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Joseph T. Barbieri

Bio: Joseph T. Barbieri is an academic researcher from Medical College of Wisconsin. The author has contributed to research in topics: Pertussis toxin & Protein subunit. The author has an hindex of 55, co-authored 181 publications receiving 8898 citations. Previous affiliations of Joseph T. Barbieri include Children's Hospital of Wisconsin & Emory University.


Papers
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Journal ArticleDOI
TL;DR: Results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis, and abolishes adenylate cyclase activity in Pseudomonas aeruginosa.
Abstract: The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

460 citations

Journal ArticleDOI
27 Oct 1989-Science
TL;DR: A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussedis, B. parapertussis, and B. bronchiseptica to avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussi toxin.
Abstract: Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.

335 citations

Journal ArticleDOI
TL;DR: Arginine 146 of ExoS is identified to be essential for the stimulation of GTPase activity of Rho proteins and identifies ExoS as a GTP enzyme-activating protein for Rho GTPases.

296 citations

Journal ArticleDOI
TL;DR: The role of the actin cytoskeleton and the Rho GTPases in host–pathogen interactions are discussed, and the mode of actions of bacterial protein toxins that target these components are reviewed.
Abstract: Many bacterial cytotoxins act on eukaryotic cells by targeting the regulators that are involved in controlling the cytoskeleton or by directly modifying actin, with members of the Rho GTPase family being particularly important targets. The actin cytoskeleton, and especially the GTPase 'molecular switches' that are involved in its control, have crucial functions in innate and adaptive immunity, and have pivotal roles in the biology of infection. In this review, we briefly discuss the role of the actin cytoskeleton and the Rho GTPases in host-pathogen interactions, and review the mode of actions of bacterial protein toxins that target these components.

240 citations

Book ChapterDOI
TL;DR: Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.
Abstract: ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains. The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42. Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis. Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity. Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates. While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins. Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.

223 citations


Cited by
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Journal ArticleDOI
31 Aug 2000-Nature
TL;DR: It is proposed that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.
Abstract: Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

4,220 citations

Journal ArticleDOI
TL;DR: A comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp.
Abstract: Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

2,456 citations

Journal ArticleDOI
24 Dec 2004-Science

1,949 citations

Journal ArticleDOI
23 Jan 2004-Cell
TL;DR: This work will describe how the activity of Rho proteins is regulated downstream from growth factor receptors and cell adhesion molecules by guanine nucleotide exchange factors and GTPase activating proteins.

1,792 citations

Journal ArticleDOI
09 Nov 2001-Science
TL;DR: Underlying principles of guanosine nucleotide–binding proteins regulate a variety of processes, including sensual perception, protein synthesis, various transport processes, and cell growth and differentiation are defined.
Abstract: Guanine nucleotide-binding proteins regulate a variety of processes, including sensual perception, protein synthesis, various transport processes, and cell growth and differentiation. They act as molecular switches and timers that cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Recent structural studies show that the switch apparatus itself is a conserved fundamental module but that its regulators and effectors are quite diverse in their structures and modes of interaction. Here we will try to define some underlying principles.

1,754 citations