Biomedical subjects

David J McGee

Publications and source records attributed to David J McGee.

11 recordsLinked to original sources

PubMed2006

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site.

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.

Animals↗

PubMed2006

Differential expression and transcriptional analysis of the alpha-2,3-sialyltransferase gene in pathogenic Neisseria spp.

Alpha-2,3-sialyltransferase (Lst) is expressed on the outer membrane of Neisseria gonorrhoeae and Neisseria meningitidis and sialylates surface lipooligosaccharide (LOS), facilitating resistance to complement-mediated killing. The enzyme is constitutively expressed from a single gene (lst) and does not undergo antigenic or phase variation. We observed that Triton X-100 extracts of N. gonorrhoeae strain F62 contain about fivefold more sialyltransferase (Stase) activity than extracts of N. meningitidis strain MC58 [symbol: see text]3 a serogroup B acapsulate mutant. We confirmed and expanded upon this observation by showing that extracts of 16 random N. gonorrhoeae isolates contain various amounts of Stase activity, but, on average, 2.2-fold-more Stase activity than extracts of 16 N. meningitidis clinical isolates, representing several serogroups and nongroupable strains. Northern and real-time reverse transcription-PCR analysis of lst transcript levels in N. gonorrhoeae and N. meningitidis revealed that N. gonorrhoeae strains express more lst transcript than N. meningitidis strains. Although transcript levels correlate with average Stase activity observed in the two species, there was not a direct correlation between lst transcript levels and Stase activity among individual isolates of each species. Comparison of lst upstream (5'lst) regions of N. gonorrhoeae and N. meningitidis revealed striking sequence differences characteristic of the two pathogens. N. gonorrhoeae 5'lst regions possess 30-bp and 13-bp elements present as single elements or as tandem repeats that exist only as single elements in the 5'lst regions of N. meningitidis isolates. In addition, the 5'lst regions of N. meningitidis strains have 105-bp transposon-like Correia elements which are absent in N. gonorrhoeae. Chromosomal N. gonorrhoeae 5'lst::lacZ translational fusions expressed 4.75 +/- 0.09-fold (n = 4) higher beta-galactosidase (beta-gal) activity than N. meningitidis 5'lst::lacZ fusions in a host-independent manner, indicating differential expression is governed at least in part by sequence variations in the 5'lst regions. Reporter fusion assays and promoter-mapping analysis revealed that N. gonorrhoeae and N. meningitidis use different promoters with different strengths to transcribe lst. In N. gonorrhoeae, a strong sigma 70 promoter 80 bp upstream of the translational start site is used to transcribe lst, whereas this promoter is inactive in N. meningitidis. In N. meningitidis, a weak sigma 70 promoter at the 3' terminus of a 105-bp Correia repeat-enclosed element 99 bp upstream of the translational start site is used to transcribe lst. We conclude that differential Stase expression between N. gonorrhoeae and N. meningitidis is due at least in part to differential lst gene transcription.

Base Sequence↗

PubMed2006

Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined media.

The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined, while almost nothing is known about nutritional requirements of other Helicobacter spp. Although previous data showed that H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined, the specific components responsible for growth were not entirely understood. Here we describe the optimization of amino acids, metals, and sodium chloride for H. pylori. Iron, zinc, and magnesium were critical for growth; copper was not required. Optimization of sodium chloride was further beneficial. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter spp. revealed that all except H. felis grew in serum-free, unsupplemented F-12. All Helicobacter spp. were resistant to at least six antimicrobial agents when cultured in the presence of serum. However, in the absence of serum, H. pylori, H. mustelae, and H. muridarum became sensitive to polymyxin B and/or trimethoprim. Much of the data were obtained using a convenient ATP assay to quantify growth. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter spp. are not as fastidious as previously thought. The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells.

Adenosine Triphosphate↗

PubMed2005

Helicobacter pylori thioredoxin is an arginase chaperone and guardian against oxidative and nitrosative stresses.

The gastric human pathogen Helicobacter pylori faces formidable challenges in the stomach including reactive oxygen and nitrogen intermediates. Here we demonstrate that arginase activity, which inhibits host nitric oxide production, is post-translationally stimulated by H. pylori thioredoxin (Trx) 1 but not the homologous Trx2. Trx1 has chaperone activity that renatures urea- or heat-denatured arginase back to the catalytically active state. Most reactive oxygen and nitrogen intermediates inhibit arginase activity; this damage is reversed by Trx1, but not Trx2. Trx1 and arginase equip H. pylori with a "renox guardian" to overcome abundant nitrosative and oxidative stresses encountered during the persistence of the bacterium in the hostile gastric environment.

Arginase↗

PubMed2005

Colonization and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemotaxis mutants.

Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.

Animals↗

PubMed2004

Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta).

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3zeta-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.

Apoptosis↗

PubMed2004

Star-shaped azo-based dipolar chromophores: design, synthesis, matrix compatibility, and electro-optic activity.

Three new azo-benzene-based push-pull chromophores with dendritic architecture were synthesized as active materials for electro-optic applications. These chromophores were synthesized in six or seven synthetic steps with an overall yield of around 80% per step and high purity. UV-vis spectroscopy showed significant influence of the transient dipole moment on the observed r(33) values. The chromophores were stable to photochemical oxidation in ambient light and air. The electrical poling conditions were optimized for each chromophore as the T(g) of the composite material varied significantly. The highest EO coefficient achieved was 22-25 pm/V at 1550 nm wavelength. STEM analysis of the blends enabled the correlation of the activity of these large chromophores with the blend morphology. An amorphous polycarbonate host effectively disperses the chromophores in 2-20 nm aggregates in the active materials. However, macrophase separation into 200-500 nm aggregates was observed in a methacrylate host matrix.

Journal Article↗

PubMed2004

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.

Arginase↗

PubMed2002

Broadband modulation of light by using an electro-optic polymer.

A major challenge to increasing bandwidth in optical telecommunications is to encode electronic signals onto a lightwave carrier by modulating the light up to very fast rates. Polymer electro-optic materials have the necessary properties to function in photonic devices beyond the 40-GHz bandwidth currently available. An appropriate choice of polymers is shown to effectively eliminate the factors contributing to an optical modulator's decay in the high-frequency response. The resulting device modulates light with a bandwidth of 150 to 200 GHz and produces detectable modulation signal at 1.6 THz. These rates are faster than anticipated bandwidth requirements for the foreseeable future.

Journal Article↗

PubMed2002

Fixed three-dimensional holographic images.

Three-dimensional holograms were recorded in a cerium-doped, strontium barium niobate (SBN:75) photorefractive crystal. These holograms are shown to not degrade after more than one week of continuous readout and to reconstruct reproductions of the original object with an observable field of view of approximately 35 degrees.

Journal Article↗

PubMed2002

In vivo behavior of a Helicobacter pylori SS1 nixA mutant with reduced urease activity.

Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n = 75) and BALB/c (n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n = 35) and BALB/c (n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.

Animals↗