scholarly journals A novel FK-506-binding-like protein that lacks peptidyl-prolyl isomerase activity is involved in intracellular infection and in vivo virulence of Burkholderia pseudomallei

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2629-2638 ◽  
Author(s):  
Isobel H. Norville ◽  
Katrin Breitbach ◽  
Kristin Eske-Pogodda ◽  
Nicholas J. Harmer ◽  
Mitali Sarkar-Tyson ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Pathogenic Bacteria ◽  
Plaque Assay ◽  
Bacterial Pathogen ◽  
Virulence Determinants ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Fk 506 ◽  
Peptidyl Prolyl Isomerase ◽  
Intracellular Infection

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.

scholarly journals FK506-binding protein FklB is involved in biofilm formation through its peptidyl-prolyl isomerase activity

2020 ◽  
Author(s):  
Chrysoula Zografou ◽  
Maria Dimou ◽  
Panagiotis Katinakis
Keyword(s):  
Biofilm Formation ◽  
Negative Regulator ◽  
Cell Length ◽  
Wild Type ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Knock Out ◽  
E Coli ◽  
The Mean

AbstractFklB is a member of the FK506-binding proteins (FKBPs), a family that consists of five genes in Escherichia coli. Little is known about the physiological and functional role of FklB in bacterial movement. In the present study, FklB knock-out mutant ΔfklB presented an increased swarming and swimming motility and biofilm formation phenotype, suggesting that FklB is a negative regulator of these cellular processes. Complementation with Peptidyl-prolyl isomerase (PPIase)-deficient fklB gene (Y181A) revealed that the defects in biofilm formation were not restored by Y181A, indicating that PPIase activity of FklB is modulating biofilm formation in E. coli. The mean cell length of ΔfklB swarming cells was significantly smaller as compared to the wild-type BW25113. Furthermore, the mean cell length of swarming and swimming wild-type and ΔfklB cells overexpressing fklB or Y181A was considerably larger, suggesting that PPIase activity of FklB plays a role in cell elongation and/or cell division. A multi-copy suppression assay demonstrated that defects in motility and biofilm phenotype were compensated by overexpressing sets of PPIase-encoding genes. Taken together, our data represent the first report demonstrating the involvement of FklB in cellular functions of E. coli.

scholarly journals Cyclophilin A Peptidyl-Prolyl Isomerase Activity Promotes Zpr1 Nuclear Export

2002 ◽  
Vol 22 (20) ◽  
pp. 6993-7003 ◽  
Author(s):  
Husam Ansari ◽  
Giampaolo Greco ◽  
Jeremy Luban
Keyword(s):  
Nuclear Export ◽  
Biological Function ◽  
Zinc Finger Protein ◽  
Cyclophilin A ◽  
Kinetic Studies ◽  
Wild Type ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity

ABSTRACT The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Δ Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1α (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1 + cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Δ cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Δ cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1α, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity.

scholarly journals Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

2021 ◽  
Author(s):  
Kanghyun Lee ◽  
Aye C. Thwin ◽  
Eric Tse ◽  
Stephanie N. Gates ◽  
Daniel R. Southworth
Keyword(s):  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Recognition Element ◽  
Middle Domain ◽  
Ppiase Domain ◽  
Hsp90 Chaperone ◽  
Client Proteins ◽  
Maturation State ◽  
Tpr Domain

SummaryThe Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.

scholarly journals The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin.

1992 ◽  
Vol 176 (3) ◽  
pp. 751-760 ◽  
Author(s):  
F J Dumont ◽  
M J Staruch ◽  
S L Koprak ◽  
J J Siekierka ◽  
C S Lin ◽  
...  
Keyword(s):  
T Cell ◽  
Phosphatase Activity ◽  
T Cell Activation ◽  
Cell Activation ◽  
Toxic Effects ◽  
Ppiase Activity ◽  
Fk 506 ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity ◽  
Calcineurin Phosphatase

FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.

scholarly journals Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90

2015 ◽  
Vol 35 (5) ◽  
Author(s):  
Elizabeth A. Blackburn ◽  
Martin A. Wear ◽  
Vivian Landré ◽  
Vikram Narayan ◽  
Jia Ning ◽  
...  
Keyword(s):  
Heat Shock Protein 90 ◽  
Prolyl Isomerase ◽  
Temperature Dependent ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity ◽  
C Terminus ◽  
Hsp 90 ◽  
Cyclophilin 40 ◽  
Tpr Domain

Binding the C-terminus of heat shock protein 90 (Hsp 90) to the tetratricopeptide repeat (TPR) domain of cyclophilin 40 (Cyp40) allosterically changes the dynamics of the cyclophilin-active site and reduces peptidyl-prolyl isomerase (PPIase) activity.

scholarly journals Characterisation of SEQ0694 (PrsA/PrtM) of Streptococcus equi as a functional peptidyl-prolyl isomerase affecting multiple secreted protein substrates

2015 ◽  
Vol 11 (12) ◽  
pp. 3279-3286 ◽  
Author(s):  
Felicia Ikolo ◽  
Meng Zhang ◽  
Dean J. Harrington ◽  
Carl Robinson ◽  
Andrew S. Waller ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Direct Evidence ◽  
Secreted Protein ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Protein Substrates ◽  
Streptococcus Equi ◽  
Factor Secretion

We present the first direct evidence that a streptococcal parvulin lipoprotein has PPIase activity, which likely affects virulence factor secretion.

scholarly journals The Peptidyl-Prolyl Isomerase Activity of SlyD Is Not Required for Maturation of Escherichia coli Hydrogenase

2007 ◽  
Vol 189 (21) ◽  
pp. 7942-7944 ◽  
Author(s):  
Jie Wei Zhang ◽  
Michael R. Leach ◽  
Deborah B. Zamble
Keyword(s):  
Escherichia Coli ◽  
Metal Cluster ◽  
The Other ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity ◽  
Hydrogenase Enzymes

ABSTRACT Escherichia coli SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.

scholarly journals Dictyostelium discoideum as a Model System for Identification of Burkholderia pseudomallei Virulence Factors

2011 ◽  
Vol 79 (5) ◽  
pp. 2079-2088 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Maharsh K. Patel ◽  
Mark A. Schell
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Burkholderia Pseudomallei ◽  
Bacterial Pathogen ◽  
Model System ◽  
Virulence Determinants ◽  
Content Type ◽  
Throughput Model ◽  
Human Infections ◽  
High Throughput Assay

ABSTRACTBurkholderia pseudomalleiis an emerging bacterial pathogen and category B biothreat. Human infections withB. pseudomallei(called melioidosis) present as a range of manifestations, including acute septicemia and pneumonia. Although melioidosis can be fatal, little is known about the molecular basis ofB. pseudomalleipathogenicity, in part because of the lack of simple, genetically tractable eukaryotic models to facilitateen masseidentification of virulence determinants or explore host-pathogen interactions. Two assays, one high-throughput and one quantitative, were developed to monitor levels of resistance ofB. pseudomalleiand the closely related nearly avirulent speciesBurkholderia thailandensisto predation by the phagocytic amoebaDictyostelium discoideum. The quantitative assay showed that levels of resistance to, and survival within, amoeba by these bacteria and their known virulence mutants correlate well with their published levels of virulence in animals. Using the high-throughput assay, we screened a 1,500-memberB. thailandensistransposon mutant library and identified 13 genes involved in resistance to predation byD. discoideum. Orthologs of these genes were disrupted inB. pseudomallei, and nearly all mutants had similarly decreased resistance to predation byD. discoideum. For some mutants, decreased resistance also correlated with reduced survival in and cytotoxicity toward macrophages, as well as attenuated virulence in mice. These observations suggest that some factors required byB. pseudomalleifor resistance to environmental phagocytes also aid in resistance to phagocytic immune cells and contribute to disease in animals. Thus,D. discoideumprovides a novel, high-throughput model system for facilitating inquiry intoB. pseudomalleivirulence.

scholarly journals Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

2006 ◽  
Vol 74 (6) ◽  
pp. 3576-3586 ◽  
Author(s):  
Sabine Pilatz ◽  
Katrin Breitbach ◽  
Nadine Hein ◽  
Beate Fehlhaber ◽  
Jessika Schulze ◽  
...  
Keyword(s):  
Life Cycle ◽  
Burkholderia Pseudomallei ◽  
Plaque Assay ◽  
Bacterial Pathogen ◽  
Hypothetical Protein ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Endocytic Vesicles

ABSTRACT The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

Effect of FKBP65, a putative elastin chaperone, on the coacervation of tropoelastin in vitro

2010 ◽  
Vol 88 (6) ◽  
pp. 917-925 ◽  
Author(s):  
Kevin L.Y. Cheung ◽  
Matthew Bates ◽  
Vettai S. Ananthanarayanan
Keyword(s):  
Self Assembly ◽  
Secretory Pathway ◽  
Molar Ratio ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Maturation Stages ◽  
Turbidimetric Assay

FKBP65 is a protein of the endoplasmic reticulum that is relatively abundant in elastin-producing cells and is associated with tropoelastin in the secretory pathway. To test an earlier suggestion by Davis and co-workers that FKBP65 could act as an intracellular chaperone for elastin, we obtained recombinant FKBP65 (rFKBP65) by expressing it in E. coli and examined its effect on the coacervation characteristics of chicken aorta tropoelastin (TE) using an in vitro turbidimetric assay. Our results reveal that rFKBP65 markedly promotes the initiation of coacervation of TE without significantly affecting the temperature of onset of coacervation. This effect shows saturation at a 1:2 molar ratio of TE to rFKBP65. By contrast, FKBP12, a peptidyl prolyl isomerase, has a negligible effect on TE coacervation. Moreover, the effect of rFKBP65 on TE coacervation is unaffected by the addition of rapamycin, an inhibitor of peptidyl prolyl isomerase (PPIase) activity. These observations rule out the involvement of the PPIase activity of rFKBP65 in modulating the coacervation of TE. Additional experiments using a polypeptide model of TE showed that rFKBP65, while promoting coacervation, may retard the maturation of this model polypeptide into larger aggregates. Based on these results, we suggest that FKBP65 may act as an elastin chaperone in vivo by controlling both the coacervation and the maturation stages of its self-assembly into fibrils.

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