MP
Engineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.
Lyu Y, Fu C, Ma H, Su Z, Sun Z, Zhou X.
Nat Commun. 2024 Aug 2;15(1):6511.
doi: 10.1038/s41467-024-50934-5.
PMID: 39095428.
Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.
Use of zebrafish to identify host responses specific to type VI secretion system mediated interbacterial antagonism.
Virgo M, Mostowy S, Ho BT
PLoS Pathog (2024) 20(7): e1012384.
https://doi.org/10.1371/journal.ppat.1012384
Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.
An In-depth Examination of Per- and Polyfluoroalkyl (PFAS) Effects on Transporters, with Emphasis on the ABC Superfamily: A Critical Review.
Collier GE, Lavado R.
Toxicology. 2024 Jul 31:153901.
doi: 10.1016/j.tox.2024.153901. Epub ahead of print.
PMID: 39094918.
Per- and polyfluoroalkyl (PFAS) substances are a type of chemical compound unique for their multiple carbon-fluorine bonds, imbuing them with strength and environmental permanence. While legacy substances have been phased out due to human health risks, short-chain and alternative PFAS remain omnipresent. However, a detailed explanation for the pathways through which PFAS interact on a cellular and molecular level is still largely unknown, and the human health effects remain mechanistically unexplained. Of particular interest when focusing on this topic are the interactions between these exogenous chemicals and plasma and membrane proteins. Such proteins include serum albumin which can transport PFAS throughout the body, solute carrier proteins (SLC) and ATP binding cassette (ABC) transporters which are able to move PFAS into and out of cells, and proteins and nuclear receptors which interact with PFAS intracellularly. ABC transporters as a family have little available human data despite being responsible for the export of endogenous substances and drugs throughout the body. The multifactorial regulation of these crucial transporters is affected directly and indirectly by PFAS. Changes, which can include alterations to membrane transport activity and differences in protein expression, vary greatly depending on the specific PFAS and protein of interest. Together, the myriad of changes caused by understudied PFAS exposure to a class of understudied proteins crucial to cellular function and drug treatments has not been fully explored regarding human health and presents room for further exploration. This critical work aims to provide a novel framework of existing human data on PFAS and ABC transporters, allowing for future advancement and investigation into human transporter activity, mechanisms of regulation, and interactions with emerging contaminants.
Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore.
Gao X, Yeh HI, Yang Z, Fan C, Jiang F, Howard RJ, Lindahl E, Kappes JC, Hwang TC.
Nat Commun. 2024 Aug 6;15(1):6668.
doi: 10.1038/s41467-024-50641-1.
PMID: 39107303.
Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR’s activity through binding to a site also comprising TM8.
The impact of nanobodies on GPCR structural biology and their potential as therapeutic agents.
Salom D, Wu A, Liu CC, Palczewski K.
Mol Pharmacol. 2024 Aug 6:MOLPHARM-MR-2024-000974.
doi: 10.1124/molpharm.124.000974. Epub ahead of print.
PMID: 39107078.
The family of human G protein-coupled receptors (GPCRs) is comprised of about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or an allosteric modulator of GPCR signaling. In this review we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective.
Significance Statement G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs, by forming complexes directly with the receptors or indirectly through protein partners.
Role of AcrAB-TolC and Its Components in Influx-Efflux Dynamics of QAC Drugs in Escherichia coli Revealed Using SHG Spectroscopy.
Singh D, Kumar D, Gayen A, Chandra M.
J Phys Chem Lett. 2024 Aug 8;15(31):7832-7839.
doi: 10.1021/acs.jpclett.4c01189. Epub 2024 Jul 25.
PMID: 39052610.
Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.
Membranes
Nanodisc assembly from bacterial total lipid extracts.
Llewellyn TR, Pimentel ORC, Lenz KD, Montoya MM, Kubicek-Sutherland JZ.
Chem Phys Lipids. 2024 Aug 5;264:105425.
doi: 10.1016/j.chemphyslip.2024.105425. Epub ahead of print.
PMID: 39111725.
Nanodiscs are discoidal lipoproteins that have often been used as vehicles to study membrane proteins in their native configuration. Nanodiscs have been primarily made from synthetic lipids. However, nanodiscs also offer a format by which native lipids can be studied in their natural configuration. Here, we present a method to synthesize nanodiscs from bacterial total lipid extracts using the biothreat agent, Yersinia pestis, as a proof-of-concept. The creation of nanoparticles entirely composed of bacterial lipids supports membrane characterization and vaccine antigen discovery without the inherent safety concerns associated with live bacterial cells of this Tier 1 select agent pathogen.
Milk Exosome-Liposome Hybrid Vesicles with Self-Adapting Surface Properties Overcome the Sequential Absorption Barriers for Oral Delivery of Peptides.
Xiao P, Wang H, Liu H, Yuan H, Guo C, Feng Y, Qi P, Yin T, Zhang Y, He H, Tang X, Gou J.
ACS Nano. 2024 Aug 4.
doi: 10.1021/acsnano.4c02560. Epub ahead of print.
PMID: 39099105.
Milk exosomes (mExos) have demonstrated significant promise as vehicles for the oral administration of protein and peptide drugs owing to their superior capacity to traverse epithelial barriers. Nevertheless, certain challenges persist due to their intrinsic characteristics, including suboptimal drug loading efficiency, inadequate mucus penetration capability, and susceptibility to membrane protein loss. Herein, a hybrid vesicle with self-adaptive surface properties (mExos@DSPE-Hyd-PMPC) was designed by fusing functionalized liposomes with natural mExos, aiming to overcome the limitations associated with mExos and unlock their full potential in oral peptide delivery. The surface property transformation of mExos@DSPE-Hyd-PMPC was achieved by introducing a pH-sensitive hydrazone bond between the highly hydrophilic zwitterionic polymer and the phospholipids, utilizing the pH microenvironment on the jejunum surface. In comparison to natural mExos, hybrid vesicles exhibited a 2.4-fold enhancement in the encapsulation efficiency of the semaglutide (SET). The hydrophilic and neutrally charged surfaces of mExos@DSPE-Hyd-PMPC in the jejunal lumen exhibited improved preservation of membrane proteins and efficient traversal of the mucus barrier. Upon reaching the surface of jejunal epithelial cells, the highly retained membrane proteins and positively charged surfaces of the hybrid vesicle efficiently overcame the apical barrier, the intracellular transport barrier, and the basolateral exocytosis barrier. The self-adaptive surface properties of the hybrid vesicle resulted in an oral bioavailability of 8.7% and notably enhanced the pharmacological therapeutic effects. This study successfully addresses some limitations of natural mExos and holds promise for overcoming the sequential absorption barriers associated with the oral delivery of peptides.
The Pathway of a Transmembrane Helix Insertion into the Membrane Assisted by Sec61α Channel.
Gao J, Zhang YW.
Langmuir. 2024 Aug 6;40(31):16454-16462.
doi: 10.1021/acs.langmuir.4c01776. Epub 2024 Jul 24.
PMID: 39046853.
The significant inconsistency between the experimental and simulation results of the free energy for the translocon-assisted insertion of the transmembrane helix (TMH) has not been reasonably explained. Understanding the mechanism of TMH insertion through the translocon is the key to solving this problem. In this study, we performed a series of coarse-grained molecular dynamics simulations and calculated the potential mean forces (PMFs) for three insertion processes of a hydrophobic TMH. The simulations reveal the pathway of the TMH insertion assisted by a translocon. The results indicate that the TMH contacts the top of the lateral gate first and then inserts down the lateral gate, which agrees with the sliding model. The TMH begins to transfer laterally to the bilayer when it is blocked by the plug and reaches the exit of the lateral gate, where there is a free energy minimum point. We also found that the connecting section between TM2 and TM3 of Sec61α prevented TMH from leaving the lateral gate and directly transitioning to the surface-bound state. These findings provide insight into the mechanism of the insertion of TMH through the translocon.
Photophysics in Biomembranes: Computational Insight into the Interaction between Lipid Bilayers and Chromophores.
Osella S, Knippenberg S.
Acc Chem Res. 2024 Aug 6.
doi: 10.1021/acs.accounts.4c00153. Epub ahead of print.
PMID: 39105728.
Light is ubiquitously available to probe the structure and dynamics of biomolecules and biological tissues. Generally, this cannot be done directly with visible light, because of the absence of absorption by those biomolecules. This problem can be overcome by incorporating organic molecules (chromophores) that show an optical response in the vicinity of those biomolecules. Since those optical properties are strongly dependent on the chromophore’s environment, time-resolved spectroscopic studies can provide a wealth of information on biosystems at the molecular scale in a nondestructive way. In this work, we give an overview on the multiscale computational strategy developed by us in the last eight years and prove that theoretical studies and simulations are needed to explain, guide, and predict observations in fluorescence experiments. As we challenge the accepted views on existing probes, we discover unexplored abilities that can discriminate surrounding lipid bilayers and their temperature-dependent as well as solvent-dependent properties. We focus on three archetypal chromophores: diphenylhexatriene (DPH), Laurdan, and azobenzene. Our method shows that conformational changes should not be neglected for the prototype rod-shaped molecule DPH. They determine its position and orientation in a liquid-ordered (Lo) sphingomyelin/cholesterol (SM/Chol) bilayer and are responsible for a strong differentiation of its absorption spectra and fluorescence decay times in dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) membranes, which are at room temperature in liquid-disordered (Ld) and solid-gel (So) phases, respectively. Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. Finally, the conformational versatility of azobenzene in saturated Ld lipid bilayers is simulated, along with its photoisomerization pathways. By means of nonadiabatic QM/MM surface hopping analyses (QM/MM-SH), a dual mechanism is found with a torsional mechanism and a slow conversion for trans-to-cis. For cis-to-trans, simulations show a much higher quantum yield and a so-called “pedal-like” mechanism. The differences are related to the different potential energy surfaces as well as the interactions with the surrounding alkyl chains. When tails of increased length are attached to this probe, cis is pushed toward the polar surface, while trans is pulled toward the center of the membrane.
Methods
Rescaling protein-protein interactions improves Martini 3 for flexible proteins in solution.
Thomasen FE, Skaalum T, Kumar A, Srinivasan S, Vanni S, Lindorff-Larsen K.
Nat Commun. 2024 Aug 5;15(1):6645.
doi: 10.1038/s41467-024-50647-9.
PMID: 39103332.
Multidomain proteins with flexible linkers and disordered regions play important roles in many cellular processes, but characterizing their conformational ensembles is difficult. We have previously shown that the coarse-grained model, Martini 3, produces too compact ensembles in solution, that may in part be remedied by strengthening protein-water interactions. Here, we show that decreasing the strength of protein-protein interactions leads to improved agreement with experimental data on a wide set of systems. We show that the ‘symmetry’ between rescaling protein-water and protein-protein interactions breaks down when studying interactions with or within membranes; rescaling protein-protein interactions better preserves the binding specificity of proteins with lipid membranes, whereas rescaling protein-water interactions preserves oligomerization of transmembrane helices. We conclude that decreasing the strength of protein-protein interactions improves the accuracy of Martini 3 for IDPs and multidomain proteins, both in solution and in the presence of a lipid membrane.
Protein Binder Toolbox for Studies of Solute Carrier Transporters.
Gelová Z, Ingles-Prieto A, Bohstedt T, Frommelt F, Chi G, Chang YN, Garcia J, Wolf G, Azzollini L, Tremolada S, Scacioc A, Hansen JS, Serrano I, Droce A, Bernal JC, Burgess-Brown NA, Carpenter EP, Dürr KL, Kristensen P, Geertsma ER, Štefanić S, Scarabottolo L, Wiedmer T, Puetter V, Sauer DB, Superti-Furga G.
J Mol Biol. 2024 Aug 15;436(16):168665.
doi: 10.1016/j.jmb.2024.168665. Epub 2024 Jun 13.
PMID: 38878854.
Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.
A whole-cell platform for discovering synthetic cell adhesion molecules in bacteria.
Chen PY, Chen YC, Chen PP, Lin KT, Sargsyan K, Hsu CP, Wang WL, Hsia KC, Ting SY.
Nat Commun. 2024 Aug 3;15(1):6568.
doi: 10.1038/s41467-024-51017-1.
PMID: 39095377.
Developing programmable bacterial cell-cell adhesion is of significant interest due to its versatile applications. Current methods that rely on presenting cell adhesion molecules (CAMs) on bacterial surfaces are limited by the lack of a generalizable strategy to identify such molecules targeting bacterial membrane proteins in their natural states. Here, we introduce a whole-cell screening platform designed to discover CAMs targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library. Leveraging the potency of the bacterial type IV secretion system-a contact-dependent DNA delivery nanomachine-we have established a positive feedback mechanism to selectively enrich for bacteria displaying nanobodies that target antigen-expressing cells. Our platform successfully identified functional CAMs capable of recognizing three distinct outer membrane proteins (TraN, OmpA, OmpC), demonstrating its efficacy in CAM discovery. This approach holds promise for engineering bacterial cell-cell adhesion, such as directing the antibacterial activity of programmed inhibitor cells toward target bacteria in mixed populations.
An NMR Approach for Investigating Membrane Protein-Lipid Interactions Using Native Reverse Micelles.
Walters SH, Fuglestad B.
Bio Protoc. 2024 Jul 20;14(14):e5039.
doi: 10.21769/BioProtoc.5039.
PMID: 39100600.
This protocol describes using natural lipids to construct reverse micelles for high-resolution NMR studies of proteins.
Initial optimization of encapsulation conditions proceeds through visual assessment, with dynamic light scattering (DLS) to measure size distribution, and NMR to observe protein behavior.
Membrane-interacting proteins are encapsulated in their membrane-bound state. Proteins that do not interact with membranes are housed in their water-solubilized state.
Structural, functional, and inhibitory studies may be performed on native reverse micelle-encapsulated proteins.
Analysis of protein-protein and protein-membrane interactions by isotope-edited infrared spectroscopy.
Tatulian SA.
Phys Chem Chem Phys. 2024 Aug 7.
doi: 10.1039/d4cp01136h. Epub ahead of print.
PMID: 39108200.
The objective of this work is to highlight the power of isotope-edited Fourier transform infrared (FTIR) spectroscopy in resolving important problems encountered in biochemistry, biophysics, and biomedical research, focusing on protein-protein and protein membrane interactions that play key roles in practically all life processes. An overview of the effects of isotope substitutions in (bio)molecules on spectral frequencies and intensities is given. Data are presented demonstrating how isotope-labeled proteins and/or lipids can be used to elucidate enzymatic mechanisms, the mode of membrane binding of peripheral proteins, regulation of membrane protein function, protein aggregation, and local and global structural changes in proteins during functional transitions. The use of polarized attenuated total reflection FTIR spectroscopy to identify the spatial orientation and the secondary structure of a membrane-bound interfacial enzyme and the mode of lipid hydrolysis is described. Methods of production of site-directed, segmental, and domain-specific labeling of proteins by the synthetic, semisynthetic, and recombinant strategies, including advanced protein engineering technologies such as nonsense suppression and frameshift quadruplet codons are overviewed.
Integration of AlphaFold with Molecular Dynamics for Efficient Conformational Sampling of Transporter Protein NarK.
Ohnuki J, Okazaki KI.
J Phys Chem B. 2024 Aug 8;128(31):7530-7537.
doi: 10.1021/acs.jpcb.4c02726. Epub 2024 Jul 27.
PMID: 39066727.
Transporter proteins carry their substrate across the cell membrane by changing their conformation. Thus, conformational dynamics are crucial for transport function. However, clarifying the complete transport cycle is challenging even with the current structural biology approach. Molecular dynamics (MD) simulation is a computational approach that can provide the time-resolved conformational dynamics of transporter proteins in atomic details but suffers from a high computational cost. Here, we integrate state-of-the-art protein structure prediction AI, AlphaFold2 (AF2), with MD simulation to reduce the computational cost. Focusing on the transporter protein NarK, we first show that AF2 sampled broad conformations of NarK, including the inward-open, occluded, and outward-open states. We also applied the coevolution-informed mutation in AF2, identifying state-shifting mutations. Then, we show that MD simulations from AF2-generated outward-open conformation, which is experimentally unresolved, captured the essence of the conformational state. We also found that MD simulations from AF2-generated intermediates showed transient dynamics like a transition state connecting two conformational states. This study paves the way for efficient conformational sampling of transporter proteins.
ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.
Tashev SA, Euchner J, Yserentant K, Hänselmann S, Hild F, Chmielewicz W, Hummert J, Schwörer F, Tsopoulidis N, Germer S, Saßmannshausen Z, Fackler OT, Klingmüller U, Herten DP.
Nat Methods. 2024 Aug 8.
doi: 10.1038/s41592-024-02376-6. Epub ahead of print.
PMID: 39117875.
Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.
Protocol for the production and reconstitution of VDAC1 for functional assays.
Dearden GI, Ravishankar V, Sakata KT, Menon AK, Bergdoll L.
STAR Protoc. 2024 Aug 7;5(3):103240.
doi: 10.1016/j.xpro.2024.103240. Epub ahead of print.
PMID: 39116198.
The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays.
Microbio
How Pseudomonas Became A Global Pathogen
https://asm.org/Podcasts/TWiM/Episodes/How-Pseudomonas-Became-A-Global-Pathogen-TWiM-315
TWIM (« This Week In Microbiology ») explores evolution and host adaptation of Pseudomonas infections of plants, and the impact of COVID-19 on ESBL-producing E. coli on urinary tract and blood infections.
Antibiotic Potentiation Through Phytochemical-Based Efflux Pump Inhibitors to Combat Multidrug Resistance Bacteria.
Cheema HS, Maurya A, Kumar S, Pandey VK, Singh RM.
Med Chem. 2024;20(6):557-575.
doi: 10.2174/0115734064263586231022135644.
PMID: 37907487.
Antimicrobial resistance development poses a significant danger to the efficacy of antibiotics, which were once believed to be the most efficient method for treating infections caused by bacteria. Antimicrobial resistance typically involves various mechanisms, such as drug inactivation or modification, drug target modification, drug uptake restriction, and drug efflux, resulting in decreased antibiotic concentrations within the cell. Antimicrobial resistance has been associated with efflux Pumps, known for their capacity to expel different antibiotics from the cell non-specifically. This makes EPs fascinating targets for creating drugs to combat antimicrobial resistance (AMR). The varied structures of secondary metabolites (phytomolecules) found in plants have positioned them as a promising reservoir of efflux pump inhibitors. These inhibitors act as modifiers of bacterial resistance and facilitate the reintroduction of antibiotics that have lost clinical effectiveness. Additionally, they may play a role in preventing the emergence of multidrug resistant strains.
Role of the polyamine transporter PotABCD during biofilm formation by Streptococcus pneumoniae.
Vieira B, Alcantara JB, Destro G, Guerra MES, Oliveira S, Lima CA, Longato GB, Hakansson AP, Leite LC, Darrieux M, R Converso T.
PLoS One. 2024 Aug 7;19(8):e0307573.
doi: 10.1371/journal.pone.0307573.
PMID: 39110759.
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.
Tryptophan transport gene inactivation promotes the development of antibiotic resistance in Escherichia coli.
Hou B, Song J, Wang H, Ye N, Wang RW.
FEMS Microbiol Lett. 2024 Jan 9;371:fnae057. doi: 10.1093/femsle/fnae057. PMID: 39038993.
Indole serves as a signaling molecule that could regulate different bacterial physiological processes, including antibiotic resistance through biofilm formation and drug efflux pump activity. In Escherichia coli, indole is produced through the tryptophan pathway, which involves three permeases (Mtr, AroP, and TnaB) that can transport the amino acid tryptophan. Although these permeases play distinct roles in the secretion of indole biosynthesis, their impact on multidrug resistance mediated by indole remaines unclear. This study was designed to investigate the connection between the tryptophan transport system and antibiotic resistance by constructing seven gene deletion mutants from E. coli MG1655 (wild type). Our result showed that deletion of the aroP or tnaB gene led to increased antibiotic resistance as evaluated by MICs for different antibiotics. Efflux activity test results revealed that the increased antibiotic resistance was related with the AcrAB-Tolc drug efflux pump in the mutants. The transcriptome analysis further demonstrated that decreased susceptibility to kanamycin and ampicillin in E. coli was accompanied by reduced accumulation of reactive oxygen species and decreased motility. These findings highlight the substantial influence of the tryptophan transport system on antibiotic resistance in E. coli, which is crucial for developing strategies against antibiotic resistance in bacterial infections.
Miscellaneous
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