Brian Stevenson

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genomes 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non‐functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes

In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.

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.

Efficient Targeted Mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.

Analysis of Borrelia burgdorferi gene expression during life cycle phases of the tick vector Ixodes scapularis

Borrelia burgdorferi exists in nature via an enzootic cycle whereby the organism must adapt to the diverse environmental conditions provided inside the arthropod transmission vector and the mammalian reservoir hosts. B. burgdorferi genes shown to be regulated by temperature, pH and/or cell density during the organisms growth in culture medium were assayed for expression during various stages of the tick feeding cycle by reverse transcription-polymerase chain reaction (RT-PCR). ospA, ospC, flaB, erpA/I/N, erpB/J/O, rev and mlpA, were transcriptionally active following the larval and nymphal stages of feeding as determined by qualitative RT-PCR. During tick resting periods between feedings, ospC, mlpA and rev transcription were undetectable, in contrast to ospA, flaB, erpA/I/N and erpB/J/O. bba64, a gene induced by environmental changes in culture and expressed during mammalian infection, was not detectable during any of the tick life cycle phases. Quantitative PCR to determine B. burgdorferi genome equivalents in these tick samples using DNA co-purified with the RNA allowed an estimation of gene expression relative to the numbers of B. burgdorferi present in the ticks. Although the spirochete totals varied widely between individual tick pools of fed, replete nymphs, the relative expression ratios between individual target genes following a nymphal feed were comparable. Similarly, borrelial gene transcription from the larval feeding and the nymphal feeding were observed and compared. These findings analogize B. burgdorferi gene expression observed by environmental stimuli in vitro with the transcriptional activity occurring during the organisms infectious cycle within the tick.

LfhA, a Novel Factor H-Binding Protein of Leptospira interrogans

ABSTRACT The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

Functional characterization of BbCRASP-2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1.

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H‐like protein‐1 (FHL‐1) allows spirochetes to resist complement‐mediated killing. Serum‐resistant B. burgdorferi express up to five distinct complement regulator‐acquiring surface proteins (CRASPs) that bind factor H and/or FHL‐1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP‐2. BbCRASP‐2 is distinct from the four previously identified factor H/FHL‐1‐binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP‐2, cspZ, is located on the linear plasmid lp28‐3. BbCRASP‐2 is highly divergent from the factor H/FHL‐1‐binding protein BbCRASP‐1 and from members of the factor H‐binding Erp (OspE/F‐related) protein family. Peptide mapping analysis revealed that the factor H/FHL‐1 binding site is discontinuous and it was found that C‐terminal truncations abrogate factor H and FHL‐1 binding. The predominant BbCRASP‐2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL‐1 bound to BbCRASP‐2 maintain cofactor activity for factor I‐mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP‐2 in serum‐sensitive B. burgdorferi mutant B313 increased resistance to complement‐mediated lysis. The characterization of BbCRASP‐2 now provides a complete picture of the three diverse complement regulator‐binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

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.

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

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 bacteriums 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.

Immunological characterization of the complement regulator factor H-binding CRASP and Erp proteins of Borrelia burgdorferi.

Complement activation plays an important role in the elimination of invading microorganisms. Borrelia (B.) burgdorferi sensu lato the etiological agent of Lyme borreliosis, can resist complement-mediated killing. The mechanism of complement resistance of B. burgdorferi sensu stricto apparently depends on the expression of several outer surface proteins described as CRASPs (complement regulator-acquiring surface proteins). These borrelial surface proteins are able to bind components of the complement regulatory system, factor H and/or factor H-like protein 1 (FHL-1), two crucial fluid-phase negative regulators of the alternative pathway of complement. It was previously demonstrated that one CRASP is encoded by a member of the erp gene family. The purpose of the study was to use a set of monoclonal antibodies (mAb) and polyclonal antisera to characterize the relatedness of factor H-binding CRASP and Erp proteins among several B. burgdorferi sensu stricto and B. afzelii strains. Based on the observed cross-reactivities between B. burgdorferi sensu stricto strains LW2 and PKa-1, it is concluded that BbCRASP-3 is similar to ErpP, BbCRASP-4 is structurally related to ErpC, and BbCRASP-5 is similar to ErpA. The BaCRASP-2 and BaCRASP-4 proteins of B. afzelii strain EB1 reacted with both anti-ErpA and anti-ErpP antibodies whereas BaCRASP-5 of B. afzelii strain FEM1-D15 exclusively reacted with BbCRASP-3/ErpP specific antibodies. Together, these data indicate that most of the factor H-binding CRASPs are members of the Erp protein family, which represents a polymorphic class of proteins with similar or identical immunological reactivities.