scholarly journals The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus

2021 ◽  
Vol 17 (3) ◽  
pp. e1009338
Author(s):  
Junho Cho ◽  
Stephen K. Costa ◽  
Rachel M. Wierzbicki ◽  
William F. C. Rigby ◽  
Ambrose L. Cheung
Keyword(s):  
Positive Charge ◽  
Efflux Pump ◽  
Survival Strategies ◽  
Extracellular Loop ◽  
Major Question ◽  
Defense Proteins ◽  
Bacterial Membranes ◽  
Transmembrane Segments ◽  
Charge Interaction ◽  
Sense And Respond

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

scholarly journals Structural and Functional Study of the Phenicol-Specific Efflux Pump FloR Belonging to the Major Facilitator Superfamily

2005 ◽  
Vol 49 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
Martine Braibant ◽  
Jacqueline Chevalier ◽  
Elisabeth Chaslus-Dancla ◽  
Jean-Marie Pagès ◽  
Axel Cloeckaert
Keyword(s):  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Efflux Transporters ◽  
Chloramphenicol Resistance ◽  
Active Efflux ◽  
Transmembrane Segments ◽  
Efflux Mechanism ◽  
Major Facilitator

ABSTRACT The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids

2012 ◽  
Vol 442 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Wendy González ◽  
Janin Riedelsberger ◽  
Samuel E. Morales-Navarro ◽  
Julio Caballero ◽  
Jans H. Alzate-Morales ◽  
...  
Keyword(s):  
Ph Sensor ◽  
Structural Models ◽  
Voltage Sensor ◽  
Activation Mechanism ◽  
Potassium Ions ◽  
Acid Activation ◽  
Extracellular Loop ◽  
K Uptake ◽  
Transmembrane Segments ◽  
Ph Dependency

The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2–2.5 pH units) is perceived by voltage-gated inward potassium channels (Kin) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal Kin channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal Kin channel KAT1, mutations in the unique histidine exposed to the solvent (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation–π interaction. Instead, we show that Glu240 placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu240, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

scholarly journals A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Jack R. Davison ◽  
Katheryn M. Lohith ◽  
Xiaoning Wang ◽  
Kostyantyn Bobyk ◽  
Sivakoteswara R. Mandadapu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Bacterial Strains ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Membranes ◽  
Development Of Resistance ◽  
Antibiotic Compounds ◽  
Blasticidin S

ABSTRACT The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

scholarly journals Photodynamic Inactivation of Bacteria with Porphyrin Derivatives: Effect of Charge, Lipophilicity, ROS Generation, and Cellular Uptake on Their Biological Activity In Vitro

2020 ◽  
Vol 21 (22) ◽  
pp. 8716
Author(s):  
Adam Sułek ◽  
Barbara Pucelik ◽  
Marcin Kobielusz ◽  
Agata Barzowska ◽  
Janusz M. Dąbrowski
Keyword(s):  
Cellular Uptake ◽  
Efflux Pump ◽  
Theoretical Calculations ◽  
Resistant Bacteria ◽  
Ros Generation ◽  
Photodynamic Inactivation ◽  
Efflux Pump Inhibitor ◽  
E Coli ◽  
Charge Interaction

Resistance of microorganisms to antibiotics has led to research on various therapeutic strategies with different mechanisms of action, including photodynamic inactivation (PDI). In this work, we evaluated a cationic, neutral, and anionic meso-tetraphenylporphyrin derivative’s ability to inactivate the Gram-negative and Gram-positive bacteria in a planktonic suspension under blue light irradiation. The spectroscopic, physicochemical, redox properties, as well as reactive oxygen species (ROS) generation capacity by a set of photosensitizers varying in lipophilicity were investigated. The theoretical calculations were performed to explain the distribution of the molecular charges in the evaluated compounds. Moreover, logP partition coefficients, cellular uptake, and phototoxicity of the photosensitizers towards bacteria were determined. The role of a specific microbial efflux pump inhibitor, verapamil hydrochloride, in PDI was also studied. The results showed that E. coli exhibited higher resistance to PDI than S. aureus (3–5 logs) with low light doses (1–10 J/cm2). In turn, the prolongation of irradiation (up to 100 J/cm2) remarkably improved the inactivation of pathogens (up to 7 logs) and revealed the importance of photosensitizer photostability. The PDI potentiation occurs after the addition of KI (more than 3 logs extra killing). Verapamil increased the uptake of photosensitizers (especially in E. coli) due to efflux pump inhibition. This effect suggests that PDI is mediated by ROS, the electrostatic charge interaction, and the efflux of photosensitizers (PSs) regulated by multidrug-resistance (MDR) systems. Thus, MDR inhibition combined with PDI gives opportunities to treat more resistant bacteria.

Abstract 3499: Extracellular Loop (Domain I, S5-S6) of α1D L-type Ca Channel is an Antigenic Site in Autoimmune Associated Congenital Heart Block

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Eddy Karnabi ◽  
Yongxia Qu ◽  
Raj Wadgaonkar ◽  
Salvatore Mancarella ◽  
Yunkun Yue ◽  
...  
Keyword(s):  
Heart Block ◽  
Congenital Heart ◽  
Antigenic Site ◽  
Congenital Heart Block ◽  
Ca Channel ◽  
Extracellular Loop ◽  
Channel Protein ◽  
Transmembrane Segments ◽  
Electrophysiological Characterization ◽  
Domain I

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have established that anti-SSA/Ro -SSB/La autoantibodies inhibit L-type α 1D Ca current, I Ca-L and cross-react with the α 1D Ca channel protein. This study aims at identifying the possible binding sites on α 1D protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four α 1D Ca channel protein domains I–IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera from 118 mothers with CHB children and 28 control healthy sera were used in this study. Seventeen of 118 (14.4%) maternal CHB sera reacted with the extracellular loop of Domain I S5-S6 region (E1). In contrast, 2 of 28 (7%) healthy control sera reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D. reading of the positive sera establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera demonstrated inhibition (44.1%) of the α 1D I Ca-L expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 extracellular loop fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel α 1D subunit as a target for autoantibodies from mothers with CHB children. This novel finding provides insights into the development of therapeutic peptides that could bind to the pathogenic antibodies.

scholarly journals Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis

2005 ◽  
Vol 390 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Joanne C. Cheung ◽  
Jing Li ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Physiological Role ◽  
Mutant Form ◽  
Extracellular Loop ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Transmembrane Segments

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1–4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (ΔA400–A408) in TM1. These results suggest that the TM2–3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1–2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2–3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2–3 suggest that these segments form a re-entrant loop in human AE1.

Understanding membrane-active antimicrobial peptides

2017 ◽  
Vol 50 ◽  
Author(s):  
Huey W. Huang ◽  
Nicholas E. Charron
Keyword(s):  
Antimicrobial Peptides ◽  
Soft Matter ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Bacterial Resistance ◽  
Antimicrobial Agents ◽  
Molecular Structures ◽  
Membrane Domain ◽  
Defense Proteins ◽  
Bacterial Membranes

AbstractBacterial membranes represent an attractive target for the design of new antibiotics to combat widespread bacterial resistance to traditional inhibitor-based antibiotics. Understanding how antimicrobial peptides (AMPs) and other membrane-active agents attack membranes could facilitate the design of new, effective antimicrobials. AMPs, which are small, gene-encoded host defense proteins, offer a promising basis for the study of membrane-active antimicrobial agents. These peptides are cationic and amphipathic, spontaneously binding to bacterial membranes and inducing transmembrane permeability to small molecules. Yet there are often confusions surrounding the details of the molecular mechanisms of AMPs. Following the doctrine of structure–function relationship, AMPs are often viewed as the molecular scaffolding of pores in membranes. Instead we believe that the full mechanism of AMPs is understandable if we consider the interactions of AMPs with the whole membrane domain, where interactions induce structural transformations of the entire membrane, rather than forming localized molecular structures. We believe that it is necessary to consider the entire soft matter peptide-membrane system as it evolves through several distinct states. Accordingly, we have developed experimental techniques to investigate the state and structure of the membrane as a function of the bound peptide to lipid ratio, exactly as AMPs in solution progressively bind to the membrane and induce structural changes to the entire system. The results from these studies suggest that global interactions of AMPs with the membrane domain are of fundamental importance to understanding the antimicrobial mechanisms of AMPs.

scholarly journals The Current Knowledge of Invertebrate Aquaporin Water Channels with Particular Emphasis on Insect AQPs

2010 ◽  
Vol 2 (2) ◽  
pp. 91-104 ◽  
Author(s):  
Ewa Tomkowiak ◽  
Joanna Romana Pienkowska
Keyword(s):  
Current Knowledge ◽  
Water Channels ◽  
Survival Strategies ◽  
Feeding Strategies ◽  
Transmembrane Segments ◽  
Single Organism ◽  
Living Organisms ◽  
Almost All ◽  
And Function ◽  
Hostile Environments

SummaryAquaporins (AQPs) or water channels are some of the most ubiquitous integral membrane proteins, and are present in all living organisms. Their presence in the lipid bilayer of cell membranes considerably increases their permeability to water and, in some cases, to other small solutes. All AQPs, identified thus far, share the same structure, comprising of six transmembrane segments and two conserved regions forming the pore. Depending on the transported solutes, AQPs can be divided into two classes: ‘classical’ aquaporins (permeable only to water) and aquaglyceroporins (permeable also to glycerol and/or other solutes). Many subtypes of AQPs coexist in a single organism. Localization of particular subtypes of AQPs is tissue-specific. AQPs have been well characterized in almost all vertebrate classes. However, little is known about their counterparts in invertebrates. Most of the water channels characterized in invertebrates are found in insects. Therefore, the knowledge of aquaporins in invertebrates is generally limited to the information concerning water channels in this class of organism. Insects are characterized by an astonishing variety of physiological adaptations, notable in their feeding strategies or survival strategies in hostile environments. An example of such, is feeding on blood, or tolerating extreme cold or drought. It is likely that many of these adaptation patterns emerged due to the expression and regulation of particular aquaporins. Here we review the current state of knowledge of invertebrate AQPs (of insects and nematodes) and compare their structure and function with mammalian water channels

scholarly journals GraS sensory activity in S. epidermidis is modulated by the “guard loop” of VraG and the ATPase activity of VraF

2021 ◽  
Author(s):  
Stephen K. Costa ◽  
Junho Cho ◽  
Ambrose L. Cheung
Keyword(s):  
Information Processing ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Treatment Strategies ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Extracellular Loop ◽  
Sensory Activity ◽  
Sense And Respond ◽  
Considerable Morbidity

Antimicrobial peptides (AMPs) are one of the key immune responses that can eliminate pathogenic bacteria through membrane perturbation. As a successful skin commensal, Staphylococcus epidermidis can sense and respond to AMPs through the GraXRS two-component system and an efflux system comprising the VraG permease and VraF ATPase. GraS is a membrane sensor known to function in AMP resistance through a negatively charged, 9-residue extracellular loop which is predicted to be linear without any secondary structure. An important question is how GraS can impart effective sensing of AMPs through such a small unstructured sequence. In this study, we verified the role of graS and vraG in AMP sensing in S. epidermidis as demonstrated by the failure of the Δ graS or Δ vraG mutants to sense. Deletion of the extracellular loop of VraG did not affect sensing but reduced survival against polymyxin B. Importantly, a specific region within the extracellular loop, termed the guard loop (GL), has inhibitory activity since sensing of polymyxin B was enhanced in the ΔGL mutant, indicating GL may act as a gate keeper to sensing. Bacterial two-hybrid analysis demonstrated that the extracellular regions of GraS and VraG interact, but interaction appears dispensable to sensing activity. Mutation of the extracellular loop of VraG, GL and the active site of VraF suggested that an active detoxification function of VraG is necessary for AMP resistance. Altogether, we provide evidence for a unique sensory scheme that relies on the function of a permease to impart effective information processing. Importance Staphylococcus epidermidis has become an important opportunistic pathogen responsible for nosocomial and device-related infections that account for considerable morbidity worldwide. A thorough understanding of the mechanisms that enable S. epidermidis to colonize human skin successfully is essential for the development of alternative treatment strategies and prophylaxis. Here, we demonstrate the importance of an antimicrobial peptide response system in a clinically relevant S. epidermidis strain. Furthermore, we provide evidence for a unique sensory scheme that would rely on the detoxification function of a permease to effect information processing.

scholarly journals Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

2002 ◽  
Vol 46 (7) ◽  
pp. 2169-2173 ◽  
Author(s):  
Julio Ramos Aires ◽  
Jean-Claude Pechère ◽  
Christian Van Delden ◽  
Thilo Köhler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Proper Function ◽  
Amino Acid Residues ◽  
Pump System ◽  
Transmembrane Segments ◽  
Partial Activity

ABSTRACT At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.

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