Complement regulator-acquiring surface proteins of Borrelia burgdorferi: Structure, function and regulation of gene expression

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.

Download Full-text

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal–tick infection cycle

International Journal of Medical Microbiology ◽

10.1016/j.ijmm.2007.10.002 ◽

2008 ◽

Vol 298 ◽

pp. 249-256 ◽

Cited By ~ 26

Author(s):

Tomasz Bykowski ◽

Michael E. Woodman ◽

Anne E. Cooley ◽

Catherine A. Brissette ◽

Reinhard Wallich ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Expression Patterns ◽

Surface Proteins ◽

Infection Cycle ◽

Tick Infection ◽

Complement Regulator

Download Full-text

Borrelia burgdorferi Infection-Associated Surface Proteins ErpP, ErpA, and ErpC Bind Human Plasminogen

Infection and Immunity ◽

10.1128/iai.01133-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 300-306 ◽

Cited By ~ 64

Author(s):

Catherine A. Brissette ◽

Katrin Haupt ◽

Diana Barthel ◽

Anne E. Cooley ◽

Amy Bowman ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Critical Role ◽

Aminocaproic Acid ◽

Factor H ◽

Surface Proteins ◽

Primary Role ◽

Dependent Manner ◽

Lysine Residues ◽

Complement Regulator ◽

Mammalian Infection

ABSTRACT Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium's dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ε-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.

Download Full-text

Molecular Characterization of Borrelia burgdorferi erp Promoter/Operator Elements

Journal of Bacteriology ◽

10.1128/jb.186.9.2745-2756.2004 ◽

2004 ◽

Vol 186 (9) ◽

pp. 2745-2756 ◽

Cited By ~ 49

Author(s):

Kelly Babb ◽

Jason D. McAlister ◽

Jennifer C. Miller ◽

Brian Stevenson

Keyword(s):

Borrelia Burgdorferi ◽

Dna Sequences ◽

Fluorescent Protein ◽

Factor H ◽

Surface Proteins ◽

Virulence Determinants ◽

Transcriptional Initiation ◽

Bacterial Proteins ◽

Complement Regulator ◽

Green Fluorescent

ABSTRACT Many Borrelia burgdorferi Erp outer surface proteins have been demonstrated to bind the host complement regulator factor H, which likely contributes to the ability of these organisms to evade the host innate immune system. B. burgdorferi controls Erp protein synthesis throughout the bacterial infectious cycle, producing the proteins during mammalian infections but repressing their synthesis during tick infections. Defining the mechanism by which B. burgdorferi regulates the expression of these virulence determinants will provide important insight into the biological and pathogenic properties of the Lyme disease spirochete. The present study demonstrates that two highly conserved DNA sequences located 5′ of erp operons specifically bind bacterial proteins. Analyses with B. burgdorferi of transcriptional fusions between erp promoter/operator DNAs and the gene for green fluorescent protein indicated that the expression of these operons is regulated at the level of transcriptional initiation. These analyses also indicated significant differences in the promoter strengths of various erp operons, which likely accounts for reported variations in expression levels of different Erp proteins. Mutagenesis of promoter-gfp fusions demonstrated that at least one of the proteins which bind erp operator DNA functions as a repressor of transcription.

Download Full-text

A transcriptomic taxonomy of Drosophila circadian neurons around the clock

10.1101/2020.09.15.297051 ◽

2020 ◽

Author(s):

Dingbang Ma ◽

Dariusz Przybylski ◽

Katharine C. Abruzzi ◽

Matthias Schlichting ◽

Qunlong Li ◽

...

Keyword(s):

Gene Expression ◽

Synapse Formation ◽

Regulation Of Gene Expression ◽

Surface Proteins ◽

Circadian Oscillation ◽

Temporal Regulation ◽

Functional Studies ◽

Spatial Regulation ◽

Sequencing Method ◽

The Many

AbstractMany different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila circadian neurons, we optimized a single cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. We suggest that these molecules orchestrate the temporal regulation of synapse formation and/or strength. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.

Download Full-text

Coordinated Expression of Borrelia burgdorferi Complement Regulator-Acquiring Surface Proteins during the Lyme Disease Spirochete's Mammal-Tick Infection Cycle

Infection and Immunity ◽

10.1128/iai.00604-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4227-4236 ◽

Cited By ~ 61

Author(s):

Tomasz Bykowski ◽

Michael E. Woodman ◽

Anne E. Cooley ◽

Catherine A. Brissette ◽

Volker Brade ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Expression Patterns ◽

Resistance Mechanism ◽

Factor H ◽

Surface Proteins ◽

Mrna Levels ◽

Tick Infection ◽

Complement Regulator ◽

Mammalian Infection

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferi outer surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZ expression patterns throughout the B. burgdorferi infectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZ and synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZ expression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferi to control expression of this borrelial infection-associated protein.

Download Full-text

Purification and Characterization of Borrelia burgdorferi from Feeding Nymphal Ticks (Ixodes scapularis)

Infection and Immunity ◽

10.1128/iai.69.6.3536-3541.2001 ◽

2001 ◽

Vol 69 (6) ◽

pp. 3536-3541 ◽

Cited By ~ 14

Author(s):

Sivaprakash Rathinavelu ◽

Aravinda M. de Silva

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Ixodes Scapularis ◽

Gradient Centrifugation ◽

Surface Proteins ◽

Mrna Levels ◽

Percoll Density Gradient Centrifugation ◽

Percoll Density Gradient ◽

Gene Expression Studies

ABSTRACT Here we describe a protocol for purifying Borrelia burgdorferi from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels ofB. burgdorferi outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.

Download Full-text

Binding of the human Factor H-related protein (FHR-1) to Borrelia burgdorferi is mediated by complement regulator-acquiring surface proteins

Molecular Immunology ◽

10.1016/j.molimm.2006.07.084 ◽

2007 ◽

Vol 44 (1-3) ◽

pp. 182

Author(s):

Katrin Haupt ◽

Reinhard Wallich ◽

Peter Kraiczy ◽

Volker Brade ◽

Christine Skerka ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Human Factor ◽

Factor H ◽

Surface Proteins ◽

Related Protein ◽

Complement Regulator

Download Full-text

lacZ Reporter System for Use in Borrelia burgdorferi

Applied and Environmental Microbiology ◽

10.1128/aem.01389-10 ◽

2010 ◽

Vol 76 (22) ◽

pp. 7407-7412 ◽

Cited By ~ 9

Author(s):

Beth M. Hayes ◽

Mollie W. Jewett ◽

Patricia A. Rosa

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Regulation Of Gene Expression ◽

Reporter Genes ◽

Galactosidase Activity ◽

Lacz Reporter ◽

Reporter System ◽

Endogenous Gene ◽

Solid Media ◽

Infectious Cycle

ABSTRACT Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.

Download Full-text

Further Characterization of Complement Regulator-Acquiring Surface Proteins of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.69.12.7800-7809.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7800-7809 ◽

Cited By ~ 152

Author(s):

Peter Kraiczy ◽

Christine Skerka ◽

Volker Brade ◽

Peter F. Zipfel

Keyword(s):

Borrelia Burgdorferi ◽

Detailed Comparison ◽

Factor H ◽

Surface Proteins ◽

Human Complement ◽

Causative Agents ◽

Complement Regulator ◽

Low Passage ◽

The Individual

ABSTRACT The three genospecies Borrelia burgdorferi,Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B.afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674–1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived fromB. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37°C compared with those cultured at 20°C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.

Download Full-text