Caroline Ojaimi

Profiling of temperature-induced changes in Borrelia burgdorferi gene expression by using whole genome arrays.

ABSTRACT Borrelia burgdorferi is the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affect B. burgdorferi gene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putative B. burgdorferi open reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression in B. burgdorferi B31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment of B. burgdorferi to growth under diverse environmental conditions.

Activation of Cardiac Progenitor Cells Reverses the Failing Heart Senescent Phenotype and Prolongs Lifespan

Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by depletion of the cardiac progenitor cell (CPC) pool is unknown. Similarly, whether current lifespan reflects the ineluctable genetic clock or heart failure interferes with the genetically determined fate of the organ and organism is an important question. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by attenuation of the insulin-like growth factor-1/insulin-like growth factor-1 receptor and hepatocyte growth factor/c-Met systems, which do not counteract any longer the CPC renin–angiotensin system, resulting in cellular senescence, growth arrest, and apoptosis. However, pulse-chase 5-bromodeoxyuridine–labeling assay revealed that the senescent heart contains functionally competent CPCs that have the properties of stem cells. This subset of telomerase-competent CPCs have long telomeres and, following activation, migrate to the regions of damage, where they generate a population of young cardiomyocytes, reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to prolongation of maximum lifespan.

Notch1 regulates the fate of cardiac progenitor cells.

The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notch1 and raised the possibility that Notch1 regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPCs) in the niches express Notch1 receptor, and the supporting cells exhibit the Notch ligand Jagged1. The nuclear translocation of Notch1 intracellular domain (N1ICD) up-regulates Nkx2.5 in CPCs and promotes the formation of cycling myocytes in vitro. N1ICD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jagged1 activation of the Notch1 pathway. Importantly, inhibition of Notch1 in infarcted mice impairs the commitment of resident CPCs to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notch1 favors the early specification of CPCs to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notch1 may have critical implications in the control of heart homeostasis and its adaptation to pathologic states.

Disease Severity in a Murine Model of Lyme Borreliosis is Associated with the Genotype of the Infecting Borrelia burgdorferi Sensu Stricto Strain

The pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing 2 distinct ribosomal DNA spacer restriction fragment-length polymorphism genotypes (RSTs) was assessed in a murine model of Lyme disease. B. burgdorferi was recovered from 71.5% and 26.6% of specimens from mice infected with RST1 and RST3 isolates, respectively (P<.0001). The average ankle diameter and histologic scores for carditis and arthritis were significantly higher after 2 weeks of infection among mice infected with RST1 isolates than among those infected with RST3 isolates (P<.001). These clinical manifestations were associated with larger numbers of spirochetes in target tissues but not with the serum sensitivity of the individual isolates. Thus, the development and severity of disease in genetically identical susceptible hosts is determined mainly by the pathogenic properties of the infecting B. burgdorferi isolate. The RST1 genotype is genetically homogeneous and thus may represent a recently evolved clonal lineage that is highly pathogenic in humans and animals.

Impact of Genotypic Variation of Borrelia burgdorferi Sensu Stricto on Kinetics of Dissemination and Severity of Disease in C3H/HeJ Mice

ABSTRACT Various genotypes of Borrelia burgdorferi sensu stricto have been previously identified among a large collection of isolates cultured from patients with Lyme disease in the United States. Furthermore, association of specific genotypes with hematogenous dissemination early in the disease course has been observed. The present study assessed kinetics of spirochete dissemination and disease severity in C3H/HeJ mice infected with two different genotypes ofB. burgdorferi. Spirochete load in plasma and ear and other tissue samples of infected mice was measured by quantitative PCR, and these data were compared to those obtained by culture and histopathologic analysis. In mice infected with isolate BL206 (a type 1 strain), the peak number of spirochetes was observed in plasma between day 4 and 7, in heart and ear tissue on day 14, and in joints on day 28 postinoculation. There was a correlation between the peak number of spirochetes in plasma on day 4 or 7 and that in ear biopsy and joint specimens on day 14. By contrast, spirochete burdens in plasma of mice infected with isolate B356 (a type 3 strain) were 16- and 5-fold lower than those of BL206-infected mice on days 7 and 14 of infection, respectively. Similarly, approximately 6- and 13-fold fewer spirochetes were detected in the heart tissues of B356-infected mice compared to BL206-infected mice. Histopathologically, severe arthritis and aortitis were noted only in mice infected with isolate BL206. Spirochete dissemination and disease severity vary significantly in mice infected with distinct genotypes of B. burgdorferi, suggesting that genotypic differences in the infecting spirochetes play a key role in the pathogenesis and development of clinical disease.

Hyperhomocysteinemia Alters Cardiac Substrate Metabolism by Impairing Nitric Oxide Bioavailability Through Oxidative Stress

Background— Hyperhomocysteinemia (HHcy) has been considered a vascular disease associated with increased levels of oxidative stress that results in scavenging of NO. However, little is known of the impact of HHcy on cardiac function and especially myocardial metabolism. Methods and Results— L-Homocysteine was intravenously infused into conscious dogs, and the dogs were fed methionine to increase plasma homocysteine to 10 &mgr;mol/L for acute and 24 &mgr;mol/L for chronic HHcy. There was no significant change in hemodynamics with HHcy. Veratrine-induced, NO-dependent, coronary vasodilation (Bezold-Jarisch reflex) was reduced by 32% but was restored by simultaneous intravenous infusion of ascorbic acid or apocynin. Acute and chronic HHcy significantly increased uptake of glucose and lactate and decreased uptake of free fatty acid by the heart. HHcy significantly decreased bradykinin- or carbachol-induced reduction of myocardial oxygen consumption in vitro, and this effect was completely restored by coincubation with ascorbic acid, Tempol, or apocynin. Western blot analysis indicated an increase in Nox2 (82%) and a reduction in endothelial nitric oxide synthase (39%), phospho-endothelial nitric oxide synthase (39%), and superoxide dismutase-1 (45%). Microarray analysis of gene expression in heart tissue from chronic HHcy indicated a switch in cardiac phenotype to enzymes that metabolize glucose. Conclusions— HHcy directly modulates substrate use by the heart independent of changes in hemodynamics or ventricular function by reducing NO bioavailability through the generation of superoxide. The progression of cardiac or coronary heart disease associated with HHcy should be evaluated in light of the impact of alterations in the regulation of cardiac metabolism and substrate use.

Borrelia burgdorferi gene expression profiling with membrane-based arrays

Publisher Summary The chapter describes Borrelia burgdorferi membrane arrays contain PCR-amplified open reading frames (ORFs) from Borrelia burgdorferi strain B31 MI, and illustrates the strain whose genome sequence. The B. burgdorferi B31 genome is unique among fully sequenced bacterial genomes in that it consists of a linear chromosome 910,725 bp in length and a collection of 9 circular and 12 linear plasmids. Among the 1689 putative open reading frames (ORFs), 855 are chromosome encoded and 834 are plasmid encoded. More than 90% of the Borrelia burgdorferi plasmid ORFs are unrelated to any known bacterial sequences. The novel genes found on the Borrelia burgdorferi plasmids may, therefore, contribute to the ability of this pathogen to survive and maintain its complex life cycle. Since the identities of all the spots (ORFs) on the array are known, the complete gene expression profile of an organism under a given set of conditions may be analyzed with a single array.

Comparative Transcriptional Profiling of Borrelia burgdorferi Clinical Isolates Differing in Capacities for Hematogenous Dissemination

ABSTRACT Borrelia burgdorferi, the etiologic agent of Lyme disease, is genetically heterogeneous. Previous studies have shown a significant association between the frequency of hematogenous dissemination in Lyme disease patients and the genotype of the infecting B. burgdorferi strain. Comparative transcriptional profiling of two representative clinical isolates with distinct genotypes (BL206 and B356) was undertaken. A total of 78 open reading frames (ORFs) had expression levels that differed significantly between the two isolates. A number of genes with potential involvement in nutrient uptake (BB0603, BBA74, BB0329, BB0330, and BBB29) have significantly higher expression levels in isolate B356. Moreover, nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have cell surface properties that differ considerably. One of these genes, BBA74, encodes a protein of 257 amino acid residues that has been shown to possess porin activity. BBA74 transcript level was >20-fold higher in B356 than in BL206, and strain B356 contained three- to fivefold more BBA74 protein. BBA74 was disrupted by the insertion of a kanamycin resistance cassette into the coding region. The growth rates of both wild-type and mutant strains were essentially identical, and cultures reached the same final cell densities. However, the mutant strains consistently showed prolonged lags of 2 to 5 days prior to the induction of log-phase growth compared to wild-type strains. It is tempting to speculate that the absence of BBA74 interferes with the enhanced nutrient uptake that may be required for the entry of cells into log-phase growth. These studies demonstrate the value of comparative transcriptional profiling for identifying differences in the transcriptomes of B. burgdorferi clinical isolates that may provide clues to pathogenesis. The 78 ORFs identified here are a good starting point for the investigation of factors involved in the hematogenous dissemination of B. burgdorferi.

Potential Mechanisms of Low Sodium Diet–Induced Cardiac Disease: Superoxide-NO in the Heart

Rationale: Patients on a low salt (LS) diet have increased mortality. Objective: To determine whether reduction in NO bioactivity may contribute to the LS-induced cardiac dysfunction and mortality. Methods and Results: Adult male mongrel dogs were placed on LS (0.05% sodium chloride) for 2 weeks. Body weight (25.4±0.4 to 23.6±0.4 kg), left ventricular systolic pressure (137.0±3.4 to 124.0±6.7 mm Hg), and mean aortic pressure (111±3.1 to 98±4.3 mm Hg) decreased. Plasma angiotensin II concentration increased (4.4±0.7 to 14.8±3.7 pg/mL). Veratrine-induced (5 &mgr;g/kg) NO-mediated vasodilation was inhibited by 44% in LS; however, the simultaneous intravenous infusion of ascorbic acid or apocynin acutely and completely reversed this inhibition. In LS heart tissues, lucigenin chemiluminescence was increased 2.3-fold to angiotensin II (10−8 mol/L), and bradykinin (10−4 mol/L) induced reduction of myocardial oxygen consumption in vitro was decreased (40±1.3% to 16±6.3%) and completely restored by coincubation with tiron, tempol or apocynin. Switching of substrate uptake from free fatty acid to glucose by the heart was observed (free fatty acid: 8.97±1.39 to 4.53±1.12 &mgr;mol/min; glucose: 1.31±0.52 to 6.86±1.78 &mgr;mol/min). Western blotting indicated an increase in both p47phox (121%) and gp91phox (44%) as did RNA microarray analysis (433 genes changed) showed an increase in p47phox (1.6-fold) and gp91phox (2.0 fold) in the LS heart tissue. Conclusions: LS diet induces the activation of the renin–angiotensin system, which increases oxidative stress via the NADPH oxidase and attenuates NO bioavailability in the heart.

Coronary nitric oxide production controls cardiac substrate metabolism during pregnancy in the dog

The aim of this study was to examine the role of nitric oxide (NO) in the control of cardiac metabolism at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy and a statistically significant increase in cardiac output (55 +/- 4%) and in cardiac NOx production (44 +/- 4 to 59 +/- 3 nmol/min, P < 0.05). Immunohistochemistry of the left ventricle showed an increase in endothelial nitric oxide synthase staining in the endothelial cells at P60. NO-dependent coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by N(G)-nitro-l-arginine methyl ester (l-NAME). Isotopically labeled substrates were infused to measure oleate, glucose uptake, and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4 +/- 0.5 vs. 6.2 +/- 0.4 micromol/min) but greatly increased in response to l-NAME injection (to 19.9 +/- 0.9 micromol/min, P < 0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3 +/- 0.6 to 10.4 +/- 0.5 micromol/min, P < 0.05) and decreased in response to l-NAME (to 4.5 +/- 0.5 micromol/min, P < 0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, whereas genes involved in FFA oxidation were elevated. The acute inhibition of NO shifts the heart away from FFA and toward glucose metabolism despite the downregulation of the carbohydrate oxidative pathway. The increase in endothelium-derived NO during pregnancy results in a tonic inhibition of glucose oxidation and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes.