Kelly Babb

Differential binding of host complement inhibitor factor H by Borrelia burgdorferi Erp surface proteins: a possible mechanism underlying the expansive host range of Lyme disease spirochetes.

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, is capable of infecting a wide variety of vertebrates. This broad host range implies that B. burgdorferi possesses the ability to contravene the immune defenses of many potential hosts. B. burgdorferi produces multiple different Erp proteins on its outer membrane during mammalian infection. It was reported previously that one Erp protein can bind human factor H (J. Hellwage, T. Meri, T. Heikkilä, A. Alitalo, J. Panelius, P. Lahdenne, I. J. T. Seppälä, and S. Meri, J. Biol. Chem. 276:8427–8435, 2001). In this paper we report that the ability to bind the complement inhibitor factor H is a general characteristic of Erp proteins. Furthermore, each Erp protein exhibits different relative affinities for the complement inhibitors of various potential animal hosts. The data suggest that the presence of multiple Erp proteins on the surface can allow a single B. burgdorferi bacterium to resist complement-mediated killing in any of the wide range of potential hosts that it might infect. Thus, Erp proteins likely contribute to the persistence of B. burgdorferi in nature and to the ability of this bacterium to cause Lyme disease in humans and other animals.

Temporal analysis of Borrelia burgdorferi Erp protein expression throughout the mammal-tick infectious cycle.

ABSTRACT Previous immunological studies indicated that the Lyme disease spirochete, Borrelia burgdorferi, expresses Erp outer surface proteins during mammalian infection. We conducted analyses of Erp expression throughout the entire tick-mammal infectious cycle, which revealed that the bacteria regulate Erp production in vivo. Bacteria within unfed nymphal ticks expressed little to no Erp proteins. However, as infected ticks fed on mice, B. burgdorferi increased production of Erp proteins, with essentially all transmitted bacteria expressing these proteins. Mice infected with B. burgdorferi mounted rapid IgM responses to all tested Erp proteins, followed by strong immunoglobulin G responses that generally increased in intensity throughout 11 months of infection, suggesting continued exposure of Erp proteins to the host immune system throughout chronic infection. As naive tick larvae acquired B. burgdorferi by feeding on infected mice, essentially all transmitted bacteria produced Erp proteins, also suggestive of continual Erp expression during mammalian infection. Shortly after the larvae acquired bacteria, Erp production was drastically downregulated. The expression of Erp proteins on B. burgdorferi throughout mammalian infection is consistent with their hypothesized function as factor H-binding proteins that protect the bacteria from host innate immune responses.

LuxS-Mediated Quorum Sensing in Borrelia burgdorferi, the Lyme Disease Spirochete

ABSTRACT The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. The luxS gene of B. burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant. Cultured B. burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.

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.

Distinct Regulatory Pathways Control Expression of Borrelia burgdorferi Infection-Associated OspC and Erp Surface Proteins

ABSTRACT Deciphering the mechanisms by which Borrelia burgdorferi controls the synthesis of proteins associated with mammalian infection will be an important step toward understanding the pathogenic properties of Lyme disease-causing bacteria. We present results of studies indicating that B. burgdorferi senses a wide variety of environmental stimuli, including soluble chemicals, which enables it to independently control synthesis of the Erp and OspC proteins. Regulation of OspC and Erp expression appears to occur at the level of transcription. In this regard, we observed that one or more DNA-binding proteins interact specifically with erppromoter DNA but not with the ospC promoter.

Borrelia burgdorferi RevA Antigen Is a Surface-Exposed Outer Membrane Protein Whose Expression Is Regulated in Response to Environmental Temperature and pH

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, produces RevA protein during the early stages of mammalian infection. B. burgdorferi apparently uses temperature as a cue to its location, producing proteins required for infection of warm-blooded animals at temperatures corresponding to host body temperature, but does not produce such virulence factors at cooler, ambient temperatures. We have observed that B. burgdorferi regulates expression of RevA in response to temperature, with the protein being synthesized by bacteria cultivated at 34°C but not by those grown at 23°C. Tissues encountered byB. burgdorferi during its infectious cycle vary in their pH values, and the level of RevA expression was also found to be dependent upon pH of the culture medium. The cellular localization of RevA was also analyzed. Borrelial inner and outer membranes were purified by isopycnic centrifugation, and membrane fractions were conclusively identified by immunoblot analysis using antibodies raised against the integral inner membrane protein MotB and outer membrane-associated Erp lipoproteins. Immunoblot analyses indicated that RevA is located in the B. burgdorferi outer membrane. These analyses also demonstrated that an earlier report (H. A. Bledsoe et al., Infect. Immun. 176:7447–7455, 1994) had misidentified such B. burgdorferi membrane fractions. RevA was further demonstrated to be exposed to the external environment, where it could facilitate interactions with host tissues.

Cross-Reactivity of Antibodies against Leptospiral Recurrent Uveitis-Associated Proteins A and B (LruA and LruB) with Eye Proteins

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

Synthesis of Autoinducer 2 by the Lyme Disease Spirochete, Borrelia burgdorferi

Defining the metabolic capabilities and regulatory mechanisms controlling gene expression is a valuable step in understanding the pathogenic properties of infectious agents such as Borrelia burgdorferi. The present studies demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. Consistent with those observations, B. burgdorferi was shown to synthesize the end product 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). Addition of in vitro-synthesized DPD to cultured B. burgdorferi resulted in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria can utilize the other LuxS product, homocysteine, for regeneration of methionine, B. burgdorferi was found to lack such ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD and utilizes a form of that molecule as an AI-2 pheromone to control gene expression.

Quorum sensing by the Lyme disease spirochete

The Lyme disease spirochete, Borrelia burgdorferi, utilizes a LuxS/autoinducer-2-dependent quorum sensing mechanism to control a specific subset of bacterial proteins. It is hypothesized that this system facilitates transmission of B. burgdorferi from feeding ticks into warm-blooded hosts.

Borrelia burgdorferi EbfC Defines a Newly-Identified, Widespread Family of Bacterial DNA-Binding Proteins

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure.