Publications (9)31.68 Total impact
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Article: Distribution of Lyme disease spirochete cp32 prophages and natural diversity among their lipoprotein-encoding erp loci.
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ABSTRACT: Lyme disease spirochetes possess complex genomes, consisting of a main chromosome and 20 or more smaller replicons. Among those small DNAs are the cp32 elements, a family of prophages that replicate as circular episomes. All complete cp32s contain an erp locus, which encodes surface-exposed proteins. Sequences were compared for all 193 erp alleles carried by 22 different strains of Lyme spirochete, to investigate their natural diversity and evolutionary histories. These included multiple isolates from an endemic focus in the northeastern USA, and isolates from across North America and Europe. Bacteria were derived from diseased humans and from vector ticks, and included members of 5 different Borrelia genospecies. All erp operon 5' non-coding regions were found to be highly conserved, as are also the initial 70-80 bp of all erp open reading frames, indicative of a common evolutionary origin. However, the majority of the protein coding regions are highly diverse, due to numerous intra- and intergenic recombination events. Most erp alleles are chimeras derived from sequences of closely-related and distantly related erp sequences, and from unknown origins. Since known functions of Erp surface proteins involve interactions with various host tissue components, this diversity may reflect both their multiple functions and the abilities of Lyme spirochetes to successfully infect a wide variety of vertebrate host species.Applied and environmental microbiology 04/2013; · 3.69 Impact Factor -
Article: Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins.
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ABSTRACT: The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that synthesis of OspC and Erps can be stimulated in culture by shifting temperature from 23 to 34°C, leading to a hypothesis that B. burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing separate evaluation of each factor's impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase expression of the OspC and Erp infection-associated proteins.Journal of bacteriology 12/2012; · 3.94 Impact Factor -
Article: Borrelia burgdorferi cp32 BpaB modulates expression of the prophage NucP nuclease and SsbP single-stranded DNA-binding protein.
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ABSTRACT: The Borrelia burgdorferi BpaB proteins of the spirochete's ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5'-TTATA-3'. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5' noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, respectively. Chromatin immunoprecipitation indicated that BpaB binds erp, ssbP, and nucP in live B. burgdorferi. A mutant bacterium that overexpressed BpaB produced significantly higher levels of ssbP and nucP transcript than did the wild-type parent.Journal of bacteriology 06/2012; 194(17):4570-8. · 3.94 Impact Factor -
Article: EbfC (YbaB) is a new type of bacterial nucleoid-associated protein and a global regulator of gene expression in the Lyme disease spirochete.
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ABSTRACT: Nearly every known species of Eubacteria encodes a homolog of the Borrelia burgdorferi EbfC DNA-binding protein. We now demonstrate that fluorescently tagged EbfC associates with B. burgdorferi nucleoids in vivo and that chromatin immunoprecipitation (ChIP) of wild-type EbfC showed it to bind in vivo to sites throughout the genome, two hallmarks of nucleoid-associated proteins. Comparative RNA sequencing (RNA-Seq) of a mutant B. burgdorferi strain that overexpresses EbfC indicated that approximately 4.5% of borrelial genes are significantly impacted by EbfC. The ebfC gene was highly expressed in rapidly growing bacteria, but ebfC mRNA was undetectable in stationary phase. Combined with previous data showing that EbfC induces bends in DNA, these results demonstrate that EbfC is a nucleoid-associated protein and lead to the hypothesis that B. burgdorferi utilizes cellular fluctuations in EbfC levels to globally control transcription of numerous genes. The ubiquity of EbfC proteins in Eubacteria suggests that these results apply to a wide range of pathogens and other bacteria.Journal of bacteriology 04/2012; 194(13):3395-406. · 3.94 Impact Factor -
Article: Identification of novel DNA-binding proteins using DNA-affinity chromatography/pull down.
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ABSTRACT: This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.Current protocols in microbiology 02/2012; Chapter 1:Unit1F.1. -
Article: BpaB and EbfC DNA-binding proteins regulate production of the Lyme disease spirochete's infection-associated Erp surface proteins.
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ABSTRACT: Vector-borne pathogens regulate their protein expression profiles, producing factors during host infection that differ from those produced during vector colonization. The Lyme disease agent, Borrelia burgdorferi, produces Erp surface proteins throughout mammalian infection and represses their synthesis during colonization of vector ticks. Known functions of Erp proteins include binding of host laminin, plasmin(ogen), and regulators of complement activation. A DNA region immediately 5' of erp operons, the erp operator, is required for transcriptional regulation. The B. burgdorferi BpaB and EbfC proteins exhibit high in vitro affinities for erp operator DNA. In the present studies, chromatin immunoprecipitation (ChIP) demonstrated that both proteins bind erp operator DNA in vivo. Additionally, a combination of in vivo and in vitro methods demonstrated that BpaB functions as a repressor of erp transcription, while EbfC functions as an antirepressor.Journal of bacteriology 12/2011; 194(4):778-86. · 3.94 Impact Factor -
Article: Simultaneous isolation of Ixodidae and bacterial (Borrelia spp.) genomic DNA.
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ABSTRACT: Tick and tick-borne diseases have become widely distributed throughout the United States. As a result, the interest associated with tick allocation and the potential threat they may pose has increased. Efforts have expanded to understand biotic and abiotic factors which may influence tick/pathogen distribution. Thus, we have developed a procedure which allows the simultaneous isolation of both tick and bacterial DNA. Downstream applications are diverse; however, we describe the use of multiplex PCR to confirm the presence of spirochete DNA from tick samples. We suspect that this procedure is not limited to tick-bacteria systems and may be applied to a variety of arthropod-related endeavors.Current protocols in microbiology 11/2010; Chapter 1:Unit1E.2. -
Article: BpaB, a novel protein encoded by the Lyme disease spirochete's cp32 prophages, binds to erp Operator 2 DNA.
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ABSTRACT: Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5' of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNA-binding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels.Nucleic Acids Research 09/2010; 38(16):5443-55. · 8.03 Impact Factor -
Article: Functional characterization of Borrelia spielmanii outer surface proteins that interact with distinct members of the human factor H protein family and with plasminogen.
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ABSTRACT: Acquisition of complement regulator factor H (CFH) and factor H-like protein 1 (CFHL1) from human serum enables Borrelia spielmanii, one of the etiological agents of Lyme disease, to evade complement-mediated killing by the human host. Up to three distinct complement regulator-acquiring surface proteins (CRASPs) may be expressed by serum-resistant B. spielmanii, each exhibiting an affinity for CFH and/or CFHL1. Here, we describe the functional characterization of the 15-kDa CRASPs of B. spielmanii, members of the polymorphic Erp (OspE/F-related) protein family, that bind two distinct host complement regulators, CFH and factor H-related protein 1 (CFHR1), but not CFHL1. CFH bound to the B. spielmanii CRASPs maintained cofactor activity for factor I-mediated C3b inactivation. Three naturally occurring alleles of this protein bound CFH and CFHR1 while a fourth natural allele could not. Comparative sequence analysis of these protein alleles identified a single amino acid, histidine-79, as playing a significant role in CFH/CFHR1 binding, with substitution by an arginine completely abrogating ligand binding. The mutation of His-79 to Arg did not inhibit binding of plasminogen, another known ligand of this group of borrelial outer-surface proteins.Infection and immunity 10/2009; 78(1):39-48. · 4.21 Impact Factor
Top co-authors
Top Journals
Institutions
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2013
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University of Pennsylvania
- Department of Biology
Philadelphia, PA, USA
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2010–2012
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University of Kentucky
- Department of Microbiology, Immunology & Molecular Genetics
Lexington, KY, USA
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