scholarly journals Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN

2017 ◽  
Vol 114 (25) ◽  
pp. 6557-6562 ◽  
Author(s):  
Nitin Kumar ◽  
Chih-Chia Su ◽  
Tsung-Han Chou ◽  
Abhijith Radhakrishnan ◽  
Jared A. Delmar ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Burkholderia Cepacia ◽  
Integral Membrane Protein ◽  
Necrotizing Pneumonia ◽  
Transmembrane Helices ◽  
Dimeric Molecule ◽  
Burkholderia Multivorans ◽  
Outer Leaflet ◽  
Major Pathogens ◽  
Multiple Antibiotics

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

Structure and function of the NHE1 isoform of the Na+/H+ exchanger

2002 ◽  
Vol 80 (5) ◽  
pp. 499-508 ◽  
Author(s):  
Emily Slepkov ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Amino Acid Residues ◽  
Transmembrane Helices ◽  
Cytoskeletal Organization ◽  
Transmembrane Segments ◽  
And Function ◽  
Extracellular Sodium

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cyto skel etal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.Key words: cation transport, intracellular pH, membrane proteins, Na+/H+ exchanger.

scholarly journals Crystal structure of a human plasma membrane phospholipid flippase

2020 ◽  
Vol 295 (30) ◽  
pp. 10180-10194 ◽  
Author(s):  
Hanayo Nakanishi ◽  
Katsumasa Irie ◽  
Katsumori Segawa ◽  
Kazuya Hasegawa ◽  
Yoshinori Fujiyoshi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Plasma ◽  
Binding Sites ◽  
The Other ◽  
Asymmetric Distribution ◽  
Transmembrane Helices ◽  
Outer Leaflet ◽  
Extracellular Side ◽  
Phospholipid Flippase ◽  
Plasma Membrane Phospholipid

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C–CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p–Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1–CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

scholarly journals Comparative Analysis of Plant and Animal Models for Characterization of Burkholderia cepacia Virulence

2003 ◽  
Vol 71 (9) ◽  
pp. 5306-5313 ◽  
Author(s):  
Steve P. Bernier ◽  
Laura Silo-Suh ◽  
Donald E. Woods ◽  
Dennis E. Ohman ◽  
Pamela A. Sokol
Keyword(s):  
Comparative Analysis ◽  
Animal Models ◽  
Burkholderia Cepacia ◽  
Parent Strain ◽  
Lung Infection ◽  
Infection Model ◽  
Burkholderia Multivorans ◽  
Lung Infections

ABSTRACT A simple alfalfa model was developed as an alternative infection model for virulence studies of the Burkholderia cepacia complex. Symptoms of disease were observed in wounded alfalfa seedlings within 7 days following inoculation of 101 to 105 CFU of most strains of the B. cepacia complex. Strains from seven genomovars of the B. cepacia complex were tested for virulence in the alfalfa model, and the degree of virulence was generally similar in strains belonging to the same genomovar. Strains of Burkholderia multivorans and some strains of Burkholderia stabilis did not cause symptoms of disease in alfalfa seedlings. Representative strains were also tested for virulence using the rat agar bead model. Most of the strains tested were able to establish chronic lung infections; B. stabilis strains were the exception. Most of the strains that were virulent in the alfalfa infection model were also virulent in the lung infection model. The B. cepacia genomovar III mutants K56pvdA::tp and K56-H15 were significantly less virulent in the alfalfa infection model than their parent strain. Therefore, this alfalfa infection model may be a useful tool for assessing virulence of strains of the B. cepacia complex and identifying new virulence-associated genes.

scholarly journals Mucoid morphotype variation of Burkholderia multivorans during chronic cystic fibrosis lung infection is correlated with changes in metabolism, motility, biofilm formation and virulence

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3124-3137 ◽  
Author(s):  
Inês N. Silva ◽  
Ana S. Ferreira ◽  
Jörg D. Becker ◽  
James E. A. Zlosnik ◽  
David P. Speert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Phenotypic Variation ◽  
Biofilm Formation ◽  
Burkholderia Cepacia ◽  
Galleria Mellonella ◽  
Acute Infection ◽  
Lung Infection ◽  
Infection Model ◽  
Burkholderia Multivorans ◽  
Expression Of Genes

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens infecting hosts such as cystic fibrosis (CF) patients. Long-term Bcc infection of CF patients’ airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates displaying different morphotypes from a chronically infected CF patient to evaluate trait development during lung infection. Expression profiling of mucoid D2095 and non-mucoid D2214 isolates revealed decreased expression of genes encoding products related to virulence-associated traits and metabolism in D2214. Furthermore, D2214 showed no exopolysaccharide production, lower motility and chemotaxis, and more biofilm formation, particularly under microaerophilic conditions, than the clonal mucoid isolate D2095. When Galleria mellonella was used as acute infection model, D2214 at a cell number of approximately 7×106 c.f.u. caused a higher survival rate than D2095, although 6 days post-infection most of the larvae were dead. Infection with the same number of cells by mucoid D2095 caused larval death by day 4. The decreased expression of genes involved in carbon and nitrogen metabolism may reflect lower metabolic needs of D2214 caused by lack of exopolysaccharide, but also by the attenuation of pathways not required for survival. As a result, D2214 showed higher survival than D2095 in minimal medium for 28 days under aerobic conditions. Overall, adaptation during Bcc chronic lung infections gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their capacity for survival in this opportunistic human niche.

scholarly journals A Genome-Wide Association Analysis Reveals a Role for Recombination in the Evolution of Antimicrobial Resistance in Burkholderia multivorans

2018 ◽  
Author(s):  
Julio Diaz Caballero ◽  
Shawn T. Clark ◽  
Pauline W. Wang ◽  
Sylva L. Donaldson ◽  
Bryan Coburn ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Genome Wide Association Study ◽  
Genome Wide Association ◽  
Mortality And Morbidity ◽  
Burkholderia Multivorans ◽  
Post Transplant ◽  
Genome Wide ◽  
A Genome ◽  
Genomic Study ◽  
Gwas Approach

AbstractCystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomic study of 111 B. multivorans sputum isolates from a single CF patient through three stages of infection including the initial incident infection, deep sampling of a one-year period of chronic infection, and deep sampling of a post-transplant recolonization. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found that the incident isolate was more susceptible to agents from three antimicrobial classes (β-lactams, aminoglycosides, quinolones), while the chronic isolates diversified into distinct genetic lineages with reduced antimicrobial susceptibility to the same agents. The post-transplant reinfection isolates displayed genetic and phenotypic signatures that were distinct from sputum isolates from all CF lung specimens. There were numerous examples of parallel pathoadaptation, in which individual loci, or even the same codon, were independently mutated multiple times. This set of loci was enriched for functions associated with virulence and resistance. Our GWAS approach identified one variant in the ampD locus (which was independently mutated four times in our dataset) associated with resistance to β-lactams, and two non-synonymous polymorphisms associated with resistance to both aminoglycosides and quinolones, affecting an araC family transcriptional regulator, which was independently mutated three times, and an outer member porin, which was independently mutated twice. We also performed recombination analysis and identified a minimum of 14 recombination events. Parallel pathoadaptive loci and polymorphisms associated with β-lactam resistance were over-represented in these recombinogenic regions. This study illustrates the power of deep, longitudinal sampling coupled with evolutionary and lineage-corrected GWAS analyses to reveal how pathogens adapt to their hosts.

scholarly journals Crystal structures of Bax and Bak Reveal Molecular Events Initiating Apoptosis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1490-C1490
Author(s):  
Peter Czabotar ◽  
Jason Brouwer ◽  
Dana Westphal ◽  
Geoff Thompson ◽  
Peter Colman
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Molecular Mechanisms ◽  
Structural Studies ◽  
Core Domain ◽  
Hydrophobic Groove ◽  
Outer Leaflet ◽  
Molecular Events ◽  
Surface Crystal

A key event in apoptosis is the conversion of Bax or Bak from inert monomers into cytotoxic mitochondrial membrane perforating oligomers. Certain BH3-only relatives can initiate this step through direct interactions, yet the means by which conformational changes are invoked, the nature of the conformational changes themselves, the mechanism by which they insert into membranes and the process by which they perforate these barriers has largely remained a mystery. Our recent structural studies provided the first insights into this process for Bax [1]. We found that BH3 domains activate Bax by binding to a hydrophobic groove on its surface. Crystal structures of these complexes revealed an unexpected conformational change involving dissociation of a previously unrecognized "core" domain from a "latch' domain. A further structure of the freed Bax "core" domains revealed that these form dimers that possess a surface of aromatic residues which we hypothesis engage the outer leaflet of the mitochondrial membrane and induce curvature. We have now extended our studies to include structures of Bax bound to alternative BH3-only proteins providing new insights into key interactions occurring at this interface. Additionally, we have solved structures of activated Bak and of the freed Bak "core" domain dimers. These results further our understanding of the molecular mechanisms by which these highly dynamic proteins engage the mitochondrial membrane and thus control the life/death switch in cells.

scholarly journals Comparison of Clinical Manifestations, Antimicrobial Susceptibility Patterns, and Mutations of Fluoroquinolone Target Genes between Elizabethkingia meningoseptica and Elizabethkingia anophelis Isolated in Taiwan

2018 ◽  
Vol 7 (12) ◽  
pp. 538 ◽  
Author(s):  
Jiun-Nong Lin ◽  
Chung-Hsu Lai ◽  
Chih-Hui Yang ◽  
Yi-Han Huang
Keyword(s):  
Ribosomal Rna ◽  
Target Gene ◽  
Target Genes ◽  
Clinical Manifestations ◽  
Gene Mutations ◽  
Clinical Microbiology Laboratory ◽  
Elizabethkingia Meningoseptica ◽  
Major Pathogens ◽  
Susceptibility Patterns ◽  
Multiple Antibiotics

Elizabethkingia meningoseptica and Elizabethkingia anophelis are two major pathogens in the genus Elizabethkingia. Studies have revealed that Elizabethkingia anophelis is frequently misidentified as E. meningoseptica. Therefore, our aim was to explore the clinical and molecular differences between these two species. The database of a clinical microbiology laboratory in a university-affiliated hospital of Taiwan was searched to identify patients with Elizabethkingia infections between January 2005 and June 2018. Species were reidentified using 16S ribosomal RNA gene sequencing. Twenty E. meningoseptica and 72 E. anophelis samples were collected from consecutive patients. E. meningoseptica was significantly more frequently isolated from the cerebrospinal fluid than was E. anophelis. The most susceptible antibiotic for all Elizabethkingia isolates was minocycline (91.3%), followed by levofloxacin (52.2%), tigecycline (23.9%), and piperacillin tazobactam (23.9%). Compared with E. anophelis, E. meningoseptica was significantly less susceptible to piperacillin tazobactam, minocycline, and levofloxacin. Regarding nonsynonymous substitutions in the quinolone-resistance determining regions of DNA gyrase, six sites were recognized in E. meningoseptica and one site was recognized in E. anophelis. E. meningoseptica had a significantly higher rate of fluoroquinolone target gene mutations than did E. anophelis. Because of less susceptibility to multiple antibiotics than E. anophelis, empirical antimicrobial therapy of E. meningoseptica should be more rigorous.

scholarly journals Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 393-399 ◽  
Author(s):  
Nigel Unwin
Keyword(s):  
Membrane Lipids ◽  
Electrical Response ◽  
Postsynaptic Membrane ◽  
Chemical Synapse ◽  
Transmembrane Helices ◽  
Time Resolved ◽  
Presynaptic Nerve Terminal ◽  
Local Rigidity ◽  
Outer Leaflet ◽  
Lipid Distribution

Rapid communication at the chemical synapse depends on the action of ion channels residing in the postsynaptic membrane. The channels open transiently upon the binding of a neurotransmitter released from the presynaptic nerve terminal, eliciting an electrical response. Membrane lipids also play a vital but poorly understood role in this process of synaptic transmission. The present study examines the lipid distribution around nicotinic acetylcholine (ACh) receptors in tubular vesicles made from postsynaptic membranes of theTorpedoray, taking advantage of the recent advances in cryo-EM. A segregated distribution of lipid molecules is found in the outer leaflet of the bilayer. Apparent cholesterol-rich patches are located in specific annular regions next to the transmembrane helices and also in a more extended `microdomain' between the apposed δ subunits of neighbouring receptors. The particular lipid distribution can be interpreted straightforwardly in relation to the gating movements revealed by an earlier time-resolved cryo-EM study, in which the membranes were exposed briefly to ACh. The results suggest that in addition to stabilizing the protein, cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel.

scholarly journals Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates

2014 ◽  
Vol 58 (9) ◽  
pp. 5211-5219 ◽  
Author(s):  
Rebecca J. Malott ◽  
Chia-Hung Wu ◽  
Tracy D. Lee ◽  
Trevor J. Hird ◽  
Nathan F. Dalleska ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Polymyxin B ◽  
Inhalation Therapy ◽  
Pulmonary Infections ◽  
Binding Assays ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Burkholderia Multivorans ◽  
Liquid Chromatography Tandem Mass ◽  
Antimicrobial Synergy

ABSTRACTBurkholderia cepaciacomplex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc speciesB. multivoranswere highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made byB. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinicalB. multivoransisolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.

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