RESUMO
Dense bacterial suspensions display collective motion exhibiting coherent flow structures reminiscent of turbulent flows. However, in contrast to inertial turbulence, the microscopic dynamics underlying bacterial turbulence is only beginning to be understood. Here, we report experiments revealing correlations between microscopic dynamics and the emergence of collective motion in bacterial suspensions. Our results demonstrate the existence of three microscopic dynamical regimes: initial ballistic dynamics followed by an intermittent Lévy walk before the intriguing decay to random Gaussian fluctuations. Our experiments capture that the fluid correlation time earmarks the transition from Lévy to Gaussian fluctuations demonstrating the microscopic reason underlying the observation. By harnessing the flow activity via bacterial concentration, we reveal systematic control over the flow correlation timescales, which, in turn, allows controlling the duration of the Lévy walk.
Assuntos
Modelos Biológicos , Movimento , Escherichia coli , Fatores de Tempo , BactériasRESUMO
Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR.
RESUMO
Bio-spinterfaces present numerous opportunities to study spintronics across the biomolecules attached to (ferro)magnetic electrodes. While it offers various exciting phenomena to investigate, it is simultaneously challenging to make stable bio-spinterfaces as biomolecules are sensitive to many factors that it encounters during thin-film growth to device fabrication. The chirality-induced spin-selectivity effect is an exciting discovery, demonstrating an understanding that a specific electron's spin (either up or down) passes through a chiral molecule. The present work utilizes Ustilago maydis Rvb2 protein, an ATP-dependent DNA helicase (also known as Reptin), to fabricate bio-spintronic devices to investigate spin-selective electron transport through the protein. Ferromagnetic materials are well-known for exhibiting spin-polarization, which many chiral and biomolecules can mimic. We report herein spin-selective electron transmission through Rvb2 that exhibits 30% spin polarization at a low bias (+0.5 V) in a device configuration, Ni/Rvb2 protein/indium tin oxide measured under two different magnetic configurations. Our findings demonstrate that biomolecules can be put in circuit components without any expensive vacuum deposition for the top contact. The present study holds a remarkable potential to advance spin-selective electron transport in other biomolecules, such as proteins and peptides, for biomedical applications.
Assuntos
Peptídeos , Proteínas , Transporte de Elétrons , Peptídeos/química , Elétrons , EletrodosRESUMO
Three platinum(II)-N-heterocyclic carbene (NHC) compounds [Pt(L1)Cl](PF6) (1), [Pt(L2)(COD)](PF6)2 (2) and [Pt(L2)Cl2] (3) were synthesized bearing pyridyl-functionalized butenyl-tethered (L1H) and n-butyl tethered (L2H) NHC ligands, and their antibacterial activity against clinically relevant human pathogens was evaluated. Complex 1 was designed to have one of its metal coordination sites masked with a hemilabile butenyl group. The antibacterial activity spectrum against the ESKAPE panel of pathogens shows superior activity of 1 compared to 2 and 3 against the Gram-positive S. aureus pathogen. Complex 1 showed equipotent activity against clinical drug-resistant S. aureus and Enterococcus isolates. Furthermore, 1 demonstrated concentration-dependent bactericidal activity with a long post-antibiotic effect, eradicated preformed S. aureus biofilm and synergized with gentamicin and minocycline for combinatorial antimicrobial therapy. Under in vivo conditions, 1 displayed potent activity in reducing bacterial load in a murine thigh infection model, similar to vancomycin, albeit at 2.5× less dosage. An array of experiments reveals key characteristics for the hemilabile complex 1 as a potential anti-staphylococcal drug.
Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Humanos , Animais , Camundongos , Enterococcus , Staphylococcus aureus , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologiaRESUMO
Copper-based ternary metal oxide (i.e., Cu0.52Al0.01Fe0.47O4) impregnated reduced graphene oxide nanohybrid is verified for microbial and arsenic treatment. Growth inhibition of colonies are observed around 99.99% (E. coli), and 99.83% (S. aureus) at 10-20 µg/mL of hybrid dosage, respectively. The inhibition rates for both the colonies are increased to 99.9998% at 80 µg/mL. TEM images have shown insight of cell-content/lipid leakage behavior after inoculating with the hybrid. The efficient hindrance towards microbial colony growth is attributed to better charge transfer, reactive oxygen species generation, and metal-ion release. Maximum arsenic sorption capacities are observed around 248 and 314 mg/g for As(III), and As(V), respectively (Ci ~ 500 ppm). Surface morphology studies onto arsenic adsorption are reported with atomic force microscope, and FT-IR/Raman analysis. A detailed discussion onto individual spectra of As 3d spectra confirmed the occurrence of redox transformation in arsenic species [As(III)]. The variation in the quantity (at. %) of oxygen functional groups in O1s spectra (i.e., M-O, M-OH, and -OH2) onto the hybrid supported the ligand-exchange behavior. Cyclic voltammetry study in arsenic electrolytes (10 µM - 1 mM) provides the occurrence of various in-situ electrochemical reactions supporting the redox activity. A significant electromagnetic wave absorption characteristics of the present hybrid is proposed with plausible airborne antimicrobial-agent abilities.
Assuntos
Arsênio , Poluentes Químicos da Água , Purificação da Água , Arsênio/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Escherichia coli , Staphylococcus aureus , Purificação da Água/métodos , Poluentes Químicos da Água/análise , Óxidos , Água , AdsorçãoRESUMO
Zinc is a cofactor for enzymes involved in DNA replication, peptidoglycan hydrolysis, and pH maintenance, in addition to the transfer of the methyl group to thiols. Here, we discovered a new role of Zn2+ as an inhibitor for S-adenosyl methionine (SAM) binding in a mycobacterial methyltransferase. Rv1377c is annotated as a putative methyltransferase that is upregulated upon the mitomycin C treatment of Mycobacterium tuberculosis. Sequence analysis and experimental validation allowed the identification of distinct motifs responsible for SAM binding. A detailed analysis of the AlphaFold-predicted structure of Rv1377c revealed four cysteine residues capable of coordinating a Zn2+ ion located in proximity to the SAM-binding site. Further, experimental studies showed distinct conformational changes upon Zn2+ binding to the protein, which compromised its ability to bind SAM. This is the first report wherein Zn2+-driven conformational changes in a methyltransferase undermines its ability to bind SAM.
RESUMO
Antimicrobial resistance (AMR) is a global health concern. Targetting AMR, we present an in situ lactonization mechanism generating 4-nitroisobenzofuran-1(3H)-one (IITK2020), an exclusive S. aureus inhibitor at 2-4 µg mL-1 MIC including multidrug-resistant S. aureus clinical strains, that prevents peptidoglycan biosynthesis.
Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Peptidoglicano , Staphylococcus aureusRESUMO
Whole-genome sequencing (WGS) data of a bacterial strain IITK SM2 isolated from an aquifer located in the middle Indo-Gangetic plain is reported here, along with its physiological, morphological, biochemical, and redox-transformation characteristics in the presence of dissolved arsenic (As). The aquifer exhibits oxidizing conditions relative to As speciation. Analyses based on 16S rRNA and recN sequences indicate that IITK SM2 was clustered with C. youngae NCTC 13708T and C. pasteuri NCTC UMH17T. However, WGS analyses using the digital DNA-DNA hybridization and Rapid Annotations using Subsystems Technology suggest that IITK SM2 belongs to a strain of C. youngae. This strain can effectively reduce As(V) to As(III) but cannot oxidize As(III) to As(V). It exhibited high resistance to As(V) [32,000 mg L-1] and As(III) [1,100 mg L-1], along with certain other heavy metals typically found in contaminated groundwater. WGS analysis also indicates the presence of As-metabolizing genes such as arsC, arsB, arsA, arsD, arsR, and arsH in this strain. Although these genes have been identified in several As(V)-reducers, the clustering of these genes in the forms of arsACBADR, arsCBRH, and an independent arsC gene has not been observed in any other Citrobacter species or other selected As(V)-reducing strains of Enterobacteriaceae family. Moreover, there were differences in the number of genes corresponding to membrane transporters, virulence and defense, motility, protein metabolism, phages, prophages, and transposable elements in IITK SM2 when compared to other strains. This genomic dataset will facilitate subsequent molecular and biochemical analyses of strain IITK SM2 to identify the reasons for high arsenic resistance in Citrobacter youngae and understand its role in As mobilization in middle Indo-Gangetic plain aquifers.
Assuntos
Arsênio , Água Subterrânea , Arsênio/análise , Citrobacter/genética , DNA , Água Subterrânea/química , RNA Ribossômico 16S/genéticaRESUMO
Transcriptional repressor, LexA, regulates the "SOS" response, an indispensable bacterial DNA damage repair machinery. Compared to its E.coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in "SOS" regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different "SOS" boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, this study provides insights into the kinetics of the interaction between Mtb LexA and its target "SOS" boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of "SOS" regulation and activation in Mtb.
RESUMO
Transcriptional repressor, LexA, regulates the 'SOS' response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in 'SOS' regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different 'SOS' boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target 'SOS' boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of 'SOS' regulation and activation in Mtb.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Interferometria/métodos , Mycobacterium tuberculosis/enzimologia , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Cinética , Filogenia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Resposta SOS em Genética/fisiologia , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Long term multiple systemic antibiotics form the cornerstone in the treatment of bone and joint tuberculosis, often combined with local surgical eradication. Implanted carriers for local drug delivery have recently been introduced to overcome some of the limitations associated with conventional treatment strategies. In this study, we used a calcium sulphate hemihydrate (CSH)/nanohydroxyapatite (nHAP) based nanocement (NC) biomaterial as a void filler as well as a local delivery carrier of two standard of care tuberculosis drugs, Rifampicin (RFP) and Isoniazid (INH). We observed that the antibiotics showed different release patterns where INH showed a burst release of 67% and 100% release alone and in combination within one week, respectively whereas RFP showed sustained release of 42% and 49% release alone and in combination over a period of 12 weeks, respectively indicating different possible interactions of antibiotics with nHAP. The interactions were studied using computational methodology, which showed that the binding energy of nHAP with RFP was 148 kcal/mol and INH was 11 kcal/mol, thus varying substantially resulting in RFP being retained in the nHAP matrix. Our findings suggest that a biphasic ceramic based drug delivery system could be a promising treatment alternative to bone and joint TB.
Assuntos
Antituberculosos/farmacologia , Sulfato de Cálcio/farmacologia , Portadores de Fármacos/farmacologia , Durapatita/farmacologia , Isoniazida/farmacologia , Rifampina/farmacologia , Tuberculose Osteoarticular/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Regeneração Óssea/efeitos dos fármacos , Biologia Computacional , Preparações de Ação Retardada/farmacologia , Interações Medicamentosas , Quimioterapia Combinada , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacosRESUMO
During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Complexos Multiproteicos/metabolismo , Mutação , Subunidades Proteicas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologiaRESUMO
Quantitative evaluation of small molecule permeation and accumulation in Gram-negative bacteria is important for drug development against these bacteria. While these measurements are commonly performed at physiological pH, Escherichia coli and many other Enterobacteriaceae infect human gastrointestinal and urinary tracts, where they encounter different pH conditions. To understand how external pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG) spectroscopy using SHG-active antimicrobial compound malachite green as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic accumulations separately in live E. coli cells, which was never done before. Compartment-wise measurements reveal accumulation of the probe molecule in cytoplasm at physiological and alkaline pH, while entrapment in periplasm at weakly acidic pH and retention in external solution at highly acidic pH. Behind such disparity in localizations, up to 2 orders of magnitude reduction in permeability across the inner membrane at weakly acidic pH and outer membrane at highly acidic pH are found to play key roles. Our results unequivocally demonstrate the control of external pH over entry and compartment-wise distribution of small molecules in E. coli cells, which is a vital information and should be taken into account in antibiotic screening against E. coli and other Enterobacteriaceae members. In addition, our results demonstrate the ability of malachite green as an excellent SHG-indicator of changes of individual cell membrane and periplasm properties of live E. coli cells in response to external pH change from acidic to alkaline. This finding, too, has great importance, as there is barely any other molecular probe that can provide similar information.