Borrelia burgdorferi EbfC, a novel, chromosomally encoded protein, binds specific DNA sequences adjacent to erp loci on the spirochete's resident cp32 prophages

All examined isolates of the Lyme disease spirochete, Borrelia burgdorferi, naturally maintain numerous variants of a prophage family as circular cp32 episomes. Each cp32 carries a locus encoding one or two different Erp outer membrane, surface-exposed lipoproteins. Many of the Erp proteins bind a host complement regulator, factor H, which is hypothesized to protect the spirochete from complement-mediated killing. We now describe the isolation and characterization of a novel, chromosomally encoded protein, EbfC, that binds specific DNA sequences located immediately 5' of all erp loci. This is one of the first site-specific DNA-binding proteins to be identified in any spirochete. The location of the ebfC gene on the B. burgdorferi chromosome suggests that the cp32 prophages have evolved to use this bacterial host protein for their own benefit and that EbfC probably plays additional roles in the bacterium. A wide range of other bacteria encode homologs of EbfC, none of which have been well characterized, so demonstration that B. burgdorferi EbfC is a site-specific DNA-binding protein has broad implications across the eubacterial kingdom.     

Axe-Txe,a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant,clinical isolate of Enterococcus faecium

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.     

Borrelia burgdorferi adhesins identified using in vivo phage display

Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for beta(3)-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.     

Differential telomere processing by Borrelia telomere resolvases in vitro but not in vivo

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.     

HrpA, a DEAH-box RNA helicase, is involved in global gene regulation in the Lyme disease spirochete

Spirochetes causing Lyme borreliosis are obligate parasites that can only be found in a tick vector or a vertebrate host. The ability to survive in these two disparate environments requires up and downregulation of specific genes by regulatory circuits that remain largely obscure. In this work on the Lyme spirochete, B. burgdorferi, we show that a disruption of the hrpA gene, which encodes a putative RNA helicase, results in a complete loss in the ability of the spirochetes to infect mice by needle inoculation. Studies of protein expression in culture by 2D gels revealed a change in the expression of 33 proteins in hrpA clones relative to the wild-type parent. Quantitative characterization of protein expression by iTRAQ analysis revealed a total of 187 differentially regulated proteins in an hrpA background: 90 downregulated and 97 upregulated. Forty-two of the 90 downregulated and 65 of the 97 upregulated proteins are not regulated under any conditions by the previously reported regulators in B. burgdorferi (bosR, rrp2, rpoN, rpoS or rrp1). Downregulated and upregulated proteins also fell into distinct functional categories. We conclude that HrpA is part of a new and distinct global regulatory pathway in B. burgdorferi gene expression. Because an HrpA orthologue is present in many bacteria, its participation in global regulation in B. burgdorferi may have relevance in other bacterial species where its function remains obscure. We believe this to be the first report of a role for an RNA helicase in a global regulatory pathway in bacteria. This finding is particularly timely with the recent growth of the field of RNA regulation of gene expression and the ability of RNA helicases to modulate RNA structure and function.     

Simultaneous coexpression of Borrelia burgdorferi Erp proteins occurs through a specific,erp locus-directed regulatory mechanism

An individual Borrelia burgdorferi bacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed that B. burgdorferi controls expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent to erp loci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed at erp loci and not by a global, plasmid-wide mechanism.     

Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi

The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B. burgdorferi. The humoral response to these Ag occurs relatively early during the course of infection. Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa. Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B. burgdorferi. Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct. However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum. The amino terminal sequence of the 71- and 73-kDa proteins of B. burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E. coli (Hsp70). It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B. burgdorferi.     

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.     

Preparation and purification of recombinant outer surface protein A (rOspA) of Borrelia burgdorferi sensu stricto and Borrelia afzelii

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitating disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recombinant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.     

Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination.

Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-gammaR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-gamma-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-gamma and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-gamma-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-gamma-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-gammaR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.     

Examination of the Borrelia burgdorferi transcriptome in Ixodes scapularis during feeding

Borrelia burgdorferi gene expression within the guts of engorging Ixodes scapularis ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library. Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks. These data demonstrate a new approach to the global analysis of B. burgdorferi genes that are preferentially expressed within the vector during feeding.     

A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31.

We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip2l. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.