RESUMEN

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection.

Asunto(s)

Antituberculosos , Proteínas Bacterianas , Pared Celular , Mycobacterium tuberculosis , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Antituberculosos/farmacología , Antituberculosos/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Tuberculosis/tratamiento farmacológico , Oxidorreductasas/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Ácidos Micólicos/metabolismo , Oxidorreductasas de Alcohol , Proteínas de Transporte de Membrana

RESUMEN

Abscisic acid (ABA) is the primary preventing factor of seed germination, which is crucial to plant survival and propagation. ABA-induced seed germination inhibition is mainly mediated by the dimeric PYR/PYL/RCAR (PYLs) family members. However, little is known about the relevance between dimeric stability of PYLs and seed germination. Here, we reveal that stabilization of PYL dimer can relieve ABA-induced inhibition of seed germination using chemical genetic approaches. Di-nitrobensulfamide (DBSA), a computationally designed chemical probe, yields around ten-fold improvement in receptor affinity relative to ABA. DBSA reverses ABA-induced inhibition of seed germination mainly through dimeric receptors and recovers the expression of ABA-responsive genes. DBSA maintains PYR1 in dimeric state during protein oligomeric state experiment. X-ray crystallography shows that DBSA targets a pocket in PYL dimer interface and may stabilize PYL dimer by forming hydrogen networks. Our results illustrate the potential of PYL dimer stabilization in preventing ABA-induced seed germination inhibition.

Asunto(s)

Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Germinación , Semillas , Germinación/efectos de los fármacos , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Arabidopsis/genética , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Cristalografía por Rayos X , Sulfonamidas/farmacología , Sulfonamidas/química , Proteínas de Transporte de Membrana

RESUMEN

Visceral cestodiases, like cysticercoses and echinococcoses, are caused by cystic larvae from parasites of the Cestoda class and are endemic or hyperendemic in many areas of the world. Current therapeutic approaches for these diseases are complex and present limitations and risks. Therefore, new safer and more effective treatments are urgently needed. The Niemann-Pick C1 (NPC1) protein is a cholesterol transporter that, based on genomic data, would be the solely responsible for cholesterol uptake in cestodes. Considering that human NPC1L1 is a known target of ezetimibe, used in the treatment of hypercholesterolemia, it has the potential for repurposing for the treatment of visceral cestodiases. Here, phylogenetic, selective pressure and structural in silico analyses were carried out to assess NPC1 evolutive and structural conservation, especially between cestode and human orthologs. Two NPC1 orthologs were identified in cestode species (NPC1A and NPC1B), which likely underwent functional divergence, leading to the loss of cholesterol transport capacity in NPC1A. Comparative interaction analyses performed by molecular docking of ezetimibe with human NPC1L1 and cestode NPC1B pointed out to similarities that consolidate the idea of cestode NPC1B as a target for the repurposing of ezetimibe as a drug for the treatment of visceral cestodiases.

Asunto(s)

Cestodos , Ezetimiba , Proteína Niemann-Pick C1 , Ezetimiba/farmacología , Ezetimiba/uso terapéutico , Humanos , Animales , Proteína Niemann-Pick C1/metabolismo , Cestodos/metabolismo , Cestodos/efectos de los fármacos , Cestodos/genética , Filogenia , Simulación del Acoplamiento Molecular , Reposicionamiento de Medicamentos/métodos , Simulación por Computador , Colesterol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/química , Anticolesterolemiantes/farmacología , Anticolesterolemiantes/uso terapéutico

RESUMEN

Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.

Asunto(s)

Enfermedad de Alzheimer , Endosomas , Proteínas Relacionadas con Receptor de LDL , Proteínas de Transporte de Membrana , Linaje , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Endosomas/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Femenino , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación Missense , Transporte de Proteínas , Multimerización de Proteína , Anciano , Persona de Mediana Edad , Células HEK293

RESUMEN

As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.

Asunto(s)

Proteínas Bacterianas , Mycobacterium tuberculosis , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Transporte Biológico , Microscopía por Crioelectrón , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Conformación Proteica

RESUMEN

Over 50 billion cells undergo apoptosis each day in an adult human to maintain tissue homeostasis by eliminating damaged or unwanted cells. Apoptotic deficiency can lead to age-related diseases with reduced apoptotic metabolites. However, whether apoptotic metabolism regulates aging is unclear. Here, we show that aging mice and apoptosis-deficient MRL/lpr (B6.MRL-Faslpr/J) mice exhibit decreased apoptotic levels along with increased aging phenotypes in the skeletal bones, which can be rescued by the treatment with apoptosis inducer staurosporine (STS) and stem cell-derived apoptotic vesicles (apoVs). Moreover, embryonic stem cells (ESC)-apoVs can significantly reduce senescent hallmarks and mtDNA leakage to rejuvenate aging bone marrow mesenchymal stem cells (MSCs) and ameliorate senile osteoporosis when compared to MSC-apoVs. Mechanistically, ESC-apoVs use TCOF1 to upregulate mitochondrial protein transcription, resulting in FLVCR1-mediated mitochondrial functional homeostasis. Taken together, this study reveals a previously unknown role of apoptotic metabolites in ameliorating bone aging phenotypes and the unique role of TCOF1/FLVCR1 in maintaining mitochondrial homeostasis.

Asunto(s)

Envejecimiento , Apoptosis , Homeostasis , Células Madre Mesenquimatosas , Mitocondrias , Animales , Humanos , Ratones , Envejecimiento/metabolismo , Apoptosis/efectos de los fármacos , Huesos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Osteoporosis/metabolismo , Fenotipo , Estaurosporina/farmacología

RESUMEN

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1Apo), fluconazole-bound state (Cdr1Flu), and milbemycin oxime-inhibited state (Cdr1Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.

Asunto(s)

Antifúngicos , Azoles , Candida albicans , Microscopía por Crioelectrón , Proteínas Fúngicas , Proteínas de Transporte de Membrana , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/antagonistas & inhibidores , Antifúngicos/farmacología , Antifúngicos/química , Azoles/farmacología , Azoles/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Farmacorresistencia Fúngica , Fluconazol/farmacología , Sitios de Unión

RESUMEN

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Farmacorresistencia Bacteriana Múltiple , Klebsiella pneumoniae , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cloranfenicol/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Tetraciclina/farmacología , Secuenciación Completa del Genoma

RESUMEN

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus, has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis, in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 µg/mL, respectively. Baicalein at 64 µg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 µg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.

Asunto(s)

Antibacterianos , Biopelículas , Sinergismo Farmacológico , Flavanonas , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Tetraciclina , Flavanonas/farmacología , Antibacterianos/farmacología , Tetraciclina/farmacología , Staphylococcus aureus/efectos de los fármacos , Humanos , Biopelículas/efectos de los fármacos , Resistencia a la Tetraciclina , Scutellaria baicalensis/química , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Células HeLa

RESUMEN

Sorbicillinoids are a class of fungal polyketides with diverse structures and distinguished bioactivities. Although remarkable progress has been achieved in their chemistry and biosynthesis, the efflux of sorbicillinoids is poorly understood. Here, we found MFS transporter AcsorT was responsible for the biosynthesis of sorbicillinoids in Acremonium chrysogenum. Combinatorial knockout and subcellular location demonstrated that the plasma membrane-associated AcsorT was responsible for the transportation of sorbicillinol and subsequent formation of oxosorbicillinol and acresorbicillinol C via the berberine bridge enzyme-like oxidase AcsorD in the periplasm. Homology modeling and site-directed mutation revealed that Tyr303 and Arg436 were the key residues of AcsorT, which was further explained by molecular dynamics simulation. Based on our study, it was suggested that AcsorT modulates sorbicillinoid production by coordinating its biosynthesis and export, and a transport model of sorbicillinoids was proposed in A. chrysogenum.

Asunto(s)

Acremonium , Proteínas Fúngicas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Acremonium/metabolismo , Acremonium/genética , Acremonium/química , Policétidos/metabolismo , Policétidos/química , Transporte Biológico , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química

RESUMEN

Sugar content is a critical indicator of fruit quality and is mainly controlled by sugar transporters. Sugars will eventually be exported transporters (SWEET) proteins play an indispensable role in sugar allocation between and within plant organs. Sucrose is the major sugar in many fruits and the predominant form of sugar translocated in peach (Prunus persica). However, the role of the multiple peach SWEET genes in sucrose allocation to fruit remains elusive. In this study, a total of 19 SWEET candidates have been identified in the peach genome, and two Clade III SWEET genes, PpSWEET9a and PpSWEET14, are found to be highly expressed in mature source leaves and branches. Complementation assays, transgene manipulations, and protein interaction studies reveal that PpSWEET9a and PpSWEET14 serve as sucrose efflux proteins and form a heterooligomer that synergistically directs sucrose allocation from source leaves to fruits. Our findings provide insights into the effect of SWEETs on sugar accumulation in peach fruit and identify genetic candidates for improving fruit quality.

Asunto(s)

Frutas , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta , Proteínas de Plantas , Prunus persica , Sacarosa , Prunus persica/genética , Prunus persica/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Sacarosa/metabolismo , Frutas/metabolismo , Frutas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética

RESUMEN

Uptake of nucleobases and ascorbate is an essential process in all living organisms mediated by SLC23 transport proteins. These transmembrane carriers operate via the elevator alternating-access mechanism, and are composed of two rigid domains whose relative motion drives transport. The lack of large conformational changes within these domains suggests that the interdomain-linkers act as flexible tethers. Here, we show that interdomain-linkers are not mere tethers, but have a key regulatory role in dictating the conformational space of the transporter and defining the rotation axis of the mobile transport domain. By resolving a wide inward-open conformation of the SLC23 elevator transporter UraA and combining biochemical studies using a synthetic nanobody as conformational probe with hydrogen-deuterium exchange mass spectrometry, we demonstrate that interdomain-linkers control the function of transport proteins by influencing substrate affinity and transport rate. These findings open the possibility to allosterically modulate the activity of elevator proteins by targeting their linkers.

Asunto(s)

Proteínas Bacterianas , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Conformación Proteica , Dominios Proteicos , Modelos Moleculares , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Transporte Biológico , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo

RESUMEN

Based on in vitro digestion, micellar synthesis, and Caco-2 cell model, this study investigated the effects of typical flavonoids in citrus (naringenin, naringin, hesperetin, hesperidin, quercetin, and rutin) at different doses on the micellization and cellular uptake of ß-carotene. In in vitro digestion, low-dose flavonoids enhanced ß-carotene bioaccesssibility by regulating the stability and dispersibility of the intestinal medium, particularly quercetin, which significantly increased the bioaccessibility by 44.6% (p < 0.05). Furthermore, naringenin, hesperetin, hesperidin, and quercetin enhanced the micellar incorporation rate of ß-carotene; however, naringin and rutin exhibited an opposite effect, particularly naringin, which significantly reduced it by 71.3% (p < 0.05). This phenomenon could be attributed to the high solubility of naringin and rutin in micelles, resulting in a competitive inhibitory effect on ß-carotene. Besides, all treatments significantly enhanced ß-carotene cellular uptake (p < 0.05) by promoting the expression of scavenger receptor class B type I and Niemann-Pick C1-Like 1.

Asunto(s)

Citrus , Flavonoides , Micelas , beta Caroteno , Humanos , Células CACO-2 , beta Caroteno/metabolismo , beta Caroteno/química , Flavonoides/metabolismo , Flavonoides/química , Citrus/química , Citrus/metabolismo , Transporte Biológico , Digestión , Flavanonas/metabolismo , Flavanonas/química , Rutina/metabolismo , Rutina/química , Extractos Vegetales/metabolismo , Extractos Vegetales/química , Proteínas de Transporte de Membrana

RESUMEN

Thiosemicarbazones and their metal complexes have been studied for their biological activities against bacteria, cancer cells and protozoa. Short-term in vitro treatment with one gold (III) complex (C3) and its salicyl-thiosemicarbazone ligand (C4) selectively inhibited proliferation of T. gondii. Transmission Electron Microscopy (TEM) detected transient structural alterations in the parasitophorous vacuole membrane and the tachyzoite cytoplasm, but the mitochondrial membrane potential appeared unaffected by these compounds. Proteins potentially interacting with C3 and C4 were identified using differential affinity chromatography coupled with mass spectrometry (DAC-MS). Moreover, long-term in vitro treatment was performed to investigate parasitostatic or parasiticidal activity of the compounds. DAC-MS identified 50 ribosomal proteins binding both compounds, and continuous drug treatments for up to 6 days caused the loss of efficacy. Parasite tolerance to both compounds was, however, rapidly lost in their absence and regained shortly after re-exposure. Proteome analyses of six T. gondii ME49 clones adapted to C3 and C4 compared to the non-adapted wildtype revealed overexpression of ribosomal proteins, of two transmembrane proteins involved in exocytosis and of an alpha/beta hydrolase fold domain-containing protein. Results suggest that C3 and C4 may interfere with protein biosynthesis and that adaptation may be associated with the upregulated expression of tachyzoite transmembrane proteins and transporters, suggesting that the in vitro drug tolerance in T. gondii might be due to reversible, non-drug specific stress-responses mediated by phenotypic plasticity.

Asunto(s)

Proteínas Ribosómicas , Tiosemicarbazonas , Toxoplasma , Toxoplasma/efectos de los fármacos , Toxoplasma/metabolismo , Tiosemicarbazonas/farmacología , Proteínas Ribosómicas/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Adaptación Fisiológica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Regulación hacia Arriba/efectos de los fármacos , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Animales

RESUMEN

BACKGROUND: More than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli. RESULTS: Biofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^-ΔΔCT < 1 in indole-treated isolates compared to control group. CONCLUSION: The outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.

Asunto(s)

Antibacterianos , Biopelículas , Escherichia coli , Indoles , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/metabolismo , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Indoles/metabolismo , Indoles/farmacología , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Antibacterianos/farmacología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación hacia Abajo , Proteus mirabilis/genética , Proteus mirabilis/efectos de los fármacos , Proteus mirabilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Virulencia/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo

RESUMEN

Streptococcus pneumoniae is a bacterium of great global importance, responsible for more than one million deaths per year. This bacterium is commonly acquired in the first years of life and colonizes the upper respiratory tract asymptomatically by forming biofilms that persist for extended times in the nasopharynx. However, under conditions that alter the bacterial environment, such as viral infections, pneumococci can escape from the biofilm and invade other niches, causing local and systemic disease of varying severity. The polyamine transporter PotABCD is required for optimal survival of the organism in the host. Immunization of mice with recombinant PotD can reduce subsequent bacterial colonization. PotD has also been suggested to be involved in pneumococcal biofilm development. Therefore, in this study we aimed to elucidate the role of PotABCD and polyamines in pneumococcal biofilm formation. First, the formation of biofilms was evaluated in the presence of exogenous polyamines-the substrate transported by PotABCD-added to culture medium. Next, a potABCD-negative strain was used to determine biofilm formation in different model systems using diverse levels of complexity from abiotic surface to cell substrate to in vivo animal models and was compared with its wild-type strain. The results showed that adding more polyamines to the medium stimulated biofilm formation, suggesting a direct correlation between polyamines and biofilm formation. Also, deletion of potABCD operon impaired biofilm formation in all models tested. Interestingly, more differences between wild-type and mutant strains were observed in the more complex model, which emphasizes the significance of employing more physiological models in studying biofilm formation.

Asunto(s)

Biopelículas , Streptococcus pneumoniae , Biopelículas/crecimiento & desarrollo , Streptococcus pneumoniae/fisiología , Streptococcus pneumoniae/metabolismo , Animales , Ratones , Poliaminas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Infecciones Neumocócicas/microbiología , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Operón

RESUMEN

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L-1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 µmol h-1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters.

Asunto(s)

Proteínas Fúngicas , Kluyveromyces , Xilosa , Xilosa/metabolismo , Kluyveromyces/metabolismo , Kluyveromyces/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/química , Simulación del Acoplamiento Molecular , Mutación , Glucosa/metabolismo

RESUMEN

Antimicrobial resistance has been an increasingly serious threat to global public health. The contribution of non-antibiotic pharmaceuticals to the development of antibiotic resistance has been overlooked. Our study found that the anti-inflammatory drug phenylbutazone could protect P. aeruginosa against antibiotic mediated killing by binding to the efflux pump regulator MexR. In this study, antibiotic activity against P. aeruginosa alone or in combination with phenylbutazone was evaluated in vitro and in vivo. Resazurin accumulation assay, transcriptomic sequencing, and PISA assay were conducted to explore the underlying mechanism for the reduced antibiotic susceptibility caused by phenylbutazone. Then EMSA, ITC, molecular dynamic simulations, and amino acid substitutions were used to investigate the interactions between phenylbutazone and MexR. We found that phenylbutazone could reduce the susceptibility of P. aeruginosa to multiple antibiotics, including parts of ß-lactams, fluoroquinolones, tetracyclines, and macrolides. Phenylbutazone could directly bind to MexR, then promote MexR dissociating from the mexA-mexR intergenic region and de-repress the expression of MexAB-OprM efflux pump. The overexpressed MexAB-OprM pump resulted in the reduced antibiotic susceptibility. And the His41 and Arg21 residues of MexR were involved in the phenylbutazone-MexR interaction. We hope this study would imply the potential risk of antibiotic resistance caused by non-antibiotic pharmaceuticals.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Proteínas de Transporte de Membrana , Pruebas de Sensibilidad Microbiana , Fenilbutazona , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Fenilbutazona/farmacología , Fenilbutazona/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Simulación de Dinámica Molecular , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Fluoroquinolonas/farmacología , Fluoroquinolonas/metabolismo , Unión Proteica , Animales

RESUMEN

BACKGROUND: Biallelic variants in the major facilitator superfamily domain containing 8 gene (MFSD8) are associated with distinct clinical presentations that range from typical late-infantile neuronal ceroid lipofuscinosis type 7 (CLN7 disease) to isolated adult-onset retinal dystrophy. Classic late-infantile CLN7 disease is a severe, rare neurological disorder with an age of onset typically between 2 and 6 years, presenting with seizures and/or cognitive regression. Its clinical course is progressive, leading to premature death, and often includes visual loss due to severe retinal dystrophy. In rare cases, pathogenic variants in MFSD8 can be associated with isolated non-syndromic macular dystrophy with variable age at onset, in which the disease process predominantly or exclusively affects the cones of the macula and where there are no neurological or neuropsychiatric manifestations. METHODS: Here we present longitudinal studies on four adult-onset patients who were biallelic for four MFSD8 variants. RESULTS: Two unrelated patients who presented with adult-onset ataxia and had macular dystrophy on examination were homozygous for a novel variant in MFSD8 NM_152778.4: c.935T>C p.(Ile312Thr). Two other patients presented in adulthood with visual symptoms, and one of these developed mild to moderate cerebellar ataxia years after the onset of visual symptoms. CONCLUSIONS: Our observations expand the knowledge on biallelic pathogenic MFSD8 variants and confirm that these are associated with a spectrum of more heterogeneous clinical phenotypes. In MFSD8-related disease, adult-onset recessive ataxia can be the presenting manifestation or may occur in combination with retinal dystrophy.

Asunto(s)

Degeneración Macular , Humanos , Adulto , Masculino , Femenino , Degeneración Macular/genética , Degeneración Macular/patología , Edad de Inicio , Ataxia/genética , Ataxia/patología , Alelos , Persona de Mediana Edad , Mutación , Proteínas de Transporte de Membrana/genética , Fenotipo

RESUMEN

Efflux-mediated ß-lactam resistance is a major public health concern, reducing the effectiveness of ß-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines ß-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under ß-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.

Asunto(s)

Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Simulación de Dinámica Molecular , Resistencia betalactámica , Resistencia betalactámica/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Conformación Proteica