The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

ABSTRACT Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10−6. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10−11). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.

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An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

Canadian Journal of Microbiology ◽

10.1139/m78-070 ◽

1978 ◽

Vol 24 (4) ◽

pp. 427-432 ◽

Cited By ~ 1

Author(s):

M. L. Marceau-Day ◽

D. F Day ◽

J. M. Ingram

Keyword(s):

Pseudomonas Aeruginosa ◽

Alkaline Phosphatase ◽

Core Region ◽

Significant Activity ◽

Wild Type ◽

Glycerol Phosphate ◽

The Core ◽

Nitrophenyl Phosphate ◽

Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

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Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier

Biochemical Journal ◽

10.1042/bj3320119 ◽

1998 ◽

Vol 332 (1) ◽

pp. 119-125 ◽

Cited By ~ 19

Author(s):

Steven VAYRO ◽

Bryan LO ◽

Mel SILVERMAN

Keyword(s):

Glucose Transporter ◽

Immunogold Labelling ◽

Transport Activity ◽

Wild Type ◽

Functional Changes ◽

Functional Studies ◽

Kd Values ◽

Function Relationship ◽

Relationship Of ◽

Glucose Carrier

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent α-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92±0.08 nmol/min per mg of protein and Km was 0.98±0.13 mM; for wild-type SGLT1, Vmax was 1.98±0.47 nmol/min per mg of protein and Km was 0.36±0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55±0.18 and 1.69±0.57 pmol/mg of protein respectively); Kd values were 0.46±0.15 and 0.51±0.11 µM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.

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High-Level Pacidamycin Resistance in Pseudomonas aeruginosa Is Mediated by an Opp Oligopeptide Permease Encoded by theopp-fabIOperon

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01198-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5565-5571 ◽

Cited By ~ 19

Author(s):

Anita Mistry ◽

Mark S. Warren ◽

John K. Cusick ◽

RoxAnn R. Karkhoff-Schweizer ◽

Olga Lomovskaya ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Frequency ◽

Cross Resistance ◽

Peptide Antibiotics ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Level Resistance ◽

Resistance Mutants

ABSTRACTPacidamycins (or uridyl peptide antibiotics) possess selectivein vivoactivity againstPseudomonas aeruginosa. An important limitation for the therapeutic use of pacidamycins withP. aeruginosais the high frequency (10−6to 10−7) at which resistant mutants emerge. To elucidate the mechanism(s) of this resistance, pacidamycin-resistantP. aeruginosamutants were isolated. Two types of mutants were obtained. Type 1, or high-level resistance mutants with a pacidamycin MIC of 512 μg/ml, were more abundant, with a frequency of ∼2 × 10−6, and did not show cross-resistance with other antibiotics. Type 2, low-level resistance mutants, were isolated with a frequency of ∼10−8and had a pacidamycin MIC of 64 μg/ml (the MIC for the wild-type strain was 4 to 16 μg/ml). These mutants were cross-resistant to levofloxacin, tetracycline, and erythromycin and were shown to overexpress either the MexAB-OprM or MexCD-OprJ multidrug resistance efflux pumps. High-level resistant mutants were isolated by transposon mutagenesis and one insertion was localized tooppB, one of two periplasmic binding protein components of an oligopeptide transport system which is encoded by theopp-fabIoperon. The Opp system is required for uptake of pacidamycin across the inner membrane, since variousopp, but notfabI, mutants were resistant to high levels of pacidamycin. Both of the two putative Opp periplasmic binding proteins, OppA and OppB, were required for pacidamycin uptake. Although both impaired uptake into and efflux from the cell can cause pacidamycin resistance inP. aeruginosa, our data suggest that impaired uptake is the primary reason for the high-frequency and high-level pacidamycin resistance.

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New multiscale characterisation methodology for effective determination of isolation-structure-function relationship of extracellular vesicles

10.1101/2021.02.09.430523 ◽

2021 ◽

Author(s):

Thanh Huyen Phan ◽

Shiva Kamini Divakarla ◽

Jia Hao Yeo ◽

Qingyu Lei ◽

Priyanka Tharkar ◽

...

Keyword(s):

Extracellular Vesicles ◽

Biological Effects ◽

Dry Mass ◽

Experimental Methodology ◽

Wide Range ◽

Isolation Methods ◽

Function Relationship ◽

Relationship Of

AbstractExtracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

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Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

International Journal of Molecular Sciences ◽

10.3390/ijms22169085 ◽

2021 ◽

Vol 22 (16) ◽

pp. 9085

Author(s):

Rafał Hołubowicz ◽

Andrzej Ożyhar ◽

Piotr Dobryszycki

Keyword(s):

Wild Type ◽

Positional Vertigo ◽

Natural Variants ◽

Self Association ◽

Function Relationship ◽

The Stability ◽

And Function ◽

Relationship Of ◽

Wild Type Form ◽

Paroxysmal Positional Vertigo

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

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MUC1 ectodomain is a flagellin-targeting decoy receptor and biomarker operative during Pseudomonas aeruginosa lung infection

Scientific Reports ◽

10.1038/s41598-021-02242-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Avelino C. Verceles ◽

Pavan Bhat ◽

Zain Nagaria ◽

Destiny Martin ◽

Harsh Patel ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bronchoalveolar Lavage Fluid ◽

Wild Type Strain ◽

Lavage Fluid ◽

Airway Epithelial ◽

Wild Type ◽

Epithelial Surface ◽

Decoy Receptor ◽

Neu1 Sialidase

AbstractWe previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.

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Insight into the Structure-Function Relationship of the Bone Lamellar Unit through Finite Element Modelling Based on High-Frequency SAM Data

IFMBE Proceedings - World Congress on Medical Physics and Biomedical Engineering, September 7 - 12, 2009, Munich, Germany ◽

10.1007/978-3-642-03882-2_596 ◽

2009 ◽

pp. 2246-2249

Author(s):

K. Raum ◽

Q. Grimal ◽

A. Gerisch

Keyword(s):

Finite Element ◽

Structure Function ◽

High Frequency ◽

Finite Element Modelling ◽

Element Modelling ◽

Structure Function Relationship ◽

Function Relationship ◽

Relationship Of ◽

Insight Into

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Listeria monocytogenes 6-Phosphogluconolactonase Mutants Induce Increased Activation of a Host Cytosolic Surveillance Pathway

Infection and Immunity ◽

10.1128/iai.01511-08 ◽

2009 ◽

Vol 77 (7) ◽

pp. 3014-3022 ◽

Cited By ~ 10

Author(s):

Gregory T. Crimmins ◽

Michael W. Schelle ◽

Anat A. Herskovits ◽

Peggy P. Ni ◽

Benjamin C. Kline ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Pentose Phosphate Pathway ◽

Deletion Mutant ◽

Minimal Medium ◽

Pentose Phosphate ◽

Wild Type ◽

Transposon Insertion ◽

Excess Glucose ◽

Insertion Mutants

ABSTRACT Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-β). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-β expression. One mutant from this screen induced elevated levels of IFN-β and harbored a Tn917 insertion upstream of lmo0558. This study identified lmo0558 as the 6-phosphogluconolactonase gene (pgl), which encodes the second enzyme in the pentose phosphate pathway. pgl mutant L. monocytogenes accumulated and secreted large amounts of gluconate, likely derived from labile 6-phosphogluconolactone, the substrate of Pgl. The pgl deletion mutant had decreased growth in glucose-limiting minimal medium but grew normally when excess glucose was added. Microarray analysis revealed that the pgl deletion mutant had increased expression of several β-glucosidases, consistent with known inhibition of β-glucosidases by 6-phosphogluconolactone. While growth in macrophages was indistinguishable from that of wild-type bacteria, pgl mutant L. monocytogenes exhibited a 15- to 30-fold defect in growth in vivo. In addition, L. monocytogenes harboring an in-frame deletion of pgl was more sensitive to oxidative stress. This study identified L. monocytogenes pgl and provided the first link between the bacterial pentose phosphate pathway and activation of host IFN-β expression.

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