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James A Carroll

Publications and source records attributed to James A Carroll.

14 recordsLinked to original sources
PubMed2006

Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG.

Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.

Bacterial Proteins↗
PubMed2006

Regulation and expression of bba66 encoding an immunogenic infection-associated lipoprotein in Borrelia burgdorferi.

When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT-PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian-like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb-infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out-compete for binding. bba66 promoter mapping revealed putative sigma70 and sigmaS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the -35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either sigmaN or sigmaS resulted in loss of BBA66. Promoter-GFP fusion analysis indicated that the sigma70 and/or sigmaS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.

Animals↗
PubMed2006

Delineating the requirement for the Borrelia burgdorferi virulence factor OspC in the mammalian host.

We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Serologic proteome analysis of Borrelia burgdorferi membrane-associated proteins.

Lyme disease, a global health concern, is caused by infection with Borrelia burgdorferi, B. afzelii, or B. garinii. The spirochete responsible for the disease in the United States is B. burgdorferi and is spread by the bite of an infected Ixodes tick. We utilized multiple two-dimensional gel techniques combined with proteomics to reveal the full humoral immune response of mice and Lyme patients to membrane-associated proteins isolated from Borrelia burgdorferi. Our studies indicated that a subset of immunogenic membrane-associated proteins (some new and some previously identified) was recognized by mice experimentally infected with Borrelia burgdorferi either by low-dose needle inoculation or by tick infestation. Moreover, the majority of these immunogenic membrane-associated proteins were recognized by sera from patients diagnosed with early-disseminated Lyme disease. These included RevA, ErpA, ErpP, DbpA, BmpA, FtsZ, ErpB, LA7, OppA I, OppA II, OppA IV, FlhF, BBA64, BBA66, and BB0323. Some immunogens (i.e., BBI36/38) were more reactive with sera from mice than Lyme patients, while additional membrane proteins (i.e., FlaB, P66, LA7, and Hsp90) were recognized more strongly with sera from patients diagnosed with early-localized, early-disseminated, or late (chronic)-stage Lyme disease. We were able to examine the humoral response in Lyme patients in a temporal fashion and to identify the majority of immunoreactive proteins as the disease progresses from early to late stages. This serologic proteome analysis enabled the identification of novel membrane-associated proteins that may serve as new diagnostic markers and, more importantly, as second-generation vaccine candidates for protection against Lyme disease.

Animals↗
PubMed2005

Host limits to accurate human growth hormone production in multiple plant systems.

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.

Cells, Cultured↗
PubMed2004

Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi.

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

Amino Acid Sequence↗
PubMed2003

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi.

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5' of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

Antigens, Bacterial↗
PubMed2003

Five-member gene family of Bartonella quintana.

Bartonella quintana, the agent of trench fever and an etiologic agent of bacillary angiomatosis, has an extraordinarily high hemin requirement for growth compared to other bacterial pathogens. We previously identified the major hemin receptor of the pathogen as a 30-kDa surface protein, termed HbpA. This report describes four additional homologues that share approximately 48% amino acid sequence identity with hbpA. Three of the genes form a paralagous cluster, termed hbpCAB, whereas the other members, hbpD and hbpE, are unlinked. Secondary structure predictions and other evidence suggest that Hbp family members are beta-barrels located in the outer membrane and contain eight transmembrane domains plus four extracellular loops. Homologs from a variety of gram-negative pathogens were identified, including Bartonella henselae Pap31, Brucella Omp31, Agrobacterium tumefaciens Omp25, and neisserial opacity proteins (Opa). Family members expressed in vitro-synthesized proteins ranging from ca. 26.5 to 35.1 kDa, with the exception of HbpB, an approximately 55.9-kDa protein whose respective gene has been disrupted by a approximately 510 GC-rich element containing variable-number tandem repeats. Transcription analysis by quantitative reverse transcriptase-PCR (RT-PCR) indicates that all family members are expressed under normal culture conditions, with hbpD and hbpB transcripts being the most abundant and the rarest, respectively. Mutagenesis of hbpA by allelic exchange produced a strain that exhibited an enhanced hemin-binding phenotype relative to the parental strain, and analysis by quantitative RT-PCR showed elevated transcript levels for the other hbp family members, suggesting that compensatory expression occurs.

Amino Acid Sequence↗
PubMed2003

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species.

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.

Animals↗
PubMed2002

Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides.

A highly active oxygen-evolving photosystem II (PSII) complex was purified from the HT-3 strain of the widely used cyanobacterium Synechocystis sp. PCC 6803, in which the CP47 polypeptide has been genetically engineered to contain a polyhistidine tag at its carboxyl terminus [Bricker, T. M., Morvant, J., Masri, N., Sutton, H. M., and Frankel, L. K. (1998) Biochim. Biophys. Acta 1409, 50-57]. These purified PSII centers had four manganese atoms, one calcium atom, and two cytochrome b(559) hemes each. Optical absorption and fluorescence emission spectroscopy as well as western immunoblot analysis demonstrated that the purified PSII preparation was devoid of any contamination with photosystem I and phycobiliproteins. A comprehensive proteomic analysis using a system designed to enhance resolution of low-molecular-weight polypeptides, followed by MALDI mass spectrometry and N-terminal amino acid sequencing, identified 31 distinct polypeptides in this PSII preparation. We propose a new nomenclature for the polypeptide components of PSII identified after PsbZ, which proceeds sequentially from Psb27. During this study, the polypeptides PsbJ, PsbM, PsbX, PsbY, PsbZ, Psb27, and Psb28 proteins were detected for the first time in a purified PSII complex from Synechocystis 6803. Five novel polypeptides were also identified in this preparation. They included the Sll1638 protein, which shares significant sequence similarity to PsbQ, a peripheral protein of PSII that was previously thought to be present only in chloroplasts. This work describes newly identified proteins in a highly purified cyanobacterial PSII preparation that is being widely used to investigate the structure, function, and biogenesis of this photosystem.

Arabidopsis Proteins↗
PubMed2002

Temporal generation of multiple antifungal proteins in primed seeds.

A drastic increase of antifungal activity was demonstrated during plant seed germination and in seed protein extract in vitro. Multiple antifungal proteins with a wide spectrum of activity were generated and identified. Chromatographic and electrophoretic analysis demonstrated that during seed germination, more fractions with potent antifungal activity were generated, and the antifungal activity shifted from small molecules to high molecular proteins. This germination-related increase of antifungal activity were observed in all three plants tested, i.e., cheeseweed, cigar tree and wheat. This rapid increase of antifungal activity was also observed with incubation of seed proteins in vitro, suggesting that at least part of the antifungal protein generation is independent of gene expression. Seven antifungal proteins with activities against five different plant pathogens were isolated from the active fractions. However, random digestion of purified seed protein with multiple proteinases failed to generate any antifungal proteins. It is suggested that during plant seed germination, a regulated biochemical process takes place that results in the generation of multiple peptides or proteins with antifungal activities. This onset of antifungal proteins is transitional in nature, but could play an important role in the protection of plants in early stage of development when the more sophisticated defense system has yet to develop.

Amino Acid Sequence↗
PubMed2002

Myeloperoxidase produces nitrating oxidants in vivo.

Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Animals↗
PubMed2001

Surface exposure and protease insensitivity of Borrelia burgdorferi Erp (OspEF-related) lipoproteins.

Borrelia burgdorferi can encode numerous lipoproteins of the Erp family. Although initially described as outer surface proteins, the technique used in that earlier study has since been demonstrated to disrupt bacterial membranes and allow labelling of subsurface proteins. Data are now presented from additional analyses indicating that Erp proteins are indeed surface exposed in the outer membrane. Surface localization of these infection-associated proteins indicates the potential for interactions of Erp proteins with vertebrate tissues. Some Erp proteins were resistant to in situ digestion by certain proteases, suggesting that those proteins fold in manners which hide protease cleavage sites, or that they interact with other protective membrane components. Additionally, cultivation of B. burgdorferi in the presence of antibodies directed against Erp proteins inhibited bacterial growth.

Animals↗