Integrative Inducer Intervention and Transcriptomic Analyses Reveal the Metabolism of Paralytic Shellfish Toxins in Azumapecten farreri.

Paralytic shellfish toxins (PSTs) are widely distributed neurotoxins, and the PST metabolic detoxification mechanism in bivalves has received increasing attention. To reveal the effect of phase I (cytochrome P450)-II (GST)-III (ABC transport) metabolic systems on the PST metabolism in Azumapecten farreri, this study amplified stress on the target systems using rifampicin, dl-α-tocopherol, and colchicine; measured PST levels; and conducted transcriptomic analyses. The highest toxin content reached 1623.48 µg STX eq/kg in the hepatopancreas and only 8.8% of that in the gills. Inducer intervention significantly decreased hepatopancreatic PST accumulation. The proportional reductions in the rifampicin-, dl-α-tocopherol-, and colchicine-induced groups were 55.3%, 50.4%, and 36.1%, respectively. Transcriptome analysis showed that 11 modules were significantly correlated with PST metabolism (six positive/five negative), with phase I CYP450 and phase II glutathione metabolism significantly enriched in negatively correlated pathways. Twenty-three phase I-II-III core genes were further validated using qRT-PCR and correlated with PST metabolism, revealing that CYP46A1, CYP4F6, GSTM1, and ABCF2 were significantly correlated, while CYP4F11 and ABCB1 were indirectly correlated. In conclusion, phase I-II-III detoxification enzyme systems jointly participate in the metabolic detoxification of PSTs in A. farreri. This study provides key data support to profoundly elucidate the PST metabolic detoxification mechanism in bivalves.

Changes of Mycobacterium tuberculosis specific antigen-stimulated CD27-CD38+IFN-γ+CD4+ T cells before and after anti-tuberculosis treatment.

BACKGROUND: The aim of the study was to investigate whether the expression of CD27-CD38+ in interferon (IFN)-γ+CD4+ T cells stimulated by the specific antigen early secreted antigenic target-6 (ESAT-6)/culture filter protein-10 (CFP-10) could be a potential new therapeutic evaluation indicator for anti-tuberculosis (TB) treatment. METHODS: Newly diagnosed active pulmonary TB patients, latent TB infection (LTBI) and healthy controls were enrolled from January 2021 to December 2021. PTB patients were treated by standard anti-TB regimen 2HREZ/4HR (2 months of isoniazid (H), rifampin (R), ethambutol (E), and pyrazinamide (Z) followed by 4 months of isoniazid (H) and rifampin (R)). The difference of CD27-CD38+ expression in IFN-γ+CD4+ T cells before treatment, 2 months after treatment, and 6 months after treatment were compared. RESULTS: Total 45 PTB patients, 38 LTBI cases and 43 healthy controls were enrolled. The expression of CD27-CD38+ decreased significantly after anti-TB treatment and was comparable with that in LTBI and healthy controls when the 6-month anti-TB treatment course was completed. The decline rate of CD27-CD38+ between 6 months after treatment and baseline was positively correlated with erythrocyte sedimentation rate (r = 0.766, P < 0.0001), C-reactive protein (r = 0.560, P = 0.003) and chest computerized tomography severity score (r = 0.632, P = 0.0005). The area under receiver operator characteristic curve of CD27-CD38+ in distinguish pulmonary TB patients before and after treatment was 0.779. CONCLUSION: The expression of CD27-CD38+ in ESAT-6/CFP-10 stimulated IFN-γ+CD4+T cells can well reflect the changes of the disease before and after anti-TB treatment, which is expected to be a potential new therapeutic evaluation index. Clinical Registry number chiCTR1800019966.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

Mechanistic Static Model based Prediction of Transporter Substrate Drug-Drug Interactions Utilizing Atorvastatin and Rifampicin.

OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.

High-resolution landscape of an antibiotic binding site.

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Quantitative Prediction of OATP-Mediated Disposition and Biliary Clearance Using Human Liver Chimeric Mice.

Drug biliary clearance (CLbile) in vivo is among the most difficult pharmacokinetic parameters to predict accurately and quantitatively because biliary excretion is influenced by metabolic enzymes, transporters, and passive diffusion across hepatocyte membranes. The purpose of this study is to demonstrate the use of Hu-FRG mice [Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice transplanted with human-derived hepatocytes] to quantitatively predict human organic anion transporting polypeptide (OATP)-mediated drug disposition and CLbile To predict OATP-mediated disposition, six OATP substrates (atorvastatin, fexofenadine, glibenclamide, pitavastatin, pravastatin, and rosuvastatin) were administered intravenously to Hu-FRG and Mu-FRG mice (FRG mice transplanted with mouse hepatocytes) with or without rifampicin as an OATP inhibitor. We calculated the hepatic intrinsic clearance (CLh,int) and the change of hepatic clearance (CLh) caused by rifampicin (CLh ratio). We compared the CLh,int of humans with that of Hu-FRG mice and the CLh ratio of humans with that of Hu-FRG and Mu-FRG mice. For predicting CLbile, 20 compounds (two cassette doses of 10 compounds) were administered intravenously to gallbladder-cannulated Hu-FRG and Mu-FRG mice. We evaluated the CLbile and investigated the correlation of human CLbile with that of Hu-FRG and Mu-FRG mice. We found good correlations between humans and Hu-FRG mice in CLh,int (100% within threefold) and CLh ratio (R2 = 0.94). Moreover, we observed a much better relationship between humans and Hu-FRG mice in CLbile (75% within threefold). Our results suggest that OATP-mediated disposition and CLbile can be predicted using Hu-FRG mice, making them a useful in vivo drug discovery tool for quantitatively predicting human liver disposition. SIGNIFICANCE STATEMENT: OATP-mediated disposition and biliary clearance of drugs are likely quantitatively predictable using Hu-FRG mice. The findings can enable the selection of better drug candidates and the development of more effective strategies for managing OATP-mediated DDIs in clinical studies.

Rhus chinensis Mill. fruits alleviate liver injury induced by isoniazid and rifampicin through regulating oxidative stress, apoptosis, and bile acid transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhus chinensis Mill. is a species of the genus Rhus belonging to the family Anacardiaceae. Its fruits used to treat/prevent liver related diseases (e.g., jaundice and hepatitis) in folk medicine. Otherwise, the effects and underlying mechanisms of the fruits on the prevention of isoniazid and rifampicin-caused liver injury have not been investigated. AIM OF THE STUDY: To study the preventive effects and mechanisms of the Rhus chinensis Mill. fruits on isoniazid and rifampicin-caused liver injury. MATERIALS AND METHODS: This experiment was based on rifampicin (75 mg/kg/day) and isoniazid (75 mg/kg/day)-induced liver damage model to explain the pharmacological effects of Rhus chinensis Mill. fruits. The prevention of the extract from Rhus chinensis Mill. fruits on isoniazid and rifampicin-caused liver injury were evaluated using biochemical parameters, histopathological analysis, and immunofluorescence technique. Apart from that, the potential molecular mechanisms were elucidated by analyzing the expression of such crucial proteins participated in oxidative stress, apoptosis, and bile acid transport. RESULTS: The extract from Rhus chinensis Mill. fruits significantly reduced the levels of ALT, AST, TBIL, ALP and MDA. Besides, the extract, especially 800 mg/kg b.w., was remarkably decreased the content of TNF-α,IL-6 and IL-1ß, restored the levels of GSH and SOD. The results of Western blot also presented that the extract could activate the Nrf2 protein pathway and inhibit the expression of CYP2E1 to reduce oxidative stress. Meanwhile, the extract significantly up-regulated the expressions of BSEP and Mrp2 to regulate the transport of bile acid, and alleviated the cellular apoptosis via adjusting the expression of Bax and Bcl-2 proteins. CONCLUSIONS: Rhus chinensis Mill. fruits can prevent the liver injury induced by isoniazid and rifampicin in mice through adjusting the expressions of multiple proteins in oxidative stress, apoptosis, and bile acid transport pathways. This paper may provide scientific basis for the fruits as a Chinese medicine to prevent/cure liver injury.

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction.

Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

Hepatocyte Transplantation Rebalances Cytokines for Hepatic Regeneration in Rats with Ataxia Telangiectasia Mutated Pathway-Related Acute Liver Failure.

Inadequate DNA damage response related to ataxia telangiectasia mutated gene restricts hepatic regeneration in acute liver failure. Resolving mechanistic gaps in liver damage and repair requires additional animal models that are unconstrained by ultrarapid and unpredictable mortalities or substantial divergences from human pathology. This study used Fischer 344 rats primed with the antitubercular drug, rifampicin, plus phenobarbitone, and monocrotaline, a DNA adduct-forming alkaloid. Rifampicin and monocrotaline can cause liver failure in people. This regimen resulted in hepatic oxidative stress, necrosis, DNA double-strand breaks, liver test abnormalities, altered serum cytokine expression, and mortality. Healthy donor hepatocytes were transplanted ectopically in the peritoneal cavity to study whether they could supply metabolic support and rebalance inflammatory or protective cytokines affecting liver regeneration events. Hepatocyte transplantation increased candidate cytokine levels (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, IL-10, and IL-12), leading to Atm, Stat3, and Akt signaling in hepatocytes and nonparenchymal cells, lowering of inflammation, and improvements in intermediary metabolism, DNA repair, and hepatocyte proliferation. Such control of DNA damage and inflammation, along with stimulation of hepatic growth, offers paradigms for cell signaling to restore hepatic homeostasis and regeneration in acute liver failure. Further studies of molecular pathways of high pathobiological impact will advance the knowledge of liver regeneration.

Quantitative Cytochrome P450 3A4 Induction Risk Assessment Using Human Hepatocytes Complemented with Pregnane X Receptor-Activating Profiles.

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.

Folic acid protects against tuberculosis-drug-induced liver injury in rats and its potential mechanism by metabolomics.

Observational study indicated that folic acid (FA) supplementation may protect against tuberculosis-drug-induced liver injury (TBLI). The aim is to investigate the effect and mechanism of FA on TBLI in rats. Liver injury was induced by a daily gavage of isoniazid (INH) and rifampicin (RIF) in the model and FA groups. Rats in the FA group were also treated with 2.5 mg/kg body weight FA. Rats in the control group were not treated. Eight rats were used in each group. The severity of liver injury was measured by the serum levels of hepatic enzymes and histological score. The metabolites in serum and liver tissues were analyzed by HPLC-Q-TOF-MS/MS. FA treatment significantly reduced alanine aminotransferase and liver necrosis. Seventy-nine differential metabolites in the serum and liver tissues were identified among the three groups. N-acylethanolamines, INH and RIF metabolites, phosphatidylcholines, lysophosphatidylcholines, monoglycerides, diglycerides and bile acids were regulated by FA treatment, involving key metabolic pathways, such as N-acylethanolamine metabolism, INH and RIF metabolism, liver regeneration, inflammation alleviation and bile acid metabolism. RT-PCR and western blotting results confirmed the altered N-acylethanolamine metabolism and improved drug metabolism by FA. In conclusion, FA was protective against TBLI, which may be related to the regulation of N-acylethanolamine metabolism and drug detoxification by FA.

Naringenin protects hepato-renal tissues against antituberculosis drugs induced toxic manifestations by modulating interleukin-6, insulin like growth factor-1, biochemical and ultra-structural integrity.

BACKGROUND: The antituberculosis drugs (ATDs), isoniazid, rifampicin, pyrazinamide and ethambutol prompt extreme hepatic and renal damage during treatment of tuberculosis. The present study aimed to investigate protective potential of naringenin against ATDs induced hepato-renal injury. METHODS: Rats were administered with ATDs (pyrazinamide; 210, ethambutol; 170, isoniazid; 85, rifampicin; 65 mg/kg b.wt) orally for 8 weeks (3 days/week) followed by naringenin at three different doses (10, 20 and 40 mg/kg b.wt) conjointly for 8 weeks (3 days/week alternately to ATDs administration) and silymarin (50 mg/kg b.wt) as positive control. RESULTS: Exposure to ATDs caused significant increase in interleukin-6 (IL-6), triglycerides, cholesterol, bilirubin whereas depletion in insulin like growth factor-1 (IGF-1), albumin and glucose in serum. Endogenous antioxidant enzymes glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate-dehydrogenase (G-6-PDH) were diminished in liver and kidney tissues with parallel increase in triglycerides, cholesterol, microsomal LPO and aniline hydroxylase (CYP2E1 enzyme). Ultra-structural observations of liver and kidney showed marked deviation in plasma membranes of various cellular and sub-cellular organelles after 8 weeks of exposure to ATDs. CONCLUSIONS: Conjoint treatment of naringenin counteracted ATDs induced toxic manifestations by regulating IL-6, IGF-1, CYP2E1, biochemical and ultra-structural integrity in a dose dependent manner. Naringenin has excellent potential to protect ATDs induced hepato-renal injury by altering oxidative stress, modulation of antioxidant enzymes, serum cytokines and ultra-structural changes.

Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxin-rifampicin combination against Pseudomonas aeruginosa.

BACKGROUND: Understanding the mechanism of antimicrobial action is critical for improving antibiotic therapy. For the first time, we integrated correlative metabolomics and transcriptomics of Pseudomonas aeruginosa to elucidate the mechanism of synergistic killing of polymyxin-rifampicin combination. METHODS: Liquid chromatography-mass spectrometry and RNA-seq analyses were conducted to identify the significant changes in the metabolome and transcriptome of P. aeruginosa PAO1 after exposure to polymyxin B (1 mg/L) and rifampicin (2 mg/L) alone, or in combination over 24 h. A genome-scale metabolic network was employed for integrative analysis. RESULTS: In the first 4-h treatment, polymyxin B monotherapy induced significant lipid perturbations, predominantly to fatty acids and glycerophospholipids, indicating a substantial disorganization of the bacterial outer membrane. Expression of ParRS, a two-component regulatory system involved in polymyxin resistance, was increased by polymyxin B alone. Rifampicin alone caused marginal metabolic perturbations but significantly affected gene expression at 24 h. The combination decreased the gene expression of quorum sensing regulated virulence factors at 1 h (e.g. key genes involved in phenazine biosynthesis, secretion system and biofilm formation); and increased the expression of peptidoglycan biosynthesis genes at 4 h. Notably, the combination caused substantial accumulation of nucleotides and amino acids that last at least 4 h, indicating that bacterial cells were in a state of metabolic arrest. CONCLUSION: This study underscores the substantial potential of integrative systems pharmacology to determine mechanisms of synergistic bacterial killing by antibiotic combinations, which will help optimize their use in patients.

Hepatoprotective Effects of Phloridzin against Isoniazid-Rifampicin Induced Liver Injury by Regulating CYP450 and Nrf2/HO-1 Pathway in Mice.

The protective effect of phloridzin (PHL) and its potential mechanism were examined in mice with liver injury induced by isoniazid (INH) and rifampicin (RFP). The mice were randomly divided into normal control group, model group, low (80 mg/kg), medium (160 mg/kg) and high (320 mg/kg) phloridzin-treated groups. After 28 d treatment, blood and liver tissue were collected and analysed. The results revealed that PHL regulated liver function related indicators and reduced the pathological tissue damage, indicating that PHL significantly alleviated the liver injury. Furthermore, the level of CYP450 enzyme, the expression of CYP3A4, CYP2E1, heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein were inhibited by PHL. These results indicated that PHL exerts a protecting effect against liver injury induced by combination of RFP and INH. The potential mechanisms may be concerned with the activation of Nrf2/HO-1 signaling pathway containing its key antioxidant enzymes and regulation of CYP3A4 and CYP2E1.

Activation of the SigE-SigB signaling pathway by inhibition of the respiratory electron transport chain and its effect on rifampicin resistance in Mycobacterium smegmatis.

Using a mutant of Mycobacterium smegmatis lacking the major aa3 cytochrome c oxidase of the electron transport chain (Δaa3), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa3 mutant, leading to upregulation of the SigB-dependent genes in the mutant. The pho U2 (MSMEG_1605) gene implicated in a phosphate-signaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa3 mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain.

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

The role of the farnesoid X receptor in quadruple anti-tuberculosis drug-induced liver injury.

Anti-tuberculosis drugs-induced liver injury may be associated with the hepatic farnesoid X receptor (FXR). However, the relationship between isoniazid, rifampicin, pyrazinamide and ethambutol (HRZE) coadministration-induced liver injury and FXR has not been clarified. The purpose of this study was to clarify the role of FXR in HRZE-induced liver injury. To measure indices of liver injury, blood samples were collected from clinical tuberculosis patients who had taken HRZE for approximately two months; in these patients serum total bile acids were increased, while other hepatic biochemical indexes showed no significant changes. When Wistar rats were orally administered isoniazid (30 or 60 mg/kg) + rifampicin (45 or 90 mg/kg) + pyrazinamide (150 or 300 mg/kg) + ethambutol (75 or 150 mg/kg) in combination for 15 days, the expression and function of FXR was up-regulated, and hepatic bile acids were decreased. However, following 30 days of HRZE treatment the expression and function of FXR was down-regulated and bile acids accumulated in the liver, suggestive of hepatotoxicity. Treatment of HepaRG cells with HRZE lead to time- and dose- dependent cytotoxicity, with the expression of FXR up-regulated in early stage, but down-regulated with prolonged HRZE treatment, consistent with the results of animal experiments. In summary, HRZE may upregulate FXR with short-term administration, but more prolonged treatment appears to suppress FXR function, resulting in hepatic bile acid accumulation.

Thyroid Hormone Transporters in a Human Placental Cell Model.

Background: Fetal brain development in the first half of pregnancy is dependent on maternal thyroid hormone (TH), highlighting the importance of trans-placental TH transport. It is yet unclear which transporters are involved in this process. We aimed to identify the major TH transporters in a human placental cell model (BeWo cells). Methods: Messenger RNA expression of the known TH transporters (the monocarboxylate transporter [MCT]8, MCT10, the L-type amino acid transporter [LAT]1, LAT2, the organic anion transporting peptide [OATP]1A2 and OATP4A1) in BeWo cells and human placenta were determined by quantitative PCR. To determine the specificity and efficacy of transporter inhibitors, we first determined TH uptake at different inhibitor concentrations in African green monkey kidney fibroblast-like cells (COS1 cells) overexpressing TH transporters. We then tested TH uptake in BeWo cells in the presence or absence of the optimal inhibitor concentrations. Results: All tested TH transporters were expressed in human term placentas, whereas MCT8 was absent in BeWo cells. Both 2-amino-2-norbornanecarboxylic acid (BCH) and L-tryptophan at 1 mM inhibited LATs, whereas at the highest concentration (10 mM) L-tryptophan also inhibited MCT10. Verapamil inhibited OATP1A2 and less efficiently both MCTs, but not LATs. Both rifampicin and naringin reduced OATP1A2 activity. Finally, silychristin inhibited MCT8 at submicromolar concentrations and OATP1A2 partially only at the highest concentration tested (10 µM). In BeWo cells, verapamil reduced triiodothyronine (T3) uptake by 24%, BCH by 31%, and 1 mM L-tryptophan by 41%. The combination of BCH and verapamil additively decreased T3 uptake by 53% and the combination of BCH and 10 mM L-tryptophan by 60%, suggesting a major role for MCT10 and LATs in placental T3 uptake. Indeed, transfection of BeWo cells with MCT10-specific small interfering RNA significantly reduced T3 uptake. Only the combination of BCH and verapamil significantly reduced thyroxine (T4) uptake in BeWo cells, by 32%. Conclusions: Using pharmacological inhibitors, we show that MCT10 and LATs play a major role in T3 uptake in BeWo cells. T4 uptake appears independent of known TH transporters, suggesting the presence of, currently unknown, alternative transporter(s).

Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi.

Covering: 2016 to 2022RNA polymerase (RNAP) is the central enzyme in bacterial gene expression representing an attractive and validated target for antibiotics. Two well-known and clinically approved classes of natural product RNAP inhibitors are the rifamycins and the fidaxomycins. Rifampicin (Rif), a semi-synthetic derivative of rifamycin, plays a crucial role as a first line antibiotic in the treatment of tuberculosis and a broad range of bacterial infections. However, more and more pathogens such as Mycobacterium tuberculosis develop resistance, not only against Rif and other RNAP inhibitors. To overcome this problem, novel RNAP inhibitors exhibiting different target sites are urgently needed. This review includes recent developments published between 2016 and today. Particular focus is placed on novel findings concerning already known bacterial RNAP inhibitors, the characterization and development of new compounds isolated from bacteria and fungi, and providing brief insights into promising new synthetic compounds.