Sylvia A. McPherson

Genetically improved potatoes: protection from damage by Colorado potato beetles.

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

Human Immunodeficiency Virus Type-1 Reverse Transcriptase CONTRIBUTION OF MET-184 TO BINDING OF NUCLEOSIDE 5′-TRIPHOSPHATE

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-β-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine 5′-triphosphate (1-β-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-β-L-FTCTP was 19-fold greater than that of wt RT; the kp values of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5′-triphosphate.

Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance

Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlAE85K, GrlAS81F/E85K, GrlAE85A, GrlAS81F/E85A and GrlAS81F Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg2+ concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein–quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.

The Spore-Specific Alanine Racemase of Bacillus anthracis and Its Role in Suppressing Germination during Spore Development

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Deltaalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Deltaalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Deltaalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.

Immune responses induced by administration of encapsidated poliovirus replicons which express HIV-1 gag and envelope proteins

Several viruses have been exploited for the development of recombinant vaccine vectors in which to express foreign proteins. Recently, we have described a system utilizing the RNA virus, poliovirus. We have constructed poliovirus genomes in which regions of the capsid have been substituted with gene fragments of the HIV gag and env genes. A complementation system has been designed to encapsidate defective genomes by providing the capsid protein in trans from a recombinant vaccinia virus (VV-P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with these encapsidated replicons resulted in the expression of the recombinant protein as a fusion protein with the poliovirus capsid proteins VP4 and VP1. In this study, we have utilized encapsidated replicons which express the HIV-1-gag capsid protein (p24) as well as 1.5 kb of the HIV-1 env gene. Stocks of these encapsidated replicons were obtained by 20 serial passages in the presence of VV-P1. In addition, passage of the encapsidated replicons in the presence of poliovirus type 2 Lansing resulted in the encapsidation of the replicons by the capsid proteins provided by poliovirus. The administration of the type 2 Lansing/encapsidated replicons expressing HIV-1 gag in BALB/c mice by intramuscular, intrarectal, or intragastric routes resulted in the generation of antibodies in the serum and secretions against both poliovirus and HIV-1 gag. To prove that the replicons alone are immunogenic, we administered replicons expressing either HIV-1 gag or env to transgenic mice which expressed the receptor for poliovirus type 1. Immunization of these mice by the intramuscular route resulted in the generation of serum antibodies specific for poliovirus as well as for HIV-1 antigens. The results obtained led us to the conclusion that the replicons are immunogenic when given alone or in the presence of poliovirus. These results are important for the use of the poliovirus replicons as a recombinant vaccine vector.

Drug Interactions with Bacillus anthracis Topoisomerase IV: Biochemical Basis for Quinolone Action and Resistance

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.

Cytokine production in motor neurons by poliovirus replicon vector gene delivery.

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-α). We detected high-level expression of mTNF-α in the spinal cords of these animals at 8–12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-α revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.

Anthrose Biosynthetic Operon of Bacillus anthracis

The exosporium of Bacillus anthracis spores consists of a basal layer and an external hair-like nap. The nap is composed primarily of the glycoprotein BclA, which contains a collagen-like region with multiple copies of a pentasaccharide side chain. This oligosaccharide possesses an unusual terminal sugar called anthrose, followed by three rhamnose residues and a protein-bound N-acetylgalactosamine. Based on the structure of anthrose, we proposed an enzymatic pathway for its biosynthesis. Examination of the B. anthracis genome revealed six contiguous genes that could encode the predicted anthrose biosynthetic enzymes. These genes are transcribed in the same direction and appear to form two operons. We introduced mutations into the B. anthracis chromosome that either delete the promoter of the putative upstream, four-gene operon or delete selected genes in both putative operons. Spores produced by strains carrying mutations in the upstream operon completely lacked or contained much less anthrose, indicating that this operon is required for anthrose biosynthesis. In contrast, inactivation of the downstream, two-gene operon did not alter anthrose content. Additional experiments confirmed the organization of the anthrose operon and indicated that it is transcribed from a sigma(E)-specific promoter. Finally, we demonstrated that anthrose biosynthesis is not restricted to B. anthracis as previously suggested.

Overcoming target-mediated quinolone resistance in topoisomerase IV by introducing metal-ion-independent drug-enzyme interactions.

Quinolones, which target gyrase and topoisomerase IV, are the most widely prescribed antibacterials worldwide. Unfortunately, their use is threatened by the increasing prevalence of target-mediated drug resistance. Greater than 90% of mutations that confer quinolone resistance act by disrupting enzyme-drug interactions coordinated by a critical water-metal ion bridge. Quinazolinediones are quinolone-like drugs but lack the skeletal features necessary to support the bridge interaction. These compounds are of clinical interest, however, because they retain activity against the most common quinolone resistance mutations. We utilized a chemical biology approach to determine how quinazolinediones overcome quinolone resistance in Bacillus anthracis topoisomerase IV. Quinazolinediones that retain activity against quinolone-resistant topoisomerase IV do so primarily by establishing novel interactions through the C7 substituent, rather than the drug skeleton. Because some quinolones are highly active against human topoisomerase IIα, we also determined how clinically relevant quinolones discriminate between the bacterial and human enzymes. Clinically relevant quinolones display poor activity against topoisomerase IIα because the human enzyme cannot support drug interactions mediated by the water-metal ion bridge. However, the inclusion of substituents that allow quinazolinediones to overcome topoisomerase IV-mediated quinolone resistance can cause cross-reactivity against topoisomerase IIα. Therefore, a major challenge in designing drugs that overcome quinolone resistance lies in the ability to identify substituents that mediate strong interactions with the bacterial, but not the human, enzymes. On the basis of our understanding of quinolone-enzyme interactions, we have identified three compounds that display high activity against quinolone-resistant B. anthracis topoisomerase IV but low activity against human topoisomerase IIα.

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.