John F. Bruno

Wide Distribution of a High-Virulence Borrelia burgdorferi Clone in Europe and North America

We found substantial population differentiation and recent trans-Atlantic dispersal of a high-virulence B. burgdorferi clone.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

BackgroundLyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1).ResultsRobust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases.ConclusionsIntra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.

Profiling the humoral immune response to Borrelia burgdorferi infection with protein microarrays

To determine the cell envelope proteins of Borrelia burgdorferi recognized by immune sera of patients with late Lyme disease, we developed a Borrelia microarray containing proteins encoded by 90 cell envelope genes and their homologs described in the annotated genomic sequence of B. burgdorferi, strain B31. The protein microarray was used to profile the humoral immune response using sera from 13 patients with late Lyme disease and four normal controls. Although there was considerable heterogeneity in the individual sera responses, 25 of the cell envelope proteins were recognized by seven or more samples. Sera from non-infected individuals lacked reactivity against any of the proteins on the array. Among the most antigenic envelope proteins, BLAST search revealed little sequence homology to known microbial proteins from other species. The proteins that were highly seropositive included several members of the Erp gene families, BBA24 (decorin binding protein A (DbpA)) and members of the Borrelia gene family Pfam113 that code for the Mlp lipoprotein gene family. Several novel, uncharacterized B. burgdorferi antigens identified in this study were BBA14, BBG23, BB0108, BB0442 and BBQ03. The accurate diagnosis of Lyme disease depends on correlating objective clinical abnormalities with serological evidence of exposure to B. burgdorferi. A protein array of the envelope proteins of Borrelia burgdorferi may be very useful in specifically identifying patients with Lyme disease. This approach could contribute to a more rapid discovery of antigens not expressed in vitro that may be useful for the development of vaccine and diagnostics.

Pituitary and hypothalamic insulin-like growth factor-I (IGF-I) and IGF-I receptor expression in food-deprived rats

The present study was designed to evaluate a possible role for the insulin-like growth factor-I (IGF-I) system in mediating the suppression of growth hormone (GH) secretion observed in food-deprived rats by measuring IGF-I mRNA, receptor concentration and receptor mRNA in neuroendocrine tissues (hypothalamus and pituitary). Rats were deprived of food (food-deprived) for 72 h or had free access to food (fed). Tissues were processed for measurement of steady-state levels of: (a) IGF-I and IGF-I receptor mRNA (by solution hybridization/RNase protection assay); (b) IGF-I in serum and tissue extracts (by RIA) and (c) IGF-I displaceable [125I]IGF-I binding to plasma membrane preparations. Food deprivation resulted in decreased serum and liver levels of IGF-I. Kidney IGF-I mRNA levels were reduced 80% in food-deprived rats with a concomitant increase in IGF-I receptor concentration and mRNA levels. Refeeding of food-deprived rats fully normalized these perturbations. Pituitary IGF-I content was reduced 50% in food-deprived rats while IGF-I mRNA levels were unaffected. A modest increase was seen in pituitary IGF-I receptor concentration; however, IGF-I receptor mRNA levels were not changed. Hypothalamic IGF-I mRNA content was reduced in 72 h food-deprived rats while IGF-I receptor binding capacity and mRNA were unaffected. In conclusion, IGF-I mRNA levels are decreased in liver, kidney and hypothalamus together with a reduction in plasma IGF-I in food-deprived rats but is unaffected in anterior pituitary. IGF-I receptor gene expression and binding capacity are coordinately regulated in kidney and hypothalamus, but not in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand Binding and Functional Properties of the Rat Somatostatin Receptor SSTR4 Stably Expressed in Chinese Hamster Ovary Cells.

Somatostatin (SS14) is an important regulator of endocrine and brain function exerting its action after binding to high-affinity membrane receptor subtypes. Its diverse physiological activities include inhibition of hormone secretion from pituitary, pancreas, and gut. In the CNS, SS14 acting as a neurotransmitter/neuromodulator exerts inhibitory effects on neural function. Recently, three SS14 receptor genes, SSTR1, SSTR2, and SSTR3, have been cloned and characterized. We have cloned and characterized a novel fourth member of this gene family from a rat genomic library, SSTR4, which is expressed predominantly in neural tissue. When stably expressed in CHO-K1 cells, SSTR4 binds SS14 and SS28 with high affinity; however, the SS14 analogs SMS 201-995 and MK 678 failed to displace specific binding. High-affinity agonist binding was diminished by prior exposure to both GTPgammaS and pertussis toxin (PTX) but was not effected following agonist pretreatment, indicating that SSTR4 is coupled to a PTX-sensitive G-protein but does not desensitize. SSTR4 expressed in CHO cells is coupled by a PTX-sensitive G-protein to inhibition of adenylyl cyclase since treatment of transfected cells with SS14 resulted in the inhibition of forskolin-stimulated cAMP accumulation, an effect that was abolished by PTX treatment. The cloning of four SS14 receptor subtypes provide molecular probes for structure-function studies and for identifying those particular subtypes responsible for mediating the diverse physiological action of SS14.

Detection of genetic diversity in linear plasmids 28-3 and 36 in Borrelia burgdorferi sensu stricto isolates by subtractive hybridization.

Recent studies based on sequence divergence in the ospC gene have identified limited subpopulations of B. burgdorferi associated with invasive human disease. Spirochetes with certain OspC types never cause human disease, while some others cause local infection at the primary skin site but do not hematogenously disseminate. Only four OspC genotypes (A, B, I and K) are responsible for disseminated disease and are found in the blood and cerebrospinal fluid, and hence are termed invasive strains. Subtractive hybridization was carried out between a prototype of a low passage invasive type, strain B31, and a strain associated only with local infection, group E, to identify genes associated with hematogenous dissemination. Two clones isolated from the subtraction library were unique to the B31 genome and mapped to locus BBH26 located on linear plasmid 28-3 (lp28-3) and to locus BBK48 located on linear plasmid 36 (lp36). Sequence analysis of the BBH26 locus revealed an amino acid repeat motif in the group E DNA that was absent in the B31 genome. This in-frame repeat motif was present yet variable in DNA isolated from several major OspC groups. However, no consistent sequence diversity was noted when other invasive and non-invasive strains were compared. In contrast, analysis of the BBK48 locus revealed a striking distinction between invasive and non-invasive spirochetes. PCR and Southern blot analysis indicated this locus was only present in invasive groups A, B, I, and K. BBK48 is a member of a gene family clustered on lp36. Therefore, these findings indicate that this genetic loci may participate in differentiating pathogens from non-pathogens and that its presence, which is correlated with ospC type, may play a role determining infectivity in humans.

Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

Localization of prepro-growth hormone releasing factor mRNA in rat brain and regulation of its content by food deprivation and experimental diabetes

Growth hormone releasing factor (GRF) is the principal stimulatory hypothalamic neuropeptide controlling growth hormone (GH) secretion from pituitary somatotrophs. In this study we examined the hypothalamic and extrahypothalamic sites of preproGRF mRNA expression using both in situ hybridization and nuclease protection techniques. Each of these techniques confirmed that the principal site of GRF expression in CNS is within the hypothalamic arcuate nucleus with an additional population of neurons lying in the periventromedial region of the hypothalamus. PreproGRF mRNA was also detected at low levels in paraventricular and supraoptic nuclei, in piriform and neocortex, hippocampus, olfactory bulb, striatum, and brain stem, but not in anterior pituitary. However, the levels of preproGRF mRNA in these latter brain regions were approximately 20- to 50-fold lower than levels in whole hypothalamus. To extend previous observations, we then determined which brain loci modulate preproGRF mRNA levels in response to food deprivation or experimental diabetes mellitus. PreproGRF mRNA levels in both arcuate and periventromedial hypothalamic neurons declined 3- to 5-fold following food deprivation or streptozotocin-induced diabetes mellitus while the number of preproGRF expressing neurons was relatively unchanged in each experimental protocol; however, hybridization signal in piriform cortex and hippocampus was not affected by either experimental protocol. These data implicate two hypothalamic neuronal populations in modulating GH secretion and suggest specific modulation of hypothalamic preproGRF mRNA levels by food deprivation and diabetes.

Polypeptide synthesis in enucleated mouse fibroblasts

The polypeptides synthesized in cytoplasts prepared from mouse L929 cells by cytochalasin-induced enucleation were analyzed by two-dimensional gel electrophoresis. Nearly all detectable polypeptides made in parental whole cells were likewise made in cytoplasts, though in decreasing amounts, for up to at least 12 hours after enucleation. A similar analysis of cells treated with actinomycin D, an inhibitor of transcription, showed that such cells initially synthesized all of the polypeptides made by untreated cells. However, by 12 hours after treatment, the electrophoretic patterns produced by preparations of radiolabeled cells were highly aberrant, with some polypeptides apparently greatly overproduced relative to others. The results are consistent with the notion that physical removal of the nucleus disrupts the mechanisms regulating mRNA degradation in eukaryotic cells.

Metabolic Regulation of Growth Hormone Secretagogue Gene Expression

Growth hormone (GH), secreted from somatotrophs in the anterior pituitary, is a highly regulated hormone that stimulates longitudinal growth and has multiple effects on cellular metabolism. Plasma GH levels vary widely at different times of the day and night, with profound influences of diet, exercise, sleep, and stress in a given individual. GH levels also differ with sex, age, and body habits, providing a challenge to those defining “normal” GH physiology (1–3).