly decrease the colonization capacity of Salmonella in the pig intestine. Although the impact of the exposure of humans to Fusarium toxins on infectious diseases is less well known, extrapolation from animal models suggests possible exacerbation of, for instance, colibacillosis and salmonellosis in humans, as well.

T-2 toxin impairs antifungal activities of chicken macrophages against Aspergillus fumigatus conidia but promotes the pro-inflammatory responses.

Aspergillosis is the most common fungal disease of the avian respiratory tract and is caused primarily by Aspergillus fumigatus. The respiratory macrophages provide important defence against aspergillosis. T-2 toxin (T-2), a trichothecene mycotoxin produced by Fusarium spp. in improperly stored agricultural products, has immunomodulatory effects. We studied the impact of T-2 on the antifungal response of the chicken macrophage cell line HD-11 against A. fumigatus infection. The macrophages were first exposed to 0.5 to 10 ng/ml T-2 for 24 h, and then their viability, antifungal activity, and cytokine expression in response to A. fumigatus conidial infection were determined. The viability of macrophages decreased when exposed to T-2 at concentrations higher than 1 ng/ml. One hour after conidial infection, phagocytosed conidia were observed in 30% of the non-T-2-exposed macrophages, but in only 5% of the macrophages exposed to 5 ng/ml T-2. Seven hours after infection, 24% of the conidia associated with non-T-2-exposed macrophages germinated, in contrast to 75% of those with macrophages exposed to 5 ng/ml T-2. A. fumigatus infection induced upregulation of interleukin (IL)-1ß, CXCLi1, CXCLi2 and IL-12ß, and downregulation of transforming growth factor-ß4 in macrophages. Exposure of A. fumigatus-infected macrophages to T-2 at 1 to 5 ng/ml further upregulated the expression of IL-1ß, IL-6, CCLi2, CXCLi1, CXCLi2, IL-18 (at 1 and 2 ng/ml) and IL-12ß, and further downregulated that of transforming growth factor-ß4 (at 5 ng/ml). In conclusion, T-2 impaired the antifungal activities of chicken macrophages against A. fumigatus conidia, but might stimulate immune response by upregulating the expression of pro-inflammatory cytokines, chemokines and T-helper 1 cytokines.

Germination of Aspergillus fumigatus inside avian respiratory macrophages is associated with cytotoxicity.

Although aspergillosis is one of the most common diseases in captive birds, the pathogenesis of avian aspergillosis is poorly known. We studied the role of avian respiratory macrophages as a first line of defense against avian aspergillosis. The phagocytic and killing capacities of avian respiratory macrophages were evaluated using pigeon respiratory macrophages that were inoculated with Aspergillus fumigatus conidia. On average, 25% of macrophage-associated conidia were phagocytosed after one hour. Sixteen percents of these cell-associated conidia were killed after 4 h and conidial germination was inhibited in more than 95% of the conidia. A. fumigatus conidia were shown to be cytotoxic to the macrophages. Intracellularly germinating conidia were located free in the cytoplasm of necrotic cells, as shown using transmission electron microscopy. These results suggest that avian respiratory macrophages may prevent early establishment of infection, unless the number of A. fumigatus conidia exceeds the macrophage killing capacity, leading to intracellular germination and colonization of the respiratory tract.