Drug Extrusion Research Articles

IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells.

Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer. The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction. Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription. We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.

MiRNA-449 family is epigenetically repressed and sensitizes to doxorubicin through ACSL4 downregulation in triple-negative breast cancer

Despite progress in breast cancer treatment, a significant portion of patients still relapse because of drug resistance. The involvement of microRNAs in cancer progression and chemotherapy response is well established. Therefore, this study aimed to elucidate the dysregulation of the microRNA-449 family (specifically, microRNA-449a, microRNA-449b-5p, and microRNA-449c-5p) and its impact on resistance to doxorubicin, a commonly used chemotherapeutic drug for the treatment of triple-negative breast cancer. We found that the microRNA-449 family is downregulated in triple-negative breast cancer and demonstrated its potential as a diagnostic biomarker. Besides, our findings indicate that the downregulation of the microRNA-449 family is mediated by the microRNAs-449/SIRT1-HDAC1 negative feedback loop. Moreover, it was found that the microRNA-449 family dysregulates the fatty acid metabolism by targeting ACSL4, which is a potential prognostic biomarker that mediates doxorubicin response through regulation of the drug extrusion pump ABCG2. Altogether, our results suggest that the microRNA-449 family might be a potential therapeutic target for the treatment of triple-negative breast cancer since it is implicated in doxorubicin response through ACSL4/ABCG2 axis regulation. Ultimately, our results also highlight the value of microRNAs-449 and ACSL4 as diagnostic and prognostic biomarkers in triple-negative breast cancer.Proposed model of miRNAs-449 downregulation in TNBC and doxorubicin response. MiRNAs-449 are downregulated in TNBC through a negative feedback loop with SIRT1 and HDAC1. Moreover, ACSL4 increases ABCG2 expression, thus diminishing the intracellular doxorubicin concentration and promoting doxorubicin resistance. MiRNAs-449 overexpression downregulates the ACSL4/ABCG2 axis and sensitizes doxorubicin-resistant cells to doxorubicin. Created with BioRender. TNBC: triple-negative breast cancer; DOX: doxorubicin; SIRT1: Sirtuin 1; HDAC1: Histone deacetylase 1; ACSL4: Acyl-CoA Synthetase Long-Chain Family Member 4; ABCG2: ATP-binding cassette superfamily G member 2.

Modulators of Candida albicans Membrane Drug Transporters: A LucrativePortfolio for the Development of Effective Antifungals.

The escalating prevalence of membrane drug transporters and drug efflux pumps in pathogenic yeast like Candida albicans necessitates a comprehensive understanding of their roles in MDR. The overexpression of drug transporter families, ABC and MFS, implicated in MDR through drug efflux and poses a significant challenge in the diagnosis and treatment of fungal infection. Various mechanisms have been proposed for MDR; however, the upregulation of ABC and MFS superfamily transporters is most noticeable in MDR. The direct inhibition of these transporters seems an efficient strategy to overcome this problem. The goal of the article is to present an overview of the prospect of utilizing these modulators of C. albicans drug transports as effective antifungal molecules against MDR addressing a critical gap in the field. The review tries to address to prevent drug extrusion by modulating the expression of drug transporters of C. albicans. The review discussed the progress in identifying potent, selective, and non-toxic modulators of these transporters to develop some effective antifungals and overcome MDR. We reviewed major studies in this area and found that recent work has shifted toward the exploration of natural compounds as potential modulators to restore drug sensitivity in MDR fungal cells. The focus of this review is to survey and interpret current research information on modulators of C. albicans drug transporters from natural sources emphasizing those compounds that are potent antifungal agents.

Progress on mechanisms of antibiotic resistance in Clostridioides difficile.

Clostridioides difficile (CD) is one of the most common pathogens causing health-care-associated infectious diarrhea and is listed by the U.S. Centers for Disease Control and Prevention as an urgent antibiotic resistance (AR) threat. Many resistance genes can be transferred between different CD strains present in the clinical setting, community, and environment. The antimicrobial resistance (AMR) of CD continues to evolve with the emergence and acquisition of new drug resistance mechanisms. CD has developed diverse drug resistance mechanisms, such as drug alteration, modification of the target site, and extrusion of drugs via efflux pumps. Researches have provided comprehensive knowledge about resistance mechanisms of macrolides and quinolones in CD. However, the mechanisms of resistance for metronidazole, vancomycin, and other therapeutic antibiotics against Clostridioides difficile infection (CDI) are only beginning to be elucidated. Some previously unfound mechanisms, such as plasmid-mediated drug resistance in CD, may also play an important role. In this review, we summarize the research progress on drug resistance mechanisms of CD with antimicrobial drugs already used clinically, such as metronidazole, vancomycin, and fidaxomicin, thereby providing the references for the clinical treatment and prevention of CDI, as well as the development of new antibacterial drugs and detection kits for drug resistant bacteria.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.

Human brain microvascular endothelial cells release different types of P-glycoprotein-containing extracellular vesicles upon exposure to doxorubicin

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of microvascular endothelial cells (BMECs) that form the blood-brain barrier. In addition, Pgp is localized in intracellular organelles involved in Pgp traffic and cycling and, by the release of extracellular vesicles (EVs), in intercellular Pgp transfer to cells with low Pgp expression. We recently described that drug exposure of a human BMEC line (hCMEC/D3) induces the release of Pgp-EGFP-containing EVs; however, the nature of the Pgp-enriched vesicles was not characterized. The two main categories of EVs are exosomes and microvesicles, which differ in origin, size, and molecular cargo. In the present study, we performed similar experiments with hCMEC/D3 cells in the absence and presence of doxorubicin and isolated and characterized the EVs released by the cells during the experiments by differential ultracentrifugation with/without subsequent sucrose gradient fractionation of EV pellets, proteomic profiling, EV size analysis, and confocal fluorescence microscopy. Using cocultures of hCMEC/D3 wildtype cells and cells transduced with MDR1-EGFP or monocultures of hCMEC/D3-MDR1-EGFP cells, we found release of both Pgp-enriched exosomes and microvesicles but analysis of the exosomal marker protein Rab7 indicated that doxorubicin increased particularly the release of exosomes. Transfer experiments with isolated EVs demonstrated EV endocytosis by recipient cells. EV release from BMECs in response to anticancer drugs such as doxorubicin likely serves different functions, including non-genetic intercellular transfer of a resistance phenotype to neighboring BMECs and a mechanism of drug extrusion that contributes to brain protection against potentially toxic chemotherapeutic drugs.

Association of Antibacterial Susceptibility Profile with the Prevalence of Genes Encoding Efflux Proteins in the Bangladeshi Clinical Isolates of Staphylococcus aureus

Expelling antibiotic molecules out of the cell wall through multiple efflux pumps is one of the potential mechanisms of developing resistance against a wide number of antibiotics in Staphylococcus aureus. The aim of this study was to investigate the association between the antibiotic susceptibility profile and the prevalence of different efflux pump genes i.e., norA, norB, norC, mepA, sepA, mdeA, qacA/B, and smr in the clinical isolates of S. aureus. Sixty clinical isolates were collected from a tertiary level hospital in Bangladesh. The disc diffusion method using ten antibiotics of different classes was used to discern the susceptibility profile. polymerase chain reaction (PCR) was employed to observe the resistance patterns and to detect the presence of plasmid and chromosomal encoded genes. Among the clinical isolates, 60% (36 out of 60) of the samples were Methicillin-resistant Staphylococcus aureus (MRSA), whereas 55% (33 out of 60) of the bacterial samples were found to be multi-drug resistant. The bacteria showed higher resistance to vancomycin (73.33%), followed by ciprofloxacin (60%), cefixime (53.33%), azithromycin (43.33%), and amoxicillin (31.67%). The prevalence of the chromosomally-encoded efflux genes norA (91.67%), norB (90%), norC (93.33%), mepA (93.33%), sepA (98.33%), and mdeA (93.33%) were extremely high with a minor portion of them carrying the plasmid-encoded genes qacA/B (20%) and smr (8.33%). Several genetic combinations of efflux pump genes were revealed, among which norA + norB + norC + mepA + sepA + mdeA was the most widely distributed combination among MRSA and MSSA bacteria that conferred resistance against ciprofloxacin and probably vancomycin. Based on the present study, it is evident that the presence of multiple efflux genes potentiated the drug extrusion activity and may play a pivotal role in the development of multidrug resistance in S. aureus.

Phosphorothioated amino-AS1411 aptamer functionalized stealth nanoliposome accelerates bio-therapeutic threshold of apigenin in neoplastic rat liver: a mechanistic approach

Hepatocellular carcinoma (HCC) is a leading cause of death globally. Even though the progressive invention of some very potent therapeutics has been seen, the success is limited due to the chemotherapeutic resistance and recurrence in HCC. Advanced targeted treatment options like immunotherapy, molecular therapy or surface-engineered nanotherapeutics could offer the benefits here owing to drug resistance over tumor heterogenicity. We have developed tumor-sensing phosphorothioate and amino-modified aptamer (AS1411)-conjugated stealth nanoliposomes, encapsulating with apigenin for precise and significant biodistribution of apigenin into the target tumor to exploit maximum bio-therapeutic assistances. The stable aptamer functionalized PEGylated nanoliposomes (Apt-NLCs) had an average vesicle size of 100–150 nm, a smooth surface, and an intact lamellarity, as ensured by DLS, FESEM, AFM, and Cryo-TEM. This study has specified in vitro process of optimum drug (apigenin) extrusion into the cancer cells by nucleolin receptor-mediated cellular internalization when delivered through modified AS1411 functionalized PEGylated nanoliposomes and ensured irreversible DNA damage in HCC. Significant improvement in cancer cell apoptosis in animal models, due to reduced clearance and higher intratumor drug accumulation along with almost nominal toxic effect in liver, strongly supports the therapeutic potential of aptamer-conjugated PEGylated nanoliposomes compared to the nonconjugated formulations in HCC. The study has established a robust superiority of modified AS1411 functionalized PEGylated nanoliposomes as an alternative drug delivery approach with momentous reduction of HCC tumor incidences.Graphical

Antifungal chemicals promising function in disease prevention, method of action and mechanism.

The increasing use of antimicrobial drugs has been linked to the rise of drug-resistant fungus in recent years. Antimicrobial resistance is being studied from a variety of perspectives due to the important clinical implication of resistance. The processes underlying this resistance, enhanced methods for identifying resistance when it emerges, alternate treatment options for infections caused by resistant organisms, and so on are reviewed, along with strategies to prevent and regulate the formation and spread of resistance. This overview will focus on the action mechanism of antifungals and the resistance mechanisms against them. The link between antibacterial and antifungal resistance is also briefly discussed. Based on their mechanism action, antifungals are divided into three distinct categories: azoles, which target the ergosterol synthesis; 5-fluorocytosine, which targets macromolecular synthesis and polyenes, which interact physiochemically with fungal membrane sterols. Antifungal resistance can arise through a wide variety of ways. Overexpression of the target of the antifungal drug, changes to the drug target, changes to sterol biosynthesis, decreased intercellular concentration of the target enzyme, and other processes. A correlation exists between the mechanisms of resistance to antibacterial and antifungals, despite the fact that the comparison between the two is inevitably constrained by various parameters mentioned in the review. Drug extrusion via membrane pumps has been thoroughly documented in both prokaryotic and eukaryotic cells, and development of new antifungal compounds and strategies has also been well characterized.

Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori.

ABSTRACTBacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction.

Expression and Function of ABC Transporters in Human Alveolar Epithelial Cells

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolarTM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

Roles of SmeYZ, SbiAB, and SmeDEF Efflux Systems in Iron Homeostasis of Stenotrophomonas maltophilia.

ABSTRACTStenotrophomonas maltophilia, a nonfermenting Gram-negative rod, is frequently isolated from the environment and is emerging as a multidrug-resistant global opportunistic pathogen. S. maltophilia harbors eight RND-type efflux pumps that contribute to multidrug resistance and physiological functions. Among the eight efflux pumps, SmeYZ pump is constitutively highly expressed. In our previous study, we demonstrated that loss-of-function of the SmeYZ pump results in pleiotropic phenotypes, including abolished swimming motility, decreased secreted protease activity, and compromised tolerance to oxidative stress and antibiotics. In this study, we attempted to elucidate the underlying mechanisms responsible for ΔsmeYZ-mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that smeYZ mutant experienced an iron starvation response because the genes involved in the synthesis and uptake of stenobactin, the sole siderophore of S. maltophilia, were significantly upregulated. We further verified that smeYZ mutant had low intracellular iron levels via inductively coupled plasma mass spectrometry (ICP-MS). Also, KJΔYZ was more sensitive to 2,2′-dipyridyl (DIP), a ferrous iron chelator, in comparison with the wild type. The contribution of SmeYZ, SmeDEF, and SbiAB pumps to stenobactin secretion was suggested by qRT-PCR and further verified by Chrome Azurol S (CAS) activity, iron source utilization, and cell viability assays. We also demonstrated that loss-of-function of SmeYZ led to the compensatory upregulation of SbiAB and SmeDEF pumps for stenobactin secretion. The overexpression of the SbiAB pump resulted in a reduction in intracellular iron levels, which may be the key factor responsible for the ΔsmeYZ-mediated pleiotropic phenotypes, except for antibiotic extrusion.IMPORTANCE Efflux pumps display high efficiency of drug extrusion, which underlies their roles in multidrug resistance. In addition, efflux pumps have physiological functions, and their expression is tightly regulated by various environmental and physiological signals. Functional redundancy of efflux pumps is commonly observed, and mutual regulation occurs among these functionally redundant pumps in a bacterium. Stenotrophomonas maltophilia is an opportunistic pathogen that shows intrinsic multi-drug resistance. In this study, we demonstrated that SmeYZ, SbiAB, and SmeDEF efflux pumps of S. maltophilia display functional redundancy in siderophore secretion. Inactivation of smeYZ led to the upregulation of smeDEF and sbiAB. Unexpectedly, sbiAB overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in smeYZ mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.

Role of the Two-Component System CiaRH in the Regulation of Efflux Pump SatAB and Its Correlation with Fluoroquinolone Susceptibility.

ABSTRACTStreptococcus suis is an important pathogen in both pigs and humans. Although the diseases associated with S. suis can typically be treated with antibiotics, such use has resulted in a sustained increase in drug resistance. Bacteria can sense and respond to antibiotics via two-component systems (TCSs). In this study, the TCS CiaRH was identified as playing an important role in the susceptibility of S. suis to fluoroquinolones (FQs). We found that a ΔciaRH mutant possessed lower susceptibility to FQs than the wild-type strain, with no observed growth defects at the tested concentrations and lower levels of intracellular drugs and dye. Proteomic data revealed that the levels of SatA and SatB expression were upregulated in the ΔciaRH mutant compared with their levels in the wild-type strain. The satA and satB genes encode a narrow-spectrum FQ efflux pump. The phenomena associated with combined ciaRH-and-satAB deletion mutations almost returned the ΔciaRH ΔsatAB mutant to the phenotype of the wild-type strain compared to the phenotype of the ΔciaRH mutant, suggesting that the resistance of the ΔciaRH strain to FQs could be attributed to satAB overexpression. Moreover, SatAB expression was regulated by CiaR (a response regulator of CiaRH) and SatR (a regulator of the MarR family). The ciaRH genes were consistently downregulated in response to antibiotic stress. The results of electrophoretic mobility shift assays (EMSAs) and affinity assays revealed that both regulator proteins directly controlled the ABC transporter proteins SatAB. Together, the results show that cascade-mediated regulation of antibiotic export by CiaRH is crucial for the ability of S. suis to adapt to conditions of antibiotic pressure. Our study may provide a new target for future antibiotic research and development.IMPORTANCE Streptococcus suis is a zoonotic pathogen with high incidence and mortality rates in both swine and humans. Following antibiotic treatment, the organism has evolved many resistance mechanisms, among which efflux pump overexpression can promote drug extrusion from the cell. This study clarified the role of CiaRH in fluoroquinolone resistance. A mutant with the ciaRH genes deleted showed decreased susceptibility to the antibiotics tested, an invariant growth rate, and reduced intracellular efflux pump substrates. This research also demonstrated that overexpression of the efflux pump SatAB was the main cause of ΔciaRH resistance. In addition, CiaR could combine with the promoter region of satAB to further directly suppress target gene transcription. Simultaneously, satAB was also directly regulated by SatR. Our findings may provide novel insights for the development of drug targets and help to exploit corresponding inhibitors to combat bacterial multidrug resistance.

Efficient Synthesis of Acylated, Dialkyl α-Hydroxy-Benzylphosphonates and Their Anticancer Activity.

An efficient method applying acyl chlorides as reagents was developed for the acylation of the hindered hydroxy group of dialkyl α-hydroxy-benzylphosphonates. The procedure did not require any catalyst. A few acylations were also performed with the SC-enantiomer of dimethyl α-hydroxy-benzylphosphonate, and the optical purity was retained. A part of the acyloxyphosphonates was tested against eight tumor cell lines of different tissue origin at c = 50 μM concentration. The compounds elicited moderate cytostatic effect against breast, skin, prostate, colon, and lung carcinomas; a melanoma cell line; and against Kaposi’s sarcoma cell lines. Then, dose-dependent cytotoxicity was assayed, and benzoylation of the α-hydroxy group was identified as a moiety that increases anticancer cytotoxicity across all cell lines. Surprisingly, a few analogues were more toxic to multidrug resistant cancer cell lines, thus evading P-glycoprotein mediated drug extrusion.

Zap1 is required for Candida glabrata response to fluconazole

The increasing prevalence of fluconazole-resistant clinical isolates of Candida spp. strongly hinders the widespread use of the drug. To tackle this problem, great efforts have been made to fully understand the fungal response to fluconazole. In this work, we show that the role of Zap1 in Candida glabrata goes beyond regulating yeast adaptation to zinc deficiency. In line with our previous observation that deletion of ZAP1 makes yeast cells more sensitive to fluconazole, we found that the mutant CgΔzap1 accumulates higher levels of the drug, which correlates well with its lower levels of ergosterol. Surprisingly, Zap1 is a negative regulator of the drug efflux transporter gene CDR1 and of its regulator, PDR1. The apparent paradox of drug accumulation in cells where genes encoding transporters relevant for drug extrusion are being overexpressed led us to postulate that their activity could be impaired. In agreement, Zap1-depleted cells present, in addition to decreased ergosterol levels, an altered composition of membrane phospholipids, which together should impact membrane function and impair the detoxification of fluconazole. Overall, our study brings to light Zap1 as an important hub in Candida glabrata response to fluconazole.

Simulated Drug Efflux for the AbgT Family of Membrane Transporters.

Drug extrusion through molecular efflux pumps is an important mechanism for the survival of many pathogenic bacteria by removing drugs, providing multidrug resistance (MDR). Understanding molecular mechanisms for drug extrusion in multidrug efflux pumps is important for the development of new antiresistance drugs. The AbgT family of transporters involved in the folic acid biosynthesis pathway represents one such important efflux pump system. In addition to the transport of the folic acid precursor p-amino benzoic acid (PABA), members of this family are involved in the efflux of several sulfa drugs, conferring drug resistance to the bacteria. With the availability of structures for two members of this family (YdaH and MtrF), we investigate molecular pathways for transport of PABA and a sulfa drug (sulfamethazine) particularly for the YdaH transporter using steered molecular dynamics. Our analyses reveal the probable ligand migration pathways through the transporter, which also identifies key residues along the transport pathway. In addition, simulations using both PABA and sulfamethazine show how the protein is able to transport ligands of different shapes and sizes out of the pathogen. Our observations confirm previously reported functional residues for transport along the pathways by which YdaH transporters achieve antibiotic resistance to shuttle drugs out of the cells.

Mechanisms of antibiotic resistance of Clostridioides difficile.

Clostridioides difficile (CD) is one of the top five urgent antibiotic resistance threats in USA. There is a worldwide increase in MDR of CD, with emergence of novel strains which are often more virulent and MDR. Antibiotic resistance in CD is constantly evolving with acquisition of novel resistance mechanisms, which can be transferred between different species of bacteria and among different CD strains present in the clinical setting, community, and environment. Therefore, understanding the antibiotic resistance mechanisms of CD is important to guide optimal antibiotic stewardship policies and to identify novel therapeutic targets to combat CD as well as other bacteria. Epidemiology of CD is driven by the evolution of antibiotic resistance. Prevalence of different CD strains and their characteristic resistomes show distinct global geographical patterns. Understanding epidemiologically driven and strain-specific characteristics of antibiotic resistance is important for effective epidemiological surveillance of antibiotic resistance and to curb the inter-strain and -species spread of the CD resistome. CD has developed resistance to antibiotics with diverse mechanisms such as drug alteration, modification of the antibiotic target site and extrusion of drugs via efflux pumps. In this review, we summarized the most recent advancements in the understanding of mechanisms of antibiotic resistance in CD and analysed the antibiotic resistance factors present in genomes of a few representative well known, epidemic and MDR CD strains found predominantly in different regions of the world.

Breast cancer: Muscarinic receptors as new targets for tumor therapy.

The development of breast cancer is a complex process that involves the participation of different factors. Several authors have demonstrated the overexpression of muscarinic acetylcholine receptors (mAChRs) in different tumor tissues and their role in the modulation of tumor biology, positioning them as therapeutic targets in cancer. The conventional treatment for breast cancer involves surgery, radiotherapy, and/or chemotherapy. The latter presents disadvantages such as limited specificity, the appearance of resistance to treatment and other side effects. To prevent these side effects, several schedules of drug administration, like metronomic therapy, have been developed. Metronomic therapy is a type of chemotherapy in which one or more drugs are administered at low concentrations repetitively. Recently, two chemotherapeutic agents usually used to treat breast cancer have been considered able to activate mAChRs. The combination of low concentrations of these chemotherapeutic agents with muscarinic agonists could be a useful option to be applied in breast cancer treatment, since this combination not only reduces tumor cell survival without affecting normal cells, but also decreases pathological neo-angiogenesis, the expression of drug extrusion proteins and the cancer stem cell fraction. In this review, we focus on the previous evidences that have positioned mAChRs as relevant therapeutic targets in breast cancer and analyze the effects of administering muscarinic agonists in combination with conventional chemotherapeutic agents in a metronomic schedule.

Multi-drug-resistance efflux in cisplatin-naive and cisplatin-exposed A2780 ovarian cancer cells responds differently to cell culture dimensionality.

A primary reason for chemotherapy failure is chemoresistance, which is driven by various mechanisms. Multi-drug resistance (MDR) is one such mechanism that is responsible for drug extrusion from the intracellular space. MDR can be intrinsic and thus, may pre-exist the first application of chemotherapy. However, MDR may also be acquired during tumor exposure to chemotherapeutic agents. To understand whether cell clustering can influence intrinsic and acquired MDR, the present study assessed cultured monolayers (representing individual cells) and spheroids (representing clusters) formed by cisplatin-naïve (intrinsic MDR) and cisplatin-exposed (acquired MDR) lines of ovarian cancer A2780 cells by determining the cytometry of reaction rate constant (CRRC). MDR efflux was characterized using accurate and robust cell number vs. MDR efflux rate constant (kMDR) histograms. Both cisplatin-naïve and cisplatin-exposed monolayer cells presented unimodal histograms; the histogram of cisplatin-exposed cells was shifted towards a higher kMDR value suggesting greater MDR activity. Spheroids of cisplatin-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of cisplatin-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of cisplatin-exposed cells but with a moderate shift towards greater MDR activity. A flow-cytometry assessment of multidrug resistance-associated protein 1 transporter levels in monolayers and dissociated spheroids revealed distributions similar to those of kMDR, thus, suggesting a plausible molecular mechanism for the observed differences in MDR activity. The observed greater effect of cell clustering on intrinsic rather than in acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.

Directed Mutational Strategies Reveal Drug Binding and Transport by the MDR Transporters of Candida albicans.

Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug resistance in fungi. The overproduction of these proteins enables the extrusion of incoming drugs at rates that prevent lethal effects. The promiscuity of these proteins is intriguing because they export a wide range of structurally unrelated molecules. Research in the last two decades has used multiple approaches to dissect the molecular basis of the polyspecificity of multidrug transporters. With large numbers of drug transporters potentially involved in clinical drug resistance in pathogenic yeasts, this review focuses on the drug transporters of the important pathogen Candida albicans. This organism harbors many such proteins, several of which have been shown to actively export antifungal drugs. Of these, the ABC protein CaCdr1 and the MFS protein CaMdr1 are the two most prominent and have thus been subjected to intense site-directed mutagenesis and suppressor genetics-based analysis. Numerous results point to a common theme underlying the strategy of promiscuity adopted by both CaCdr1 and CaMdr1. This review summarizes the body of research that has provided insight into how multidrug transporters function and deliver their remarkable polyspecificity.