Efflux Protein Research Articles

Inhalable nanoparticles with enhanced cuproptosis and cGAS–STING activation for synergistic lung metastasis immunotherapy

Due to the insufficient Cu+ accumulation, Cu+ efflux mechanism, and highly immunosuppressive tumor microenvironment (TME) in lung metastasis, the cuproptosis efficacy is limited. Herein, an inhalable nanodevice (CLDCu) is constructed to successfully overcome the drawbacks of cuproptosis. CLDCu consists of a Cu2+-chitosan shell and low molecular weight heparin-tocopherol succinate (LMWH-TOS, LT) core with disulfiram (DSF) loading. The prepared CLDCu can be inhaled and accumulate in large amounts in lung lesions (63.6%) with 56.5 times higher than intravenous injection. Within tumor cells, the mild acidity triggers the co-release of DSF and Cu2+, thus generating bis(diethyldithiocarbamate)-copper (CuET) to block Cu+ efflux protein ATP7B and forming toxic Cu+, leading to enhanced cuproptosis. Meanwhile, the released chitosan cooperates with CLDCu-induced cuproptosis to activate stimulator of interferon genes (STING) pathway, which significantly potentiates dendritic cells (DCs) maturation, as wells as evokes innate and adaptive immunity. In lung metastatic mice model, CLDCu is found to induce cuproptosis and reverse the immunosuppressive TME by inhalation administration. Moreover, CLDCu combined with anti-programmed cell death protein ligand-1 antibody (aPD-L1) provokes stronger antitumor immunity. Therefore, nanomedicine that combines cuproptosis with STING activation is a novel strategy for tumor immunotherapy.

Ferroptosis resists intracellular Vibrio splendidus AJ01 mediated by ferroportin in sea cucumber Apostichopus japonicus

Ferroptosis, a kind of programmed cell death, is characterized with iron-dependent lipid ROS buildup, which is considered as an important cellular immunity in resisting intracellular bacterial infection in mammalian macrophages. In this process, lipid ROS oxidizes the bacterial biofilm to inhibit intracellular bacteria. However, the function of ferroptosis in invertebrate remains unknown. In this study, the existence of ferroptosis in Apostichopus japonicus coelomocytes was confirmed, and its antibacterial mechanism was investigated. First, our results indicated that the expression of glutathione peroxidase (AjGPX4) was significantly inhibited by 0.21-fold (p < 0.01) after injecting A. japonicus with the ferroptosis inducer RSL3, and the contents of MDA (3.93-fold, p < 0.01), ferrous iron (1.40-fold, p < 0.01), and lipid ROS (3.10-fold, p < 0.01) were all significantly increased under this condition and simultaneously accompanied with mitochondrial contraction and disappearance of cristae, indicating the existence of ferroptosis in the coelomocytes of A. japonicus. Subsequently, the contents of ferrous iron (1.40-fold, p < 0.05), MDA (2.10-fold, p < 0.01), ROS (1.70-fold, p < 0.01), and lipid ROS (2.50-fold, p < 0.01) were all significantly increased, whereas the mitochondrial membrane potential and GSH/GSSG were markedly decreased by 0.68-fold (p < 0.05) and 0.69-fold (p < 0.01) under Vibrio splendidus (AJ01) infection. This process could be reversed by the iron-chelating agent deferoxamine mesylate, which indicated that AJ01 could induce coelomocytic ferroptosis. Moreover, the results demonstrated that the intracellular AJ01 load was clearly decreased to 0.49-fold (p < 0.05) and 0.06-fold (p < 0.01) after treating coelomocytes with RSL3 and ferrous iron, which indicated that enhanced ferroptosis could inhibit bacterial growth. Finally, subcellular localization demonstrated that ferrous iron efflux protein ferroportin (AjFPN) and intracellular AJ01 were co-localized in coelomocytes. After AjFPN interference (0.58-fold, p < 0.01), the signals of ferrous iron and lipid ROS levels in intracellular AJ01 were significantly reduced by 0.38-fold (p < 0.01) and 0.48-fold (p < 0.01), indicating that AjFPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via AjFPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection.

Ultrasound-Assisted Extraction of Atractylodes chinensis (DC.) Koidz. Polysaccharides and the Synergistic Antigastric Cancer Effect in Combination with Oxaliplatin.

Oxaliplatin (OXA) is recognized as a first-line drug for gastric cancer. However, low accumulation of the OXA in the target site and the development of drug resistance directly led to treatment failure. In the present study, an ultrasonic extraction method for Atractylodes chinensis (DC.) Koidz. polysaccharides (AKUs) and the combination treatment with OXA in vitro were studied. Results showed that when the pH level was 11, the ultrasound power at 450 W, the solid-liquid ratio was 1:20, and the ultrasound treatment for 30 min, the yield of AKUs was significantly increased to 13.20 ± 0.35%. The molecular weights of the AKUs ranged from 7.21 to 185.94 kDa, and its monosaccharides were mainly composed of arabinose (Ara), galactose (Gal), and glucose (Glu) with a ratio of 58.36, 16.90, and 15.49%, respectively. Cell experiments showed that, compared to OXA alone (2 μg/mL, inhibition rate of 18%), the treatment of OXA with AKUs had a significant synergistic inhibitory effect on MKN45 proliferation, which increased to 33, 41, and 45% with increasing AKUs concentrations (5-50 μg/mL), respectively, representing a 2.5-fold inhibition. Inductively coupled plasma-mass spectrometry (ICP-MS) determination confirmed that AKUs significantly increased the intracellular uptake of OXA by 29%, compared to that of OXA alone. We first demonstrated that the combined synergistic inhibitory effect of AKUs and OXA on gastric cancer cells was mediated by reducing the expression of efflux proteins (MRP1 and MRP2) and increasing the expression of ingested protein (OCT2). As a result of the above, AKUs deserved to be an effective adjuvant combined with chemotherapeutics in a clinical setting.

Zinc and manganese homeostasis closely interact in mammalian cells.

Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn-related diseases in the future.

A PadR family transcriptional repressor regulates the transcription of chromate efflux transporter in Enterobacter sp. Z1.

Chromium is a prevalent toxic heavy metal, and chromate [Cr(VI)] exhibits high mutagenicity and carcinogenicity. The presence of the Cr(VI) efflux protein ChrA has been identified in strains exhibiting resistance to Cr(VI). Nevertheless, certain strains of bacteria that are resistant to Cr(VI) lack the presence of ChrB, a known regulatory factor. Here, a PadR family transcriptional repressor, ChrN, has been identified as a regulator in the response of Enterobacter sp. Z1(CCTCC NO: M 2019147) to Cr(VI). The chrN gene is cotranscribed with the chrA gene, and the transcriptional expression of this operon is induced by Cr(VI). The binding capacity of the ChrN protein to Cr(VI) was demonstrated by both the tryptophan fluorescence assay and Ni-NTA purification assay. The interaction between ChrN and the chrAN operon promoter was validated by reporter gene assay and electrophoretic mobility shift assay. Mutation of the conserved histidine residues His14 and His50 resulted in loss of ChrN binding with the promoter of the chrAN operon. This observation implies that these residues are crucial for establishing a DNA-binding site. These findings demonstrate that ChrN functions as a transcriptional repressor, modulating the cellular response of strain Z1 to Cr(VI) exposure.

Screening of Potential Phytochemicals for the Identified Candidate Virulence Proteins in Enterobacter huaxiensis

ABSTRACT: Enterobacter huaxiensis, a novel gram-negative bacterium of the family Enterobacteriaceae, was recovered from the blood of the patients at West China Hospital. The present study aims to predict potential lead molecules against the identified virulence-associated antibiotic-resistant protein for drug designing. Two virulence-associated antibiotic-resistance proteins belonging to the OqxAB efflux protein family of the RND superfamily were identified in the pathogen using bioinformatics tools/databases. Based on the structure prediction by homology modeling and validations, the RND transporter permease subunit, OqxB was selected as the potential target for lead identification. The binding pocket of the target protein was calculated using CASTp. A total of 204 phytochemicals were screened virtually to obtain compounds that had better binding affinity, drug-likeness and pharmacokinetic potential to be used as safe ligands against the target protein. Among these, Chrysoeriol, Isopimaric acid, Baicalein and Biochanin A were found to be within the permissible range of Lipinski rule of five for drug-likeness, possessing better ADMET properties, and lower target-protein binding energy (less than -8.0 kcal/mol). Ligand-protein docking showed stable non-covalent interactions between active site residues and ligands. Thus, these compounds may be considered potential inhibitors of the target protein that may inactivate the efflux pump and restore antibiotic sensitivity.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153

BackgroundThe emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues.ObjectiveThe objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153.MethodologyEnterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation.ResultsThe Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 μg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 μg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA.ConclusionThe findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Abstract 174: Cholesterol efflux protein ABCA1 skews macrophages towards an anti-tumor phenotype and consequently impacts breast cancer progression

Abstract With a lifetime risk of approximately 1 in 8, breast cancer maintains in its position as the most prevalent form of cancer and ranks second in terms of cancer-related fatalities in women. The primary cause of mortality is often attributed to the recurrence of metastatic breast cancer. While immune therapy shows some effectiveness, many patients do not exhibit meaningful responses, likely due to immune-suppressive factors present within the tumor microenvironment. These factors include myeloid cells such as Macrophages(MΦ), therefore, there is a strong need to devise strategies to shift (MΦ) from a pro-tumor to an anti-tumor phenotype. Our previous research has uncovered the susceptibility of MΦs to disruptions in cholesterol metabolic balance. Considering these findings, coupled with epidemiological evidence linking elevated plasma cholesterol to unfavorable prognosis, the impact of cholesterol homeostasis in tumor associated MΦs on breast cancer becomes evident. An in-depth computational screen helped us identify ABCA1—a cholesterol efflux protein with elevated mRNA expression associated with increased survival. Beyond cholesterol efflux, ABCA1 also facilitates the movement of cholesterol from the inner membrane layer to the outer layer. Previous work has shown that cholesterol within the inner membrane layer can directly interact with and activate various receptors on the membrane's surface, initiating downstream signaling events. Consequently, the activities of ABCA1 in cholesterol efflux and translocation likely hold substantial regulatory implications. Underscoring the clinical significance of ABCA1 are our findings that heightened ABCA1 expression within breast tumors correlates with augmented levels of cytotoxic T cells, T cell effector enzymes (Prf-1 and Gzmb), and improved survival rates. Crucially, our in vitro investigations indicate that ABCA1 modulates multiple functions of MΦs to adopt an anti-cancer phenotype, encompassing: (1) the ability to infiltrate tumor spheroids, (2) angiogenesis, (3) efferocytosis—an immune-suppressive phagocytic process devoid of antigen presentation, and (4) the expansion, migration, and activity of cytotoxic T cells. ABCA1 transports cholesterol from macrophages to a lipid poor ApoA1 protein and facilitates the formation of nascent HDL via reverse cholesterol transport. We leveraged this mechanism to therapeutically target ABCA1 using ApoA1 mimetics 5A and 4F. Our data so far indicates that these ApoA1 phenocopy ABCA1 overexpression in MΦs. Together, our data provides compelling evidence for the involvement of ABCA1 in the pathophysiology of breast tumors. The anticipated outcomes of this research will establish the basis for the future utilization of ABCA1 and its downstream effects for therapeutic purposes. Citation Format: Shruti V. Bendre, Natalia Krawczynska, Shaunak Bhogale, Erin Weisser, Simon Han, Basel Hajyousif, Avni Singh, Rajendra K C, Yu Wang, Claire P. Schane, Adam T. Nelczyk, Anasuya DasGupta, Hashni E. Gamage, Kevin T. VanBortle, Emad Tajkhorshid, Saurabh Sinha, Wohnwa Cho, Erik R. Nelson. Cholesterol efflux protein ABCA1 skews macrophages towards an anti-tumor phenotype and consequently impacts breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 174.

Antimicrobial and Antibiofilm Activity of Highly Soluble Polypyrrole Against Methicillin-Resistant Staphylococcus aureus.

As a new promising therapeutic option, the activity of highly soluble polypyrrole (Hs-PPy), a specific formulation of a conducting polymer (polypyrrole - PPy) is evaluated against bacteria of clinical importance. The purpose was to evaluate the antimicrobial activity of Hs-PPy, alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. 10 clinical isolates of MRSA and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 μg/mL and bactericidal action at 62.5 μg/mL, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

Indole signaling enhances biofilm formation and quorum sensing in sequencing biofilm batch reactors

Indole is a heterocyclic organic compound that acts as a signaling molecule to regulate biofilm formation (BF) by microorganisms. However, the potential roles of indole in BF among microbial communities in wastewater treatment systems remain unknown. In this study, the effects of indole signaling on BF and quorum sensing (QS) of the complex microbial communities in sequencing batch biofilm reactors were systematically explored. The results showed that indole at 200 mg/L increased BF along with concentrations of homoserine lactones. High-throughput sequencing revealed the underlying mechanism of notable differences in the BF capacity of microbial communities between the indole-treated and control groups. The indole-related metabolic and QS functions of the genera in the biofilm were annotated based on the QS-related protein database and previous studies. Relative abundance analysis showed that the QS-related genera, such as Acinetobacter, that produce indole oxygenase were significantly enriched in the indole-treatment group. In addition, bioinformatics analyses revealed that indole at 200 mg/L enhanced BF, QS, and protein efflux. Based on a phylogenetic molecular ecological network, QS was positively correlated to indole treatment of Burkholderia, suggesting that indole promotes QS and BF. This study provides new insights into the effects of indole signaling on BF of microbes and sheds light on its potential regulatory mechanism in wastewater treatment plants.

Abstract A033: Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: Pharmacokinetics and exposure at the sites of efficacy and toxicity

Abstract A033: Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: Pharmacokinetics and exposure at the sites of efficacy and toxicity

Carbon sequestration reduced by the interference of nanoplastics on copper bioavailability

Microplastics (MPs) have been recognized as a serious new pollutant, especially nanoplastics (NPs) pose a greater threat to marine ecosystem than larger MPs. Within these ecosystems, phytoplankton serve as the foundational primary producers, playing a critical role in carbon sequestration. Copper (Cu), a vital cofactor for both photosynthesis and respiration in phytoplankton, directly influences their capacity to regulate atmospheric carbon. Therefore, we assessed the impact of NPs on Cu bioavailability and carbon sequestration capacity. The results showed that polystyrene nanoplastics (PS-NPs) could inhibit the growth of Thalassiosira weissflogii (a commonly used model marine diatom) and Chlorella pyrenoidosa (a standard strain of green algae). The concentration of Cu uptake by algae has a significant negative correlation with COPT1 (a Cu uptake protein), but positive with P-ATPase (a Cu efflux protein). Interestingly, PS-NPs exposure could reduce Cu uptake and carbon Cu sequestration capacity of algae, i.e., when the concentration of PS-NPs increases by 1 mg/L, the concentration of fixed carbon dioxide decreases by 0.0023 ppm. This provides a new perspective to reveal the influence mechanisms of PS-NPs on the relationship between Cu biogeochemical cycling and carbon source and sink.

The anti-staphylococcal fusidic acid as an efflux pump inhibitor combined with fluconazole against vaginal candidiasis in mouse model

BackgroundCandida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC).ObjectivesThis study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates.MethodsThe resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis.ResultsFusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue.ConclusionThe current findings highlight FA’s potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

An Insight into Synthetic Strategies, SAR Study and Anticancer Mechanism of Chalcone Derivatives: Medicinal Chemistry Perspective.

Chalcone derivatives continue to captivate medicinal chemists due to their modest chemistry, facile synthetic method, and promising biological activities. They have become wellknown in the therapeutic and pharmaceutical industries due to their diverse biological actions. These complexes offer different features and applications in a human biological system due to the flexibility of the reactions, including antimicrobial, antineoplastic, antimalarial, and other uses. As a result, chalcones have sparked much attention in malignancy research. Cancer is characterized by uncontrollably growing and spreading abnormal cells and aberrant cell behavior. These masses destroy surrounding normal tissue and can attack vital organs, leading to widespread disease. Cancer is frequently used as a warning sign for impending patient death. In the age of pharmaceutical chemistry, it is unavoidably a cause for concern and a growing weight on the populace. The pathophysiology of all malignancies is due to faulty genes that regulate the development, division, and death of cells. Various genetic and environmental variables combine to cause mutations in genes encoding essential cell-regulatory proteins, leading to the numerous alterations that characterize the evolution of cancers. Rather than directly targeting DNA synthesis, the new generation of anticancer medications target signals that promote or control the cell cycle, growth factors and their receptors, signal transduction pathways, and pathways impacting DNA repair and death. Drug hunters are focusing their attention on chalcone derivatives with varying chemical structures since they are essential in the search for anticancer drugs. Chalcone's anticancer action may be attributed to molecular changes such as drug efflux protein activities, activation of apoptosis, DNA and mitochondrial damage, inhibition of angiogenesis, tubulin inhibition, and kinase inhibition. Chalcones are used to diagnose cancer as well. The development of chalcone congeners as a prodrug or prime chemical to combat cancer necessitates a thorough investigation. This review gives an update on the different pharmacological activities of natural and synthesized chalcones in recent years. Furthermore, the structure- activity connections and processes are extensively documented, providing essential design and synthesis assistance in the future.

Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

A TetR family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae.

Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a resistance-nodulation-cell division (RND) efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB, or tolC abrogated the ARS resistance of the atrR mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a resistance-nodulation-cell division (RND) efflux pump of V. cholerae, is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.

Functional characterization of a TerC family protein of Riemerella anatipestifer in manganese detoxification and virulence.

Manganese (Mn) is an essential element for bacteria, but the overload of manganese is toxic. In a previous study, we showed that the cation diffusion facilitator protein MetA and the resistance-nodulation-division efflux pump MetB are responsible for Mn efflux in the bacterial pathogen Riemerella anatipestifer CH-1. However, whether this bacterium encodes additional manganese efflux proteins is unclear. In this study, we show that R. anatipestifer CH-1 encodes a tellurium resistance C (TerC) family protein with low similarity to other characterized TerC family proteins. Compared to the wild type (WT), the terC mutant of R. anatipestifer CH-1 (∆terC) is sensitive to Mn(II) intoxication. The ability of TerC to export manganese is higher than that of MetB but lower than that of MetA. Consistently, terC deletion (∆terC) led to intracellular accumulation of Mn2+ under excess manganese conditions. Further study showed that ∆terC was more sensitive than the WT to the oxidant hypoclorite but not to hydrogen peroxide. Mutagenesis studies showed that the mutant at amino acid sites of Glu116 (E116), Asp122 (D122), Glu245 (E245) Asp248 (D248), and Asp254 (D254) may be involved in the ability of TerC to export manganese. The transcription of terC was upregulated under excess manganese and downregulated under iron-limited conditions. However, this was not dependent on the manganese metabolism regulator MetR. In contrast to a strain lacking the manganese efflux pump MetA or MetB, the terC mutant is attenuated in virulence in a duckling model of infection due to increased sensitivity to duck serum. Finally, comparative analysis showed that homologs of TerC are distributed across the bacterial kingdom, suggesting that TerC exerts a conserved manganese efflux function.IMPORTANCERiemerella anatipestifer is a notorious bacterial pathogen of ducks and other birds. In R. anatipestifer, the genes involved in manganese efflux have not been completely identified, although MetA and MetB have been identified as two manganese exporters. Additionally, the function of TerC family proteins in manganese efflux is controversial. Here, we demonstrated that a TerC family protein helps prevent Mn(II) intoxication in R. anatipestifer and that the ability of TerC to export manganese is intermediate compared to that of MetA and MetB. Sequence analysis and mutagenesis studies showed that the conserved key amino sites of TerC are Glu116, Asp122, Glu245, Asp248, and Asp254. The transcription of terC was regulated by manganese excess and iron limitation. Finally, we show that TerC plays a role in the virulence of R. anatipestifer due to the increased sensitivity to duck serum, rather than the increased sensitivity to manganese. Taken together, these results expand our understanding of manganese efflux and the pathogenic mechanisms of R. anatipestifer.

Multixenobiotic response of Collembola to soil contamination, the physiological basis for bioindicative environmental monitoring

Collembola are well-established models in ecotoxicological research, extensively employed to investigate the effects of various contaminants, including heavy metals. The Multixenobiotic Resistance Mechanism (MXR) is a physiological response based on transmembrane efflux proteins that play a pivotal role in pumping xenobiotics and conferring resistance. This mechanism is firmly established as a biomarker of aquatic contamination and has recently shown promise as a soil biomonitoring tool. In this study, we aimed to assess the feasibility of utilizing the MXR mechanism as a biomonitoring tool, specifically by investigating the response of two Collembola species exposed to soil contaminated in a real-life situation. Soil samples were obtained from the site of Brazil's largest mine disaster, a dam rupture in Brumadinho-MG. We explored MXR activity in the model species Folsomia candida and a tropical native species, Cyphoderus sp. Our findings reveal efflux activity in both species, confirmed by model MXR protein inhibitors. Moreover, we observed distinct MXR activity levels corresponding to the degree of heavy metal contamination in the soil samples. Consequently, our results underscore the potential of combining an established soil bioindicator, such as Collembola, with the physiological response of a molecular biomarker like MXR. This approach may represent a valuable strategy for biomonitoring terrestrial ecosystems.

The arbuscular mycorrhizal fungus Rhizophagus irregularis uses the copper exporting ATPase RiCRD1 as a major strategy for copper detoxification

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with most land plants. AM fungi regulate plant copper (Cu) acquisition both in Cu deficient and polluted soils. Here, we report characterization of RiCRD1, a Rhizophagus irregularis gene putatively encoding a Cu transporting ATPase. Based on its sequence analysis, RiCRD1 was identified as a plasma membrane Cu + efflux protein of the P1B1-ATPase subfamily. As revealed by heterologous complementation assays in yeast, RiCRD1 encodes a functional protein capable of conferring increased tolerance against Cu. In the extraradical mycelium, RiCRD1 expression was highly up-regulated in response to high concentrations of Cu in the medium. Comparison of the expression patterns of different players of metal tolerance in R. irregularis under high Cu levels suggests that this fungus could mainly use a metal efflux based-strategy to cope with Cu toxicity. RiCRD1 was also expressed in the intraradical fungal structures and, more specifically, in the arbuscules, which suggests a role for RiCRD1 in Cu release from the fungus to the symbiotic interface. Overall, our results show that RiCRD1 encodes a protein which could have a pivotal dual role in Cu homeostasis in R. irregularis, playing a role in Cu detoxification in the extraradical mycelium and in Cu transfer to the apoplast of the symbiotic interface in the arbuscules.