scholarly journals Characterization of the Filamentous Hemagglutinin-Like Protein FhaS in Bordetella bronchiseptica

2005 ◽  
Vol 73 (8) ◽  
pp. 4960-4971 ◽  
Author(s):  
Steven M. Julio ◽  
Peggy A. Cotter
Keyword(s):  
Proteolytic Processing ◽  
Bordetella Bronchiseptica ◽  
Wild Type ◽  
Filamentous Hemagglutinin ◽  
Epithelial Cell Lines ◽  
Bacterial Fitness ◽  
Processing Event ◽  
Additional Level

ABSTRACT Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced Bordetella bronchiseptica genome revealed the presence of a gene, fhaS, that is nearly identical to fhaB, the FHA structural gene. We show that although fhaS expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of B. bronchiseptica to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a ΔfhaS strain was out-competed by wild-type B. bronchiseptica, indicating that fhaS is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the Bordetella infectious cycle. A survey of various Bordetella strains revealed two distinct fhaS alleles that segregate according to pathogen host range and that B. parapertussis hu most likely acquired its fhaS allele from B. pertussis horizontally, suggesting fhaS may contribute to host-species specificity.

scholarly journals Filamentous Hemagglutinin of Bordetella bronchiseptica Is Required for Efficient Establishment of Tracheal Colonization

1998 ◽  
Vol 66 (12) ◽  
pp. 5921-5929 ◽  
Author(s):  
Peggy A. Cotter ◽  
Ming H. Yuk ◽  
Seema Mattoo ◽  
Brian J. Akerley ◽  
Jeff Boschwitz ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Ectopic Expression ◽  
Etiologic Agent ◽  
Bordetella Bronchiseptica ◽  
Host Animal ◽  
Filamentous Hemagglutinin ◽  
Unanesthetized Animals ◽  
Necessary And Sufficient

ABSTRACT Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. ForBordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.

scholarly journals Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments

2000 ◽  
Vol 11 (1) ◽  
pp. 171-182 ◽  
Author(s):  
William T. Brigance ◽  
Charles Barlowe ◽  
Todd R. Graham
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Proteolytic Processing ◽  
Golgi Compartment ◽  
Specific Antisera ◽  
Sensitive Factor ◽  
Processing Event ◽  
Golgi Network

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.

scholarly journals Pseudomonas aeruginosa Gene Products PilT and PilU Are Required for Cytotoxicity In Vitro and Virulence in a Mouse Model of Acute Pneumonia

1999 ◽  
Vol 67 (7) ◽  
pp. 3625-3630 ◽  
Author(s):  
James C. Comolli ◽  
Alan R. Hauser ◽  
Leslie Waite ◽  
Cynthia B. Whitchurch ◽  
John S. Mattick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Mouse Model ◽  
Cell Lines ◽  
Twitching Motility ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
Acute Pneumonia

ABSTRACT Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. ThepilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilUmutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

scholarly journals New Insight into Filamentous Hemagglutinin Secretion Reveals a Role for Full-Length FhaB in Bordetella Virulence

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Jeffrey A. Melvin ◽  
Erich V. Scheller ◽  
Christopher R. Noël ◽  
Peggy A. Cotter
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Primary Component ◽  
Full Length ◽  
Wild Type ◽  
Content Type ◽  
Filamentous Hemagglutinin ◽  
Mutant Strains

ABSTRACTBordetellafilamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, contributes to virulence, but how it functions mechanistically is unclear. FHA is first synthesized as an ~370-kDa preproprotein called FhaB. Removal of an N-terminal signal peptide and a large C-terminal prodomain (PD) during secretion results in “mature” ~250-kDa FHA, which has been assumed to be the biologically active form of the protein. Deletion of two C-terminal subdomains of FhaB did not affect production of functional FHA, and the mutant strains were indistinguishable from wild-type bacteria for their ability to adhere to the lower respiratory tract and to suppress inflammation in the lungs of mice. However, the mutant strains, which produced altered FhaB molecules, were eliminated from the lower respiratory tract much faster than wild-typeB. bronchiseptica, suggesting a defect in resistance to early immune-mediated clearance. Our results revealed, unexpectedly, that full-length FhaB plays a critical role inB. bronchisepticapersistence in the lower respiratory tract.IMPORTANCETheBordetellafilamentous hemagglutinin (FHA) is a primary component of the acellular pertussis vaccine and an important virulence factor. FHA is initially produced as a large protein that is processed during secretion to the bacterial surface. As with most processed proteins, the mature form of FHA has been assumed to be the functional form of the protein. However, our results indicate that the full-length form plays an essential role in virulencein vivo. Furthermore, we have found that FHA contains intramolecular regulators of processing and that this control of processing is integral to its virulence activities. This report highlights the advantage of studying protein maturation and function simultaneously, as a role for the full-length form of FHA was evident only fromin vivoinfection studies and not fromin vitrostudies on the production or maturation of FHA or even fromin vitrovirulence-associated activity assays.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

2021 ◽  
pp. 1-24
Author(s):  
Juho-Matti Renko ◽  
Arun Kumar Mahato ◽  
Tanel Visnapuu ◽  
Konsta Valkonen ◽  
Mati Karelson ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Dopamine Neurons ◽  
Dopamine Depletion ◽  
Wild Type ◽  
Disease Modifying Therapy ◽  
Immortalized Cells ◽  
Midbrain Dopamine ◽  
6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 320
Author(s):  
Thaís Pereira da Silva ◽  
Fernando Jacomini de Castro ◽  
Larissa Vuitika ◽  
Nayanne Louise Costacurta Polli ◽  
Bruno César Antunes ◽  
...  
Keyword(s):  
Structural Changes ◽  
Capillary Permeability ◽  
Structural Data ◽  
Histopathological Analysis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Antigenic Properties ◽  
Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

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