Search Results (56)

Helicobacter pylori is a Gram-negative bacterium that infects almost half of the global population and is linked to gastric conditions like peptic ulcers and gastric cancer, as well as other diseases such as neurological disorders, cardiovascular problems, and iron deficiency anemia. Its survival in the acidic stomach environment is due to virulence factors like urease, flagella, and adhesion proteins (BabA, SabA). Current treatments involve a combination of antibiotics (clarithromycin, metronidazole, amoxicillin, tetracycline) and proton pump inhibitors, but increasing antibiotic resistance, especially to clarithromycin and metronidazole, poses a major challenge. Resistance mechanisms include mutations in drug targets, efflux pump overexpression, and enzymatic degradation of antibiotics. This has prompted exploration of alternative therapies targeting bacterial processes like urease activity, biofilm formation, and metabolic pathways (energy production, amino acid synthesis, iron acquisition). Natural compounds, such as chitosan and plant extracts, show promise in combating H. pylori growth and virulence. Vaccine development is also ongoing, with DNA vaccines showing potential for broad immune responses. However, no vaccine is yet close to widespread clinical use. Full article

Background: The efflux system is one of the resistance mechanisms that bacteria use to reduce the effectiveness of antibiotics, leading to the development of multidrug resistance. To evaluate other treatment choices, esomeprazole (ESO), omeprazole (OME), pantoprazole (PAN), vitamin D (VD), and vitamin K (VK) were tested for potential efflux pump (EP)-inhibiting activity. Methods: The minimum inhibitory concentrations (MICs) of the tested drugs were determined against K. pneumoniae ATCC 51503 and P. aeruginosa PAO1. Quantitative estimation of the EP-inhibiting activity of the tested medications was phenotypically investigated with a semi-automated fluorometric system and genotypically confirmed by real-time polymerase chain reaction (RT-PCR). Data were confirmed through docking study. Results: K. pneumoniae ATCC 51503 and P. aeruginosa PAO1 were positive efflux standard strains. VD and VK revealed an MIC_VD of 625–1250 µg/mL and MIC_VK of 2500–5000 µg/mL, lower than what was detected for PPIs (MIC_PPIs = 16,000–32,000 µg/mL). Vitamins showed powerful anti-efflux activity with remarkable ethidium bromide accumulation in K. pneumoniae ATCC 51503 and P. aeruginosa PAO1. Also, VD and VK significantly lowered the MIC of ciprofloxacin by 64-fold. On the molecular level, OME showed a notable decrease in the relative expression of the efflux-encoding genes acrB and mexA by 91.5% and 99.7% in ATCC 51503 and PAO1, respectively. Conclusion: This study highlights the anti-efflux activity of ESO, OME, PAN, VD, and VK against the tested Gram-negative strains. Hence, these PPIs and vitamins could be valuable adjuvant treatments to enhance the effectiveness of curing infections caused by MDR strains. Full article

Helicobacter pylori is a Gram-negative bacterium from the Epsilonproteobacteria class, associated with various gastric diseases, including gastric cancer. It infects both adults and children, with a high prevalence in developing countries due to poor health conditions. The International Agency for Research on Cancer has classified H. pylori as a class I carcinogen, linked not only to gastric cancer but also to neurological disorders. Current treatment involves proton pump inhibitors combined with antibiotics for 10 to 14 days, but patient non-compliance can lead to increased antibiotic resistance. This review examines studies from the past decade that explore flavonoids as potential future treatments for H. pylori. Flavonoids like kaempferol, rutin, quercetin, myricetin, catechin, epicatechin, eupatilin, chrysin, apigenin, and hesperetin have been shown to regulate the expression of key H. pylori genes, alter cell membrane permeability, and affect proton efflux. These biomolecules, found in various plants, have demonstrated the potential to inhibit H. pylori, even in resistant strains. Gene expression and molecular docking studies reveal how these flavonoids interact with the membrane, bacterial genes, and proteins, affecting host cell transcription, translation, and bacterial adherence. While promising, clinical trials are needed to better understand their mechanisms and efficacy. Full article

Collateral sensitivity is an evolutionary trade-off for bacteria where acquiring resistance to one antibiotic results in an increased sensitivity to another antibiotic. This study was designed to evaluate the collateral sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics induced by the evolution of resistance to apramycin. Collateral sensitivity to ampicillin, cephazolin, ceftriaxone, cefotaxime, cefepime and cefquinome occurred after MRSA were exposed to apramycin and induced to acquire resistance. This sensitivity was associated with reduced β-lactamase activity and decreased expression of the mecA gene. We also found a decrease in the proton motive force and decreased efflux activity. These results provide new insights into collateral sensitivity-based strategies for the treatment of MRSA. Full article

Cation-pumping membrane pyrophosphatases (mPPases; EC 7.1.3.1) vary in their transport specificity from obligatory H⁺ transporters found in all kingdoms of life, to Na⁺/H⁺-co-transporters found in many prokaryotes. The available data suggest a unique “direct-coupling” mechanism of H⁺ transport, in which the transported proton is generated from nucleophilic water molecule. Na⁺ transport is best rationalized by assuming that the water-borne proton propels a prebound Na⁺ ion through the ion conductance channel (“billiard” mechanism). However, the “billiard” mechanism, in its simple form, is not applicable to the mPPases that simultaneously transport Na⁺ and H⁺ without evident competition between the cations (Na⁺,H⁺-PPases). In this study, we used a pyranine-based fluorescent assay to explore the relationship between the cation transport reactions catalyzed by recombinant Bacteroides vulgatus Na⁺,H⁺-PPase in membrane vesicles. Under appropriately chosen conditions, including the addition of an H⁺ ionophore to convert Na⁺ influx into equivalent H⁺ efflux, the pyranine signal measures either H⁺ or Na⁺ translocation. Using a stopped-flow version of this assay, we demonstrate that H⁺ and Na⁺ are transported by Na⁺,H⁺-PPase in a ratio of approximately 1:8, which is independent of Na⁺ concentration. These findings were rationalized using an “extended billiard” model, whose most likely variant predicts the kinetic limitation of Na⁺ delivery to the pump-loading site. Full article

Understanding carboxylate transport through lipid membranes under physiological conditions is critical in biomedicine and biotechnology, as it allows for the emulation of biological membrane functions and can enhance the absorption of hydrophobic carboxylate-based drugs. However, the structural diversity of carboxylates has made it challenging to study their transport, and the limited available examples do not provide a comprehensive understanding of the role of the organic moiety in this process. Here, we present an in-depth analysis of the diffusion and transport of various aliphatic and aromatic carboxylates into liposomes. We assessed the influence of their size, number of carboxylate groups, and presence of hydroxyl groups. Our findings from fluorescence assays, using lucigenin and HPTS as probes, revealed that most carboxylates can spontaneously diffuse into liposomes in their protonated state, facilitated by the efflux of HNO₃ when using NaNO₃ solutions at pH 7. The Cl-ISE assay showed chloride/carboxylate exchange by a synthetic anion transporter. Clear trends were observed when the organic moiety was systematically varied, with a particular enhancement of anion transport by the presence of hydroxyl groups in the aromatic carboxylates. Our findings provide insights into the processes by which carboxylates can enter liposomes, which can contribute to understanding the transport of other biologically relevant organic anions. Full article

In the small intestine, nutrients from ingested food are absorbed and broken down by enterocytes, which constitute over 95% of the intestinal epithelium. Enterocytes demonstrate diet- and segment-dependent metabolic flexibility, enabling them to take up large amounts of glutamine and glucose to meet their energy needs and transfer these nutrients into the bloodstream. During glycolysis, ATP, lactate, and H⁺ ions are produced within the enterocytes. Based on extensive but incomplete glutamine oxidation large amounts of alanine or lactate are produced. Lactate, in turn, promotes hypoxia-inducible factor-1α (Hif-1α) activation and Hif-1α-dependent transcription of various proton channels and exchangers, which extrude cytoplasmic H⁺-ions into the intestinal lumen. In parallel, the vitamin C-dependent and duodenal cytochrome b-mediated conversion of ferric iron into ferrous iron progresses. Finally, the generated electrochemical gradient is utilized by the divalent metal transporter 1 for H⁺-coupled uptake of non-heme Fe²⁺-ions. Iron efflux from enterocytes, subsequent binding to the plasma protein transferrin, and systemic distribution supply a wide range of cells with iron, including erythroid precursors essential for erythropoiesis. In this review, we discuss the impact of vitamin C on the redox capacity of human erythrocytes and connect enterocyte function with iron metabolism, highlighting its effects on erythropoiesis. Full article

The opening of the Torpedo CLC-0 chloride (Cl⁻) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H⁺_i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H⁺_i inhibition similar to that of the channel inactivation, a resistance to the H⁺_i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H⁺_i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H⁺_i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H⁺_i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states. Full article

Advanced urothelial bladder cancer (UBC) patients are tagged by a dismal prognosis and high mortality rates, mostly due to their poor response to standard-of-care platinum-based therapy. Mediators of chemoresistance are not fully elucidated. This work aimed to study the metabolic profile of advanced UBC, in the context of cisplatin resistance. Three isogenic pairs of parental cell lines (T24, HT1376 and KU1919) and the matching cisplatin-resistant (R) sublines were used. A set of functional assays was used to perform a metabolic screening on the cells. In comparison to the parental sublines, a tendency was observed towards an exacerbated glycolytic metabolism in the cisplatin-resistant T24 and HT1376 cells; this glycolytic phenotype was particularly evident for the HT1376/HT1376R pair, for which the cisplatin resistance ratio was higher. HT1376R cells showed decreased basal respiration and oxygen consumption associated with ATP production; in accordance, the extracellular acidification rate was also higher in the resistant subline. Glycolytic rate assay confirmed that these cells presented higher basal glycolysis, with an increase in proton efflux. While the results of real-time metabolomics seem to substantiate the manifestation of the Warburg phenotype in HT1376R cells, a shift towards distinct metabolic pathways involving lactate uptake, lipid biosynthesis and glutamate metabolism occurred with time. On the other hand, KU1919R cells seem to engage in a metabolic rewiring, recovering their preference for oxidative phosphorylation. In conclusion, cisplatin-resistant UBC cells seem to display deep metabolic alterations surpassing the Warburg effect, which likely depend on the molecular signature of each cell line. Full article

Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes’ cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations. In this study, we present an inverse pH-gradient transport assay. Herein, an outward-facing proton gradient enables the characterization of proton antiporters via facile import-based transport assays. We describe two approaches for establishing sustained outward-facing proton gradients across the oocyte membrane, namely by applying alkaline external conditions or through surprisingly stable carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)-mediated acidification of the cytosol. Previously, genetic evidence has shown that DTX18 from Arabidopsis thaliana is essential for the deposition of the hydroxycinnamic acid amide p-coumaroylagmatine (coumaroylagmatine) defence compound on the leaf surface. However, direct evidence for its ability to transport coumarol-agmatine has not been provided. Here, using Xenopus oocytes as expression hosts, we demonstrate DTX18’s ability to transport coumaroyl-agmatine via both injection-based and inverse pH-gradient transport assays. Notably, by showing that DTX18 is capable of accumulating its substrate against its concentration gradient, we showcase the compatibility of the latter with mechanistic investigations. Full article

Efflux pumps play important roles in bacterial detoxification and some of them are stress-response elements that are up-regulated when the host is treated with antibiotics. However, efflux pumps that are down-regulated by stimulations are rarely discovered. Herein, we analyzed multiple transcriptome data and discovered a special (Major Facilitator Superfamily) MFS efflux pump, KpsrMFS, from Klebsiella pneumoniae, which was down-regulated when treated with antibiotics or extra carbon sources. Interestingly, overexpression of kpsrmfs resulted in halted cell growth in normal conditions, while the viable cells were rarely affected. The function of KpsrMFS was further analyzed and this efflux pump was determined to be a proton-driven transporter that can reduce the intracellular tetracycline concentration. In normal conditions, the expression of kpsrmfs was at a low level, while artificial overexpression of it led to increased endogenous reactive oxygen species (ROS) production. Moreover, by comparing the functions of adjacent genes of kpsrmfs, we further discovered another four genes that can confer similar phenotypes, indicating a special regulon that regulates cell growth. Our work provides new insights into the roles of efflux pumps and suggests a possible regulon that may regulate cell growth and endogenous ROS levels. Full article

High concentrations of sodium (Na⁺), chloride (Cl⁻), calcium (Ca²⁺), and sulphate (SO₄²⁻) are frequently found in saline soils. Crop plants cannot successfully develop and produce because salt stress impairs the uptake of Ca²⁺, potassium (K⁺), and water into plant cells. Different intracellular and extracellular ionic concentrations change with salinity, including those of Ca²⁺, K⁺, and protons. These cations serve as stress signaling molecules in addition to being essential for ionic homeostasis and nutrition. Maintaining an appropriate K⁺:Na⁺ ratio is one crucial plant mechanism for salt tolerance, which is a complicated trait. Another important mechanism is the ability for fast extrusion of Na⁺ from the cytosol. Ca²⁺ is established as a ubiquitous secondary messenger, which transmits various stress signals into metabolic alterations that cause adaptive responses. When plants are under stress, the cytosolic-free Ca²⁺ concentration can rise to 10 times or more from its resting level of 50–100 nanomolar. Reactive oxygen species (ROS) are linked to the Ca²⁺ alterations and are produced by stress. Depending on the type, frequency, and intensity of the stress, the cytosolic Ca²⁺ signals oscillate, are transient, or persist for a longer period and exhibit specific “signatures”. Both the influx and efflux of Ca²⁺ affect the length and amplitude of the signal. According to several reports, under stress Ca²⁺ alterations can occur not only in the cytoplasm of the cell but also in the cell walls, nucleus, and other cell organelles and the Ca²⁺ waves propagate through the whole plant. Here, we will focus on how wheat and other important crops absorb Na⁺, K⁺, and Cl⁻ when plants are under salt stress, as well as how Ca²⁺, K⁺, and pH cause intracellular signaling and homeostasis. Similar mechanisms in the model plant Arabidopsis will also be considered. Knowledge of these processes is important for understanding how plants react to salinity stress and for the development of tolerant crops. Full article

Monovalent cation proton antiporters (CPAs) play crucial roles in ion and pH homeostasis, which is essential for plant development and environmental adaptation, including salt tolerance. Here, 68 CPA genes were identified in soybean, phylogenetically dividing into 11 Na⁺/H⁺ exchangers (NHXs), 12 K⁺ efflux antiporters (KEAs), and 45 cation/H⁺ exchangers (CHXs). The GmCPA genes are unevenly distributed across the 20 chromosomes and might expand largely due to segmental duplication in soybean. The GmCPA family underwent purifying selection rather than neutral or positive selections. The cis-element analysis and the publicly available transcriptome data indicated that GmCPAs are involved in development and various environmental adaptations, especially for salt tolerance. Based on the RNA-seq data, twelve of the chosen GmCPA genes were confirmed for their differentially expression under salt or osmotic stresses using qRT-PCR. Among them, GmCHX20a was selected due to its high induction under salt stress for the exploration of its biological function on salt responses by ectopic expressing in Arabidopsis. The results suggest that the overexpression of GmCHX20a increases the sensitivity to salt stress by altering the redox system. Overall, this study provides comprehensive insights into the CPA family in soybean and has the potential to supply new candidate genes to develop salt-tolerant soybean varieties. Full article

While it is true that pharmacotherapy has achieved desired health outcomes, significant unmet medical needs persist in the field of central nervous system (CNS) drugs, particularly for neurodegenerative diseases such as Alzheimer’s disease, as well as ocular diseases such as diabetic retinopathy and age-related macular degeneration. Drugs cannot enter the brain from the bloodstream due to the presence of the blood–brain barrier (BBB). Similarly, they cannot enter the eyes from the bloodstream due to the blood–retina barrier (BRB), which is composed of the endothelium or the epithelium. Thus, innovative drug delivery systems that can overcome these barriers based on efflux transporters, hydrophobic lipid bilayer membranes, and tight junctions should be developed using patient-friendly techniques distinct from craniotomy procedures or intravitreal injections. Brain-penetrating CNS drugs and antihistamine drugs commonly share N-containing groups. These findings suggest that certain types of cation transporters are involved in their transportation across the cell membrane. Indeed, the proton-coupled organic cation (H⁺/OC) antiporter, whose specific characteristics remain unidentified, is responsible for transporting compounds with N-containing groups, such as clonidine and pyrilamine, at the BBB, and likely at the BRB as well. Therefore, well-designed low-molecular-weight drugs containing N-containing groups as transporter recognition units can enter the brain or the eyes through carrier-mediated transport. In this perspective review, I introduce the implementation and potential of H⁺/OC antiporter-mediated transport across the endothelium at the BBB or the BRB using drugs consciously designed with N-containing groups as their substrates. Full article

Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against E. coli, K. pneumoniae, E. cloacae, and A. baumannii. Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine–antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms. Full article