The Journal of Membrane Biology

Silva, P.; Kumar, Ayush

doi: 10.1007/s00232-017-9985-7pmid: 28866739

Acinetobacter baumannii is an important bacterial pathogen whose resistance to antibiotics is a growing concern worldwide. Among a wide array of resistance mechanisms displayed by A. baumannii, energy-dependent efflux of antibiotics by proteins belonging the resistance-nodulation-division family serves as an important one. AdeABC pump has been shown to be active in various clinical isolates. Regulation of this pump is controlled by the AdeRS two-component system. In this study, we show that the AdeRS system, in addition to modulating A. baumannii’s antibiotic susceptibility, also plays a role in biofilm formation as well as surface-associated motility. We also show that AdeRS deletion mutant is more sensitive to saline stress. In particular, motility of A. baumannii ATCC17978 on agar surface is severely hampered at higher salt concentrations when AdeRS system is absent. Therefore, our study shows that AdeRS could be part of the A. baumannii adaptation strategy to salinity stress.

Slipski, Carmine; Zhanel, George; Bay, Denice

doi: 10.1007/s00232-017-9992-8pmid: 29063140

Bacterial resistance to biocides used as antiseptics, dyes, and disinfectants is a growing concern in food preparation, agricultural, consumer manufacturing, and health care industries, particularly among Gram-negative Enterobacteriaceae, some of the most common community and healthcare-acquired bacterial pathogens. Biocide resistance is frequently associated with antimicrobial cross-resistance leading to reduced activity and efficacy of both antimicrobials and antiseptics. Multidrug resistant efflux pumps represent an important biocide resistance mechanism in Enterobacteriaceae. An assortment of structurally diverse efflux pumps frequently co-exist in these species and confer both unique and overlapping biocide and antimicrobial selectivity. TolC-dependent multicomponent systems that span both the plasma and outer membranes have been shown to confer clinically significant resistance to most antimicrobials including many biocides, however, a growing number of single component TolC-independent multidrug resistant efflux pumps are specifically associated with biocide resistance: small multidrug resistance (SMR), major facilitator superfamily (MFS), multidrug and toxin extruder (MATE), cation diffusion facilitator (CDF), and proteobacterial antimicrobial compound efflux (PACE) families. These efflux systems are a growing concern as they are rapidly spread between members of Enterobacteriaceae on conjugative plasmids and mobile genetic elements, emphasizing their importance to antimicrobial resistance. In this review, we will summarize the known biocide substrates of these efflux pumps, compare their structural relatedness, Enterobacteriaceae distribution, and significance. Knowledge gaps will be highlighted in an effort to unravel the role that these apparent “lone wolves” of the efflux-mediated resistome may offer.

Bhagirath, Anjali; Somayajula, Deepti; Li, Yanqi; Duan, Kangmin

doi: 10.1007/s00232-017-9994-6pmid: 29063141

Pseudomonas aeruginosa is an ubiquitous organism which is able to infect and colonize many types of hosts including humans. Colonization of P. aeruginosa in chronic infections leads to the formation of biofilms, which are difficult to eradicate. P. aeruginosa is capable of regulating its virulence factors in response to external environment triggers and its signaling mechanism involves two-component regulatory systems and small molecules such as bis-(3′–5′)-cyclic dimeric guanosine monophosphate. PA1611-RetS-GacS/A-RsmA/Y/Z is a key regulatory pathway in P. aeruginosa that controls several virulence factors and biofilm formation. We have previously identified a conserved cytoplasmic membrane protein cmpX (PA1775), as a regulator for PA1611 expression. In this study, we demonstrate that cmpX regulates virulence, and controls biofilm formation in P. aeruginosa as well as provide evidence showing that cmpX affects Gac/Rsm pathway, possibly by modulating intra-cellular c-di-GMP levels. A cmpX knockout showed significantly decreased promoter activity of exoS (PA1362) and increased activity of small RNA, RsmY. As compared to the wild-type PAO1, cmpX mutant had elevated intracellular c-di-GMP level as measured indirectly by cdrA (PA4625) activity, as well as increased expression of wspR (PA3702), a c-di-GMP synthase. The transcription of the major outer membrane porin gene oprF (PA1777), and sigma factor sigX (PA1776) was also significantly decreased in the cmpX mutant. Biolog phenotype microarray experiments further indicated that the cmpX knockout mutant had increased sensitivity to membrane detergents and antibiotics such as lauryl sulfobetaine, tobramycin, and vancomycin. These results point to a significant role of cmpX in P. aeruginosa virulence and colonization.

Mima, Takehiko; Gotoh, Kazuyoshi; Yamamoto, Yumiko; Maeda, Keiko; Shirakawa, Taku; Matsui, Shunsuke; Murata, Yumi; Koide, Takaki; Tokumitsu, Hiroshi; Matsushita, Osamu

doi: 10.1007/s00232-017-9991-9pmid: 28993850

Cardona, Silvia; Choy, Matthew; Hogan, Andrew

doi: 10.1007/s00232-017-9995-5pmid: 29098331

Novel therapies are urgently needed to alleviate the current crisis of multiple drug-resistant infections. The bacterial signal transduction mechanisms, known as two-component systems (TCSs), are ideal targets of novel inhibitory molecules. Highly restricted to the bacterial world, TCSs control a diverse set of cellular functions, namely virulence, response to cell envelope stress, and drug efflux. Impaired regulation of any of these aspects could affect the susceptibility of bacterial pathogens to antibiotics, which highlights the potential of TCS as targets of antibiotic adjuvant therapies. Moreover, new high-density transposon mutagenesis methods have revealed the existence of TCSs required for growth and viability. Experimental validation of gene essentiality and phenotypic characterization of knockdown mutants indicate that essential TCSs regulate aspects of the cell envelope homeostasis in coordination with cell division. In this review, we describe essential TCSs, and their potentials as targets for antibacterial molecules. We also discuss methods for the identification of small molecules that inhibit TCSs and possible reasons why antibacterial molecules targeting essential TCSs have not yet reached clinical trials.

Gao, Lang; Guo, Zisheng; Wang, Yue; Wang, Yulu; Wang, Kerong; Li, Bo; Shen, Lixin

doi: 10.1007/s00232-017-9979-5pmid: 28975384

Iron is essential for all organisms. Bacteria have devolved sophisticated systems to maintain intracellular iron homeostasis. FpvCDEF(PA2407-2410) has been reported as an ABC transporter involved in pyoverdine-Fe uptake which does not affect growth under iron-limiting condition, when it is deleted in PAO1. In this study, we proved that fpvCDEF and fpvWXYZ(PA2403-2406) constituted an ABC transporter complex containing two operons: fpvWXYZCDE and fpvF. The operon fpvWXYZCDE was regulated by iron negatively and the single gene operon fpvF was constitutively expressed. Inactivation of any one of the components, fpvW, fpvC, fpvD, fpvE, and fpvF, led to increased expression of fpvWXYZCDE suggesting that each component of fpvWXYZCDEF could be involved in iron uptake. The ABC transporter complex encoded by fpvWXYZCDEF plays important roles in growth, oxidative stress resistance, and virulence, since the deletion of fpvWXYZCDEF resulted in defective growth, increased sensitivity to H2O2, and decreased virulence compared with PAO1(ΔfpvCDEF) and the wild type PAO1 under iron-limiting condition.

Bown, Luke; Srivastava, Santosh; Piercey, Brandon; McIsaac, Clarissa; Tahlan, Kapil

doi: 10.1007/s00232-017-9997-3pmid: 29098330

Infections caused by mycobacteria are difficult to treat due to their inherent physiology, cellular structure, and intracellular lifestyle. Mycobacterium tuberculosis is a pathogen of global concern as it causes tuberculosis (TB) in humans, which requires 6–9 months of chemotherapy. The situation is further exacerbated in the case of infections caused by drug-resistant strains, which necessitate the prolonged use of agents associated with increased host toxicities. Great effort has been invested into the development of new agents for the treatment of drug-resistant infections, in addition to novel strategies to reduce treatment time. Energy production using oxidative phosphorylation is essential for the survival of M. tuberculosis, even under conditions of dormancy. Many compounds have been recently discovered that inhibit different aspects of energy metabolism in mycobacteria, some of which have been approved for human use or are currently undergoing development. The most successful examples include inhibitors of QcrB and AtpE, which are part of the cytochrome bc 1 complex and FoF1-ATP synthase, respectively. In addition, many of the discovered inhibitors are active against drug-resistant strains of M. tuberculosis, inhibit nonreplicating cells, and also show potential for the treatment of other mycobacterial infections. In the current review, we focus on the discovery of mycobacterial QcrB and AtpE inhibitors, their modes of action, and the associated mechanisms of resistance observed to date.

Rajeckaitė, Violeta; Jakštys, Baltramiejus; Rafanavičius, Arnas; Maciulevičius, Martynas; Jakutavičiūtė, Milda; Šatkauskas, Saulius

doi: 10.1007/s00232-017-0005-8pmid: 29143077

The aim of this study was to investigate the dependence of calcein extraction and cell viability on the parameters of pulsed electric field (PEF). Two different approaches concerning PEF parameters were investigated: (1) extraction efficiency and cell viability dependence on pulse number, exploiting 1200 V/cm 100 µs duration high voltage (HV) electric pulses and (2) extraction efficiency and cell viability dependence on the pulses with different duration (44–400 µs) and electric field strength (600–1800 V/cm) that result in the same amount of electric field energy delivered to Chinese hamster ovary cells. Extraction efficiency was evaluated as a percentage ratio of calcein fluorescence intensity prior and after PEF treatment. Cell viability was evaluated using PI test and cell clonogenic assay. Moreover, calcein release dynamics from cells after 600 V/cm 400 µs, 1200 V/cm 100 µs, and 1800 V/cm 44 µs was evaluated. Our results show that HV pulses induce instant calcein extraction due to reversible electroporation; however, subsequent calcein leakage over time was only observed when 9 HV pulses of 1800 V/cm 44 µs were used. The increased number of pulses resulted in more efficient total calcein extraction. With the same total energy delivered via electric pulses, the increase of calcein extraction efficiency was more dependent on pulse strength rather than pulse duration. The highest calcein extraction efficiency (84.5 ± 7.4%) was obtained using 9 electric field pulses of 1800 V/cm, 44 µs at 1 Hz. Furthermore, the extraction efficiency can be significantly enhanced if external mechanical stress (pipetting) is applied to cells. Cell viability was determined to be dependent on different PEF exposure parameters. It varied from 96.8 ± 4.8 to 31.2 ± 8.9%, implying the possibility to adjust PEF parameter combinations to maintain high cell viability.

Lloyd, Kenneth; Ojelabi, Ogooluwa; Simon, Andrew; Zutter, Julie; Carruthers, Anthony

doi: 10.1007/s00232-017-0006-7pmid: 29209831

A growing body of evidence demonstrates that GLUT1-mediated erythrocyte sugar transport is more complex than widely assumed and that contemporary interpretations of emergent GLUT1 structural data are incompatible with the available transport and biochemical data. This study examines the kinetic basis of one such incompatibility—transport allostery—and in doing so suggests how the results of studies examining GLUT1 structure and function may be reconciled. Three types of allostery are observed in GLUT1-mediated, human erythrocyte sugar transport: (1) exofacial cis-allostery in which low concentrations of extracellular inhibitors stimulate sugar uptake while high concentrations inhibit transport; (2) endofacial cis-allostery in which low concentrations of intracellular inhibitors enhance cytochalasin B binding to GLUT1 while high concentrations inhibit binding, and (3) trans-allostery in which low concentrations of ligands acting at one cell surface stimulate ligand binding at or sugar transport from the other surface while high concentrations inhibit these processes. We consider several kinetic models to account for these phenomena. Our results show that an inhibitor can only stimulate then inhibit sugar uptake if (1) the transporter binds two or more molecules of inhibitor; (2) high-affinity binding to the first site stimulates transport, and (3) low-affinity binding to the second site inhibits transport. Reviewing the available structural, transport, and ligand binding data, we propose that exofacial cis-allostery results from cross-talk between multiple, co-existent ligand interaction sites present in the exofacial cavity of each GLUT1 protein, whereas trans-allostery and endofacial cis-allostery require ligand-induced subunit–subunit interactions.

Alobeedallah, Hadeel; Cornell, Bruce; Coster, Hans

doi: 10.1007/s00232-017-0007-6pmid: 29188314

Cholesterol plays an important role in regulating the properties of phospholipid bilayers and many mechanisms have been proposed to explain why cholesterol is so ubiquitous within biological membranes of animals. Here we present the results of studies on the effect of cholesterol on the electrical/dielectric properties of lipid membranes tethered to a solid substrate. These tethered bilayer lipid membranes tBLM were formed on a commercially available chemically modified gold substrate. These lipid bilayers are very robust. Very high-resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data allowed the dielectric substructure of the lipid bilayers to be determined. The results showed that when cholesterol was present in the tethered membranes at a concentration of 10% (mol/mol); the thickness of the tBLMs increased and the membrane conductance decreased. However, when cholesterol was present in the tethered membrane at more than 30% (mol/mol) the effect of cholesterol was dramatically different; the membranes then became thinner and possessed a much larger electrical conductance. The EIS allowed a distinction to be made between a hydrophobic region in the center of the bilayer and another hydrophobic region further out towards the polar head region, in addition to the polar head region itself. Cholesterol was found to have the largest effect on the inner, hydrophobic region, although the outer hydrophobic region was also affected.