scholarly journals Functional Characterization of a Redundant Plasmodium TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test

2008 ◽  
Vol 7 (6) ◽  
pp. 1062-1070 ◽  
Author(s):  
Kirsten Heiss ◽  
Hui Nie ◽  
Sumit Kumar ◽  
Thomas M. Daly ◽  
Lawrence W. Bergman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cell Entry ◽  
Host Cells ◽  
Complementation Test ◽  
Type I ◽  
Cycle Progression ◽  
Targeted Gene Disruption ◽  
Specific Host ◽  
Host Cell Entry

ABSTRACT Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

scholarly journals BspA and Pmp proteins ofTrichomonas vaginalismediate adherence to host cells

2018 ◽  
Author(s):  
Maria R. Handrich ◽  
Sriram G. Garg ◽  
Ewen W. Sommerville ◽  
Robert P. Hirt ◽  
Sven B. Gould
Keyword(s):  
Trichomonas Vaginalis ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Host Cells ◽  
Protein Families ◽  
Sexually Transmitted ◽  
Trace Back ◽  
Oral Pathogens ◽  
Adhesion Performance

AbstractTrichomonas vaginalisis one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individualT. vaginaliscandidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing proteins are specifically expanded inT. vaginalisin comparison to other Trichomonads, and that (ii) individual proteins of both families have the ability to increase adhesion performance in a non-virulentT. vaginalisstrain andTetratrichomonas gallinarum, a parasite usually known to infect birds but not humans. Our results initiate the functional characterization of these two broadly distributed protein families, whose origin we trace back to the origin of Trichomonads themselves.

scholarly journals Characteristics of a Series of Three Bacteriophages Infecting Salmonella enterica Strains

2020 ◽  
Vol 21 (17) ◽  
pp. 6152 ◽  
Author(s):  
Katarzyna Kosznik-Kwaśnicka ◽  
Karolina Ciemińska ◽  
Michał Grabski ◽  
Łukasz Grabowski ◽  
Marcin Górniak ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Phage Therapy ◽  
Functional Characterization ◽  
Bacterial Biofilm ◽  
Host Cells ◽  
Detailed Characterization ◽  
Food Protection ◽  
Adsorption Rates ◽  
Potential Use

Molecular and functional characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, infecting various Salmonella enterica serovars and strains is presented. All these phages were able to develop lytically while not forming prophages. Moreover, they were able to survive at pH 3. The phages revealed different host ranges within serovars and strains of S. enterica, different adsorption rates on host cells, and different lytic growth kinetics at various temperatures (in the range of 25 to 42 °C). They efficiently reduced the number of cells in the bacterial biofilm and decreased the biofilm mass. Whole genome sequences of these phages have been determined and analyzed, including their phylogenetic relationships. In conclusion, we have demonstrated detailed characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, which reveal favorable features in light of their potential use in phage therapy of humans and animals, as well as for food protection purposes.

Evaluation of an enzyme immunoassay kit for estrogen receptor measurement--report II.

1988 ◽  
Vol 34 (10) ◽  
pp. 2053-2057 ◽  
Author(s):  
S Raam ◽  
D M Vrabel
Keyword(s):  
Enzyme Immunoassay ◽  
Binding Sites ◽  
Solid Phase ◽  
Functional Characterization ◽  
Quantitative Agreement ◽  
Type I ◽  
Type Ii ◽  
Immunoreactive Protein ◽  
Estrogen Binding

Abstract We present evidence to show that monoclonal antibodies to estrogen receptors (ER) in solid phase recognize the secondary estrogen binding sites with moderate to low affinity for estradiol (E2). An excellent quantitative agreement was found in five cytosols between the ER values obtained by the enzyme immunoassay (ER-EIA) and the amount of secondary estrogen binding sites measured by the assay involving dextran-coated charcoal (Clin Chem 1986;32:1496). The immunoreactive protein recognized by the antibody-coated beads, when allowed to react with ER(+) cytosols, is shown to bind [3H]estradiol only when the ligand concentration exceeds 8 nmol/L. Further biochemical and functional characterization of the immunoreactive protein is required to establish similarities/dissimilarities between this protein, high-affinity Type I ER sites, and the secondary sites such as Type II sites.

scholarly journals Shigella applies molecular mimicry to subvert vinculin and invade host cells

2006 ◽  
Vol 175 (3) ◽  
pp. 465-475 ◽  
Author(s):  
Tina Izard ◽  
Guy Tran Van Nhieu ◽  
Philippe R.J. Bois
Keyword(s):  
Host Cell ◽  
Binding Sites ◽  
Type Iii Secretion ◽  
Molecular Mimicry ◽  
Shigella Flexneri ◽  
Cell Entry ◽  
Host Cells ◽  
General Mechanism ◽  
Host Cell Entry ◽  
Cell Spread

Shigella flexneri, the causative agent of bacillary dysentery, injects invasin proteins through a type III secretion apparatus upon contacting the host cell, which triggers pathogen internalization. The invasin IpaA is essential for S. flexneri pathogenesis and binds to the cytoskeletal protein vinculin to facilitate host cell entry. We report that IpaA harbors two vinculin-binding sites (VBSs) within its C-terminal domain that bind to and activate vinculin in a mutually exclusive fashion. Only the highest affinity C-terminal IpaA VBS is necessary for efficient entry and cell–cell spread of S. flexneri, whereas the lower affinity VBS appears to contribute to vinculin recruitment at entry foci of the pathogen. Finally, the crystal structures of vinculin in complex with the VBSs of IpaA reveal the mechanism by which IpaA subverts vinculin's functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin–vinculin interaction to activate vinculin. Mimicry of vinculin's interactions may therefore be a general mechanism applied by pathogens to infect the host cell.

Molecular cloning and functional characterization of two duplicated two-cysteine containing type I interferon genes in rock bream Oplegnathus fasciatus

2012 ◽  
Vol 33 (4) ◽  
pp. 886-898 ◽  
Author(s):  
Qiang Wan ◽  
W.D. Niroshana Wicramaarachchi ◽  
Ilson Whang ◽  
Bong-Soo Lim ◽  
Myung-Joo Oh ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Type I Interferon ◽  
Functional Characterization ◽  
Type I ◽  
Oplegnathus Fasciatus ◽  
Rock Bream

scholarly journals Cysteine biosynthesis is a determinant of Brucella ovis stress survival and fitness in the intracellular niche

2020 ◽  
Author(s):  
Lydia M. Varesio ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Stationary Phase ◽  
Cell Entry ◽  
Host Cells ◽  
Cysteine Biosynthesis ◽  
Brucella Ovis ◽  
Cysteine Synthesis ◽  
Host Cell Entry ◽  
Phase Entry ◽  
Male Genital

AbstractBrucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in stationary phase cultures. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine. We provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis downstream of CysE. Deleting either cysE or both cysK1 and cysK2 leads to premature entry into stationary phase and reduced culture yield. These phenotypes are rescued by addition of cysteine or glutathione to the medium. We further show that deletion of cysE results in sensitivity to exogenous hydrogen peroxide. Finally, we demonstrate that B. ovis ΔcysE has no defect in host cell entry but is attenuated in macrophage-like cells and in ovine testis epithelial cells at one- and two-days post infection. Our study uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrated that cysteine anabolism is an important determinant of stationary phase entry in vitro and fitness in the intracellular niche.

scholarly journals Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived—From the Same Induced Pluripotent Stem Cell Source

2020 ◽  
Vol 7 (4) ◽  
pp. 133
Author(s):  
Xiufang Guo ◽  
Agnes Badu-Mensah ◽  
Michael C. Thomas ◽  
Christopher W. McAleer ◽  
James J. Hickman
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Neuromuscular Junctions ◽  
Functional Characterization ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Type I ◽  
Induced Pluripotent

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.

scholarly journals Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer

2015 ◽  
Vol 290 (34) ◽  
pp. 21007-21018 ◽  
Author(s):  
Thomas Courivaud ◽  
Nathalie Ferrand ◽  
Abdelouahid Elkhattouti ◽  
Santosh Kumar ◽  
Laurence Levy ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cell Function ◽  
Regulatory Mechanism ◽  
Human Cancer ◽  
Functional Characterization ◽  
Type I ◽  
Inhibitory Effects ◽  
Cancer Pathogenesis ◽  
Ubiquitin Ligase Activity

Although E3 ubiquitin ligases are deemed to play key roles in normal cell function and homeostasis, whether their alterations contribute to cancer pathogenesis remains unclear. In this study, we sought to investigate potential mechanisms that govern WWP1/Tiul1 (WWP1) ubiquitin ligase activity, focusing on its ability to trigger degradation of TGFβ type I receptor (TβRI) in conjunction with Smad7. Our data reveal that the WWP1 protein is very stable at steady states because its autopolyubiquitination activity is silenced due to an intra-interaction between the C2 and/or WW and Hect domains that favors WWP1 monoubiquitination at the expense of its polyubiquitination or polyubiquitination of TβRI. Upon binding of WWP1 to Smad7, this functional interplay is disabled, switching its monoubiquitination activity toward a polyubiquitination activity, thereby driving its own degradation and that of TβRI as well. Intriguingly, a WWP1 point mutation found in human prostate cancer disrupts this regulatory mechanism by relieving the inhibitory effects of C2 and WW on Hect and thereby causing WWP1 hyperactivation. That cancer-driven alteration of WWP1 culminates in excessive TβRI degradation and attenuated TGFβ cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1.

scholarly journals Characterization of the oriI andoriII Origins of Replication in Phage-Plasmid P4

1999 ◽  
Vol 73 (9) ◽  
pp. 7308-7316 ◽  
Author(s):  
Arianna Tocchetti ◽  
Gloria Galimberti ◽  
Gianni Dehò ◽  
Daniela Ghisotti
Keyword(s):  
Complementation Test ◽  
Plasmid Replication ◽  
Deletion Mapping ◽  
Type I ◽  
Rich Region ◽  
Origins Of Replication ◽  
Essential Components ◽  
Relative Arrangement

ABSTRACT In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the α andcnr genes and the ori1 and crrsites; the oriII replicon is composed of the α gene, with the internal ori2 site, and the crr region. The P4 α protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the α protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ forori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the α protein did not bind ori2. Thus, the α protein appears to act differently at the two origins of replication.

scholarly journals The Genome of Salmonella enterica Serovar Gallinarum: Distinct Insertions/Deletions and Rare Rearrangements

2005 ◽  
Vol 187 (14) ◽  
pp. 4720-4727 ◽  
Author(s):  
Kai-Yu Wu ◽  
Gui-Rong Liu ◽  
Wei-Qiao Liu ◽  
Austin Q. Wang ◽  
Sen Zhan ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Physical Map ◽  
Gene Clusters ◽  
Type I ◽  
Genomic Evolution ◽  
Specific Host ◽  
Serovar Typhimurium ◽  
Genomic Regions ◽  
Close Relatedness

ABSTRACT Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:—:—) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.

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