Active Efflux Research Articles

Quantum biochemical analysis of the TtgR regulator and effectors

The recent expansion of multidrug-resistant (MDR) pathogens poses significant challenges in treating healthcare-associated infections. Although antibacterial resistance occurs by numerous mechanisms, active efflux of the drugs is a critical concern. A single species of efflux pump can produce a simultaneous resistance to several drugs. One of the best-studied efflux pumps is the TtgABC: a tripartite resistance-nodulation-division (RND) efflux pump implicated in the intrinsic antibiotic resistance in Pseudomonas putida DOT-T1E. The expression of the TtgABC gene is down-regulated by the HTH-type transcriptional repressor TtgR. In this context, by employing quantum chemistry methods based on the Density Functional Theory (DFT) within the Molecular Fragmentation with Conjugate Caps (MFCC) approach, we investigate the coupling profiles of the transcriptional regulator TtgR in complex with quercetin (QUE), a natural polyphenolic flavonoid, tetracycline (TAC), and chloramphenicol (CLM), two broad-spectrum antimicrobial agents. Our quantum biochemical computational results show the: [i] convergence radius, [ii] total binding energy, [iii] relevance (energetically) of the ligands regions, and [iv] most relevant amino acids residues of the TtgR-QUE/TAC/CLM complexes, pointing out distinctions and similarities among them. These findings improve the understanding of the binding mechanism of effectors and facilitate the development of new chemicals targeting TtgR, helping in the battle against the rise of resistance to antimicrobial drugs. These advances are crucial in the ongoing fight against rising antimicrobial drug resistance, providing hope for a future where healthcare-associated infections can be more beneficially treated.

Lipid exchange of apolipoprotein A-I amyloidogenic variants in reconstituted high-density lipoprotein with artificial membranes.

High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

Analysis of the Cadmium Removal Mechanism of Human Gut Bacteria Enterococcus faecalis Strain ATCC19433 from a Genomic Perspective.

Cadmium (Cd) is one of the most well-known toxic metals capable of entering the human body via the food chain, leading to serious health problems. Human gut microbes play a pivotal role in controlling Cd bioavailability and toxicity within the human gastrointestinal tract, primarily due to their capacity for Cd adsorption and metabolism. In this work, a Cd-resistant bacterial strain, Enterococcus faecalis strain ATCC19433 was isolated from human gut microbiota. Cd binding assays and comprehensive characterization analyses were performed, revealing the ability of strain ATCC19433 to remove Cd from the solution. Cd adsorption primarily occurred on the bacterial cell walls, which was ascribed to the exciting of functional groups on the bacterial surfaces, containing alkyl, amide II, and phosphate groups; meanwhile, Cd could enter cells, probably through transport channels or via diffusion. These results indicated that Cd removal by the strain was predominantly dependent on biosorption and bioaccumulation. Whole-genome sequencing analyses further suggested the probable mechanisms of biosorption and bioaccumulation, including Cd transport by transporter proteins, active efflux of Cd by cadmium efflux pumps, and mitigating oxidative stress-induced cell damage by DNA repair proteases. This study evaluated the Cd removal capability and mechanism of Enterococcus faecalis strain ATCC19433 while annotating the genetic functions related to Cd removal, which may facilitate the development of potential human gut strains for the removal of Cd.

Integrated transcriptomics and metabolomics analyses reveal the aerobic biodegradation and molecular mechanisms of 2,3′,4,4′,5-pentachlorodiphenyl (PCB 118) in Methylorubrum sp. ZY-1

2,3′,4,4′,5-pentachlorodiphenyl (PCB 118), a highly representative PCB congener, has been frequently detected in various environments, garnering much attention across the scientific community. The degradation of highly chlorinated PCBs by aerobic microorganisms is challenging due to their hydrophobicity and persistence. Herein, the biodegradation and adaptation mechanisms of Methylorubrum sp. ZY-1 to PCB 118 were comprehensively investigated using an integrative approach that combined degradation performance, product identification, metabolomic and transcriptomic analyses. The results indicated that the highest degradation efficiency of 0.5 mg L−1 PCB 118 reached 75.66% after seven days of inoculation when the bacteria dosage was 1.0 g L−1 at pH 7.0. A total of eleven products were identified during the degradation process, including low chlorinated PCBs, hydroxylated PCBs, and ring-opening products, suggesting that strain ZY-1 degraded PCB 118 through dechlorination, hydroxylation, and ring-opening pathways. Metabolomic analysis demonstrated that the energy supply and redox metabolism of strain ZY-1 was disturbed with exposure to PCB 118. To counteract this environmental stress, strain ZY-1 adjusted both the fatty acid synthesis and purine metabolism. The analysis of transcriptomics disclosed that multiple intracellular and extracellular oxidoreductases (e.g., monooxygenase, alpha/beta hydrolase and cytochrome P450) participated in the degradation of PCB 118. Besides, active efflux of PCB 118 and its degradation intermediates mediated by multiple transporters (e.g., MFS transporter and ABC transporter ATP-binding protein) might enhance bacterial resistance against these substances. These discoveries provided the inaugural insights into the biotransformation of strain ZY-1 to PCB 118 stress, illustrating its potential in the remediation of contaminated environments.

Abstract LB260: Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly polymerase inhibitor

Abstract Aims: The overexpression of ABC transporters on cancer cell membranes is one of the most common causes of multidrug resistance (MDR). This study investigates the impact of ABCC1 and ABCG2 on the resistance to talazoparib (BMN-673), a potent poly (ADP-ribose) polymerase (PARP) inhibitor, in ovarian cancer treatment. Methods: The cell viability test was used to indicate the effect of talazoparib in different cell lines. Computational molecular docking analysis was conducted to simulate the interaction between talazoparib and ABCC1 or ABCG2. The mechanism of talazoparib resistance was investigated by constructing talazoparib-resistant subline A2780/T4 from A2780 through drug selection with gradually increasing talazoparib concentration. Results: Talazoparib cytotoxicity decreased in drug-selected or gene-transfected cell lines overexpressing ABCC1 or ABCG2 but can be restored by ABCC1 or ABCG2 inhibitors. Talazoparib competitively inhibited substrate drug efflux activity of ABCC1 or ABCG2. Upregulated ABCC1 and ABCG2 protein expression on the plasma membrane of A2780/T4 cells enhances resistance to other substrate drugs, which could be overcome by the knockout of either gene. In vivo experiments confirmed the retention of drug-resistant characteristics in tumor xenograft mouse models. Conclusions: The therapeutic efficacy of talazoparib in cancer may be compromised by its susceptibility to MDR, which is attributed to its interactions with the ABCC1 or ABCG2 transporters. The overexpression of these transporters can potentially diminish the therapeutic impact of talazoparib in cancer treatment. Citation Format: Shuo Fang, Xiaona Fang. Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly polymerase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB260.

Small Molecule IITR08367 Potentiates Antibacterial Efficacy of Fosfomycin against Acinetobacter baumannii by Efflux Pump Inhibition.

Fosfomycin is a broad-spectrum single-dose therapy approved for treating lower urinary tract infections. Acinetobacter baumannii, one of the five major UTI-causing pathogens, is intrinsically resistant to fosfomycin. Reduced uptake and active efflux are major reasons for this intrinsic resistance. AbaF, a major facilitator superfamily class of transporter in A. baumannii, is responsible for fosfomycin efflux and biofilm formation. This study describes the identification and validation of a novel small-molecule efflux pump inhibitor that potentiates fosfomycin efficacy against A. baumannii. An AbaF inhibitor screening was performed against Escherichia coli KAM32/pUC18_abaF, using the noninhibitory concentration of 24 putative efflux pump inhibitors. The inhibitory activity of IITR08367 [bis(4-methylbenzyl) disufide] against fosfomycin/H+ antiport was validated using ethidium bromide efflux, quinacrine-based proton-sensitive fluorescence, and membrane depolarization assays. IITR08367 inhibits fosfomycin/H+ antiport activity by perturbing the transmembrane proton gradient. IITR08367 is a nontoxic molecule that potentiates fosfomycin activity against clinical strains of A. baumannii and prevents biofilm formation by inhibiting efflux pump (AbaF). The IITR08367-fosfomycin combination reduced bacterial burden by > 3 log10 in kidney and bladder tissue in the murine UTI model. Overall, fosfomycin, in combination with IITR08367, holds the potential to treat urinary tract infections caused by A. baumannii.

Prevalence of antimicrobial resistance, biofilm formation, efflux pump activity, and virulence capabilities in multi-drug-resistant Klebsiella pneumoniae isolated from freshwater fish farms.

The present study aimed to determine the antibiotic resistance, underlying mechanisms, antibiotic residues, and virulence genes involved in 32 multi-drug-resistant Klebsiella pneumoniae isolates from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that all isolates were multi-drug-resistant, harbored tetA (96.8%), tetC (59.3%), tetD (71.9%), nfsA (59.3%), nfsB (53.1%), sul2 (68.7%), qnrC (43.7%), qnrD (50%), blaSHV (75%), blaTEM (68.7%), and blaCTX-M (93.7%) genes. Multiple antibiotic resistance index was calculated as 0.54. Sixteen isolates were confirmed to be hyper-virulent and harbored magA and rmpA genes. In total, 46.9, 31.2, and 21.9% of the isolates were categorized as strong, moderate, or weak biofilm formers, respectively. All isolates possessed an active efflux pump and harbored acrA, acrB, acrAB, and tolC genes in 94% of the isolates, followed by mdtK (56.2%). Porins such as ompK35 and ompK36 were detected in 59.3 and 62.5% of the isolates, respectively. Virulence genes fimH-1, mrkD, and entB were present in 84.3, 81.2, 87.5% of the isolates, respectively. These findings imply a potential threat that multi-drug-resistant bacterial pathogens could transmit to surrounding environments and humans through contaminated water and the aquaculture food chain.

266 Combination Drug Therapy in H3 K27-Altered Diffuse Midline Glioma Enhances Tumor Retention and Treatment Efficacy

INTRODUCTION: Diffuse midline gliomas (DMG) with H3 K27 alterations are uniformly lethal tumors for which no therapy has been found to be effective. One reason for our therapy failure is the blood-brain barrier (BBB), which severely restricts uptake and accumulation of systemically administered drugs in the brain. Convection-enhanced delivery (CED) has emerged as a technique that circumvents the BBB by directly infusing therapeutics into the tumor parenchyma through generation of a mechanical pressure gradient, allowing for homogenous drug distribution at high local concentrations with minimal systemic toxicity. However, efflux transport proteins expressed by the BBB and the tumor, including P-gp and BCRP, rapidly clear the majority of drugs given by CED, severely limiting its therapeutic potential. METHODS: A panel of FDA-approved drugs (ponatinib, topotecan, tretinoin) were evaluated alone and in combination with the known efflux transporter inhibitor everolimus. RESULTS: A high degree of formal synergy was observed in multiple DMG cell lines in vitro, which was attributed to everolimus’ ability to inhibit mTOR signaling and P-gp/BCRP. In vivo, drugs that were expected to penetrate the BBB and be less likely to undergo active efflux persisted at the site of infusion the longest following CED. Concurrent dosing with everolimus further enhanced this effect, which was particularly apparent with the dual P-gp/BCRP substrate ponatinib. CONCLUSIONS: The proclivity of many drugs to be rapidly cleared from the brain by efflux transporters generates a vulnerability that can be exploited for the development of novel therapeutic regimens in DMG, such as with concomitant dosing of P-gp/BCRP-inhibiting drugs when using CED.

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

Temocillin is a narrow spectrum β-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Zosuquidar: An Effective Molecule for Intracellular Ca2+ Measurement in P-gp Positive Cells.

Intracellular calcium, as a second messenger, is involved in multilevel cellular regulatory pathways and plays a role (among other processes) in switching between survival and initiation of cell death in neoplastic cells. The development of multidrug resistance (MDR) in neoplastic cells is associated with the ability of cells to escape programmed cell death, in which dysregulation of intracellular calcium may play an important role. Therefore, reliable monitoring of intracellular calcium levels is necessary. However, such a role might be limited by a real obstacle since several fluorescent intracellular calcium indicators are substrates of membrane ABC drug transporters. For example, Fluo-3/AM is a substrate of P-glycoprotein (ABCB1 member of the ABC family), whose overexpression is the most frequent cause of MDR. The overexpression of ABCB1 prevents MDR cell variants from retaining this tracer in the intracellular space where it is supposed to detect calcium. The solution is to use a proper inhibitor of P-gp efflux activity to ensure the retention of the tracer inside the cells. The present study showed that Zosuquidar and Tariquidar (P-gp inhibitors) are suitable for monitoring intracellular calcium, either by flow cytometry or confocal microscopy, in cells overexpressing P-gp.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

The SMAC mimetic GDC-0152 is a direct ABCB1-ATPase activity modulator and BIRC5 expression suppressor in cancer cells

Upregulation of the multidrug efflux pump ABCB1/MDR1 (P-gp) and the anti-apoptotic protein BIRC5/Survivin promotes multidrug resistance in various human cancers. GDC-0152 is a DIABLO/SMAC mimetic currently being tested in patients with solid tumors. However, it is still unclear whether GDC-0152 is therapeutically applicable for patients with ABCB1-overexpressing multidrug-resistant tumors, and the molecular mechanism of action of GDC-0152 in cancer cells is still incompletely understood. In this study, we found that the potency of GDC-0152 is unaffected by the expression of ABCB1 in cancer cells. Interestingly, through in silico and in vitro analysis, we discovered that GDC-0152 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multidrug efflux activity at sub-cytotoxic concentrations (i.e., 0.25×IC50 or less). Further investigation revealed that GDC-0152 also decreases BIRC5 expression, induces mitophagy, and lowers intracellular ATP levels in cancer cells at low cytotoxic concentrations (i.e., 0.5×IC50). Co-treatment with GDC-0152 restored the sensitivity to the known ABCB1 substrates, including paclitaxel, vincristine, and YM155 in ABCB1-expressing multidrug-resistant cancer cells, and it also restored the sensitivity to tamoxifen in BIRC5-overexpressing tamoxifen-resistant breast cancer cells in vitro. Moreover, co-treatment with GDC-0152 restored and potentiated the anticancer effects of paclitaxel in ABCB1 and BIRC5 co-expressing xenograft tumors in vivo. In conclusion, GDC-0152 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide essential information to physicians for designing a more patient-specific GDC-0152 clinical trial program in the future.

Intoxication of antibiotic persisters by host RNS inactivates their efflux machinery during infection.

The host environment is of critical importance for antibiotic efficacy. By impacting bacterial machineries, stresses encountered by pathogens during infection promote the formation of phenotypic variants that are transiently insensitive to the action of antibiotics. It is assumed that these recalcitrant bacteria-termed persisters-contribute to antibiotic treatment failure and relapsing infections. Recently, we demonstrated that host reactive nitrogen species (RNS) transiently protect persisters against the action of β-lactam antibiotics by delaying their regrowth within host cells. Here, we discovered that RNS intoxication of persisters also collaterally sensitizing them to fluoroquinolones during infection, explaining the higher efficiency of fluoroquinolones against intramacrophage Salmonella. By reducing bacterial respiration and the proton-motive force, RNS inactivate the AcrAB efflux machinery of persisters, facilitating the accumulation of fluoroquinolones intracellularly. Our work shows that target inactivity is not the sole reason for Salmonella persisters to withstand antibiotics during infection, with active efflux being a major contributor to survival. Thus, understanding how the host environment impacts persister physiology is critical to optimize antibiotics efficacy during infection.

Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research

BackgroundHuman induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged.MethodsTo solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids.ResultsWe established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org.ConclusionsWe have successfully improved the function and convenience of ELCs by utilizing organoid technology.

Distribution of quetiapine between serum and whole blood in therapeutic drug-monitoring specimens.

Quetiapine use is on the rise, leading to a corresponding increase in acute intoxications, some of which have fatal outcomes. When assessing whole-blood quetiapine concentrations during forensic autopsies, interpretations are primarily based on toxicity data from studies of serum concentrations. To our knowledge, there are only two previous studies that have attempted to establish the ratio between whole blood and serum quetiapine concentrations with limited populations and high variability of results. Paired specimens of whole blood and serum from 16 quetiapine users recruited from the Psychiatric Clinic, St. Olav University Hospital were analyzed using LC-MS-MS. Quetiapine concentrations in both matrices were determined and compared. The mean blood:serum ratio of quetiapine was 0.74 (standard deviation (SD) = 0.05, 95% confidence interval (CI) 0.71-0.76, P < 0.001), range 0.66-0.85. Simple linear regression showed strong linear correlation between quetiapine concentrations in the two matrices (B = 0.774, P > 0.001, r = 0.999). Our results imply that quetiapine occurs at lower concentrations within erythrocytes than in plasma. This is most likely due to a high degree of plasma protein binding. Other factors which may influence the distribution of quetiapine between these compartments are solubility, metabolism and passive or active efflux mechanisms. We did not observe any covariation between blood:serum ratios and serum concentrations. Quetiapine was consistently present at lower concentrations in whole blood than in serum. If so inclined to, a conversion factor of ∼0.7 may be considered for extrapolation of concentrations from serum to whole blood, at least in cases with therapeutic quetiapine concentration levels.

P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

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.

Diverse temporal and spatial mechanisms work, partially through Stanniocalcin-1, V-ATPase and senescence, to activate the extracellular ATP-mediated drug resistance in human cancer cells.

Resistance to drug therapies is associated with a large majority of cancer-related deaths. ATP-binding cassette (ABC) transporter-mediated drug efflux, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), glutathione (GSH), senescence, and vacuole-type ATPase (V-ATPase) all contribute to the resistance. We recently showed that extracellular ATP (eATP) induces and regulates EMT, CSC formation, and ABC transporters in human cancer cells and tumors. eATP also consistently upregulates Stanniocalcin-1 (STC1), a gene that significantly contributes to EMT, CSC formation, and tumor growth. We also found that eATP enhances drug resistance in cancer cells through eATP internalization mediated by macropinocytosis, leading to an elevation of intracellular ATP (iATP) levels, induction of EMT, and CSC formation. However, these factors have never been systematically investigated in the context of eATP-induced drug resistance. In this study, we hypothesized that eATP increases drug resistance via inducing ABC efflux, EMT, CSCs, STC1, and their accompanied processes such as GSH reducing activity, senescence, and V-ATPase. RNA sequencing, metabolomics, gene knockdown and knockout, and functional assays were performed to investigate these pathways and processes. Our study results showed that, in multiple human cancer lines, eATP induced genes involved in drug resistance, elevated ABC transporters' efflux activity of anticancer drugs; generated transcriptomic and metabolic profiles representing a drug resistant state; upregulated activities of GSH, senescence, and V-ATPase to promote drug resistance. Collectively, these newly found players shed light on the mechanisms of eATP-induced as well as STC1- and V-ATPase-mediated drug resistance and offer potential novel targets for combating drug resistance in cancers.