Faculty Opinions recommendation of TheN-Acetylmuramic Acid 6-Phosphate Phosphatase MupP Completes thePseudomonasPeptidoglycan Recycling Pathway Leading to Intrinsic Fosfomycin Resistance.

ABSTRACT Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from Escherichia coli and chromosomally encoded FosA from Klebsiella pneumoniae (FosAKP). The structure, molecular dynamics, catalytic activity, and fosfomycin resistance of FosA3 and FosAKP were also compared to those of FosA from Pseudomonas aeruginosa (FosAPA), for which prior crystal structures exist. E. coli TOP10 transformants expressing FosA3 and FosAKP conferred significantly greater fosfomycin resistance (MIC, >1,024 μg/ml) than those expressing FosAPA (MIC, 16 μg/ml), which could be explained in part by the higher catalytic efficiencies of the FosA3 and FosAKP enzymes. Interestingly, these differences in enzyme activity could not be attributed to structural differences at their active sites. Instead, molecular dynamics simulations and hydrogen-deuterium exchange experiments with FosAKP revealed dynamic interconnectivity between its active sites and a loop structure that extends from the active site of each monomer and traverses the dimer interface. This dimer interface loop is longer and more extended in FosAKP and FosA3 than in FosAPA, and kinetic analyses of FosAKP and FosAPA loop-swapped chimeric enzymes highlighted its importance in FosA activity. Collectively, these data yield novel insights into fosfomycin resistance that could be leveraged to develop new strategies to inhibit FosA and potentiate fosfomycin activity.

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Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1061 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1061-1066 ◽

Cited By ~ 41

Author(s):

T E McGraw ◽

K W Dunn ◽

F R Maxfield

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Surface ◽

Phorbol Ester ◽

Cho Cells ◽

Transferrin Receptor ◽

Phosphorylation Site ◽

Major Protein ◽

Kinase C ◽

Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

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Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in Stenotrophomonas maltophilia

mSystems ◽

10.1128/msystems.00077-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 5

Author(s):

Yi-Wei Huang ◽

Yu Wang ◽

Yun Lin ◽

Chin Lin ◽

Yi-Tsung Lin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutant Strain ◽

Stenotrophomonas Maltophilia ◽

Deletion Mutant ◽

Enterobacter Cloacae ◽

Underlying Mechanism ◽

Inducible Expression ◽

Citrobacter Freundii ◽

Recycling Pathway ◽

The Impact

ABSTRACT Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S. maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S. maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance. Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR–β-lactamase module–harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsI, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S. maltophilia harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampD I (the homologue of Escherichia coli ampD), nagZ, ampR, and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant ΔmrdA-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpDI, and was less related to the CreBC two-component system. Inactivation of mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamyl-meso-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain ΔmrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S. maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S. maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.

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Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2014.09.014 ◽

2014 ◽

Vol 1843 (12) ◽

pp. 2991-3003 ◽

Cited By ~ 3

Author(s):

María Gabriela Márquez ◽

Yamila Romina Brandán ◽

Edith del Valle Guaytima ◽

Carlos Humberto Paván ◽

Nicolás Octavio Favale ◽

...

Keyword(s):

Focal Adhesions ◽

Endocytic Recycling ◽

Recycling Pathway

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation

10.1101/2021.10.03.21264281 ◽

2021 ◽

Author(s):

Franziska Paul ◽

Calista Ng ◽

Shahriar Nafissi ◽

Yalda Nilipoor ◽

Ali Reza Tavasoli ◽

...

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Muscle Weakness ◽

Germline Mutations ◽

Lysosomal Degradation ◽

Mendelian Disorder ◽

Progressive Muscle Weakness ◽

Endosomal Recycling ◽

Early Endosomes ◽

Recycling Pathway

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively-acting germline alleles p.Arg180Gly and p.Gly183Arg which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate PI3P and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts exhibit accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

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