Identification and characterization of virulence determinants in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi is the etiological agent of Lyme disease, the most common tick-borne disease in the United States. Although the most frequently reported symptom is arthritis, patients can also experience severe cardiac, neurologic, and dermatologic abnormalities. The identification of virulence determinants in infectious B. burgdorferi strains has been limited by their slow growth rate, poor transformability, and general lack of genetic tools. The present study demonstrates the use of transposon mutagenesis for the identification of infectivity-related factors in infectious B. burgdorferi, examines the potential role for chemotaxis in mammalian infection, and describes the development of a novel method for the analysis of recombination events at the Ids antigenic variation locus. A pool of Himar1 mutants was isolated using an infectious B. burgdorferi clone and the transposon vector pMarGent. Clones exhibiting reduced infectivity in mice possessed insertions in virulence determinants putatively involved in host survival and dissemination. These results demonstrated the feasibility of extensive transposon mutagenesis studies for the identification of additional infectivity-related factors. mcp-5 mutants were chosen for further study to determine the role of chemotaxis during infection. Animal studies indicated that mcp-5 mutants exhibited a reduced infectivity potential, and suggested a role for mcp-5 during the early stages of infection. An in vitro phenotype for an mcp-5 mutant was not detected. Genetic complementation of an mcp-5 mutant resulted in restoration of Mcp-5 expression in the complemented clone, as demonstrated by western blotting, but the organisms were not infectious in mice. We believe this result is a consequence of differences in expression between genes located on the linear chromosome and genes present on the circular plasmid used for trans-complementation. Overall, this work implicates mcp-5 as an important determinant of mammalian infectivity. Finally, the development of a computer-assisted method for the analysis of recombination events occurring at the B. burgdorferi vls antigenic variation locus has proven highly valuable for the detailed examination of vls gene conversion. The studies described here provide evidence for the importance of chemotaxis during infection in mice and demonstrate advances in both genetic and computational approaches for the further characterization of the Lyme disease spirochete. ^

Antigenic variation in Lyme disease spirochetes by segmental recombination of VMP-like sequence cassettes

Lyme disease is a multisystemic disorder caused by tick-borne infection of humans or other mammalian hosts with Borrelia burgdorferi. If untreated, the spirochetes can persist in the mammalian host for months or years. The mechanisms by which Lyme disease spirochetes evade the immune response have not been determined. In this study, we have identified and characterized an elaborate genetic system in the Lyme disease spirochete B. burgdorferi that promotes extensive antigenic variation of a 34-kDa surface-exposed lipoprotein, VlsE. A 28-kilobase linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. The presence of lp28-1 correlates with the high-infectivity phenotype in B. burgdorferi strains tested. Segments of the 15 non-expressed (silent) vls cassette sequences located upstream of vlsE are able to recombine into the centra vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. When compared to parental VlsE, VlsE variants progressively accumulate sequence changes during the period of 4, 7, 14, 21, and 28 days post infection in C3H/HeN mice. However, no recombination was detected during the period of 28-day in vitro culture, suggesting in vivo induction of VlsE antigenic variation. Adaptive immune responses do not appear to play a significant role in this induction, since similar recombination events were also observed in immunodeficient SCID mice. The $5\sp\prime$ and $3\sp\prime$ noncassette regions of vlsE are apparently not subject to recombination and sequence variation. The structure and sequence of the silent vls cassette locus is preserved during the process of the VlsE antigenic variation, consistent with a nonreciprocal recombination mechanism. This combinatorial form of antigenic variation could potentially yield millions of VlsE variants in the mammalian host, and thereby contribute to immune evasion, long-term survival, and pathogenesis of B. burgdorferi. ^

The dynamic proteome of Lyme disease Borrelia.

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Plasmid -encoded virulence determinants of Borrelia burgdorferi

Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, infects both mammals and ticks. Its genome, sequenced in 1997, consists of one linear chromosome and over 20 linear and circular plasmids. Continuous passage of organisms in culture causes them to lose certain plasmids and also results in loss of infectivity in mammals. In this work, 19 B. burgdorferi clonal isolates were examined for infectivity in mice and for plasmid content utilizing polymerase chain reaction (PCR). Two plasmids, a 28 kilobase (kb) linear plasmid (Ip28-1) and a 25 kb linear plasmid (Ip25) were found to be required for full infectivity. Previous studies had demonstrated that Ip28-1 contains the vls locus, which is involved in antigenic variation and immune evasion. Gene BBE22 on Ip25 is predicted to encode the nicotinamidase PncA, an enzyme that converts nicotinamide to nicotinic acid as part of a pathway for NAD synthesis. To examine the potential role of BBE22 in infectivity, a shuttle vector containing BBE22 (pBBE22) was constructed and used to transform B. burgdorferi clone 5A13, which contains all plasmids except lp25. Transformation with pBBE22 restored infectivity of clone 5A13 in mice, whereas 5A13 transformed with the shuttle vector alone was not infectious. To determine whether BBE22 acts as a nicotinamidase in vivo, a Salmonella typhimurium pncA− nadB− transposon mutant was transformed with pBBE22 or with pQE30:BBE22, which contained BBE22 in an E. coli expression vector. Both constructs complemented the Salmonella mutant, permitting growth in minimal media plus nicotinamide. Salmonella cells over-expressing BBE22 also exhibited nicotinamidase activity, as determined by ammonia production in the presence of nicotinamide. Site-directed mutagenesis of BBE22 at the predicted active site (resulting in a Cys120Ala substitution) abrogated the ability to restore infectivity to B. burgdorferi 5A13 and to complement the pncA mutation in S. typhimurium. These studies indicate that BBE22 is a nicotinamidase required for NAD synthesis and survival of B. burgdorferi in mammals. This is also the first demonstration of ‘molecular Koch's postulates’ in B. burgdorferi, i.e. that a specific gene is essential for infectivity of the Lyme disease spirochete. ^

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

The immune response to the VlsE antigenic variation protein of Borrelia burgdorferi

The spirochete Borrelia burgdorferi (Bb) is the causative agent of Lyme disease. During infection, a strong immune response is elicited towards Bb by its host; however, the organism is able to persist and to disseminate to many different tissues. The vls locus is located on the linear plasmid lp28-1, a plasmid shown to be important for virulence in the mouse model. During infection, vlsE undergoes antigenic variation through a series of gene conversions, which results in the insertion of sequences from the silent, unexpressed cassettes into the vlsE cassette. We hypothesize that this antigenic variation is important in the spirochete's ability to persist within mammals by allowing it to evade the immune system. To define the role of vls in immune evasion, the immune response against VlsE was determined by using a recombinant form of VlsE (VlsE1-His) as an antigen to screen patient sera. Lyme patients produce antibodies that recognize VlsE, and these antibodies are present throughout the course of disease. Immunization with the VlsE1-His protein provided protection against infection with Bb expressing the same variant of VlsE (VlsE1), but was only partially protective when mice were infected with organisms expressing VlsE variants; however, subsequent VlsE immunization studies yielded inconsistent protection. Successful immunizations produced different antibody reactivities to VlsE epitopes than non-protective immunizations, but the reason for this variable response is unclear. In the process of developing genetic approaches to transform infectious Bb, it was determined that the transformation barrier posed by plasmids lp25 and lp56 could be circumvented by replacing the required lp25 gene pncA. To characterize the role of vlsE in infectivity, Bb lacking lp28-1 were complemented with a shuttle plasmid containing the lp25 encoded virulence determinant pncA and vlsE. Complemented spirochetes express VlsE, but the gene does not undergo antigenic variation and infectivity in the mouse model was not restored, indicating that either antigenic variation of vlsE is necessary for survival in the mouse model or that other genes on lp28-1 are important for virulence. ^

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

A METAGENOMIC STUDY OF THE TICK MIDGUT

A Metagenomic Study of the Tick Midgut Daniel Yuan, B.S. Supervisory Professor : Steven J. Norris, Ph.D. Southern tick–associated rash illness (STARI) or Master’s disease is a Lyme-like illness that occurs following bites by Amblyomma americanum, the lone-star tick. Clinical symptoms include a bull’s eye rash similar to the erythema migrans lesions of Lyme disease, as well as fever and joint pains. Lyme disease is caused by Borrelia burgdorferi and related spirochetes. However, B. burgdorferi has not been detected in STARI patients, or in ticks in the South Central U.S. The causative agent of STARI has not been identified, although it was once thought to be caused by another Borrelia species, Borrelia lonestari. Furthermore, while adult A. americanum have up to a 5.6% Borrelia lonestari infection rate, the prevalence of all Borrelia species in Texas ticks as a whole is not known. Previous studies indicate that 6%-30% of Northern Ixodes scapularis ticks are infected by Borrelia burgdorferi while only 10% of Northern A. americanum and I. scapularis ticks are infected by Borrelia species. The first specific aim of this project was to determine the bacterial community that inhabits the midgut of Texas and Northeastern ticks by using high throughput metagenomic sequencing to sequence bacterial 16S rDNA. Through the use of massively parallel 454 sequencing, we were able to individually sequence hundreds of thousands of 16S rDNA regions of the bacterial flora from 133 ticks from the New York, Missouri and Texas. The presence of previously confirmed endosymbionts, specifically the Rickettsia spp. and Coxiella spp., that are commonly found in ticks were confirmed, as well as some highly prevalent genera that were previously undocumented. Furthermore, multiple pathogenic genera sequences were often found in the same tick, suggesting the possibility of co-infection of multiple pathogenic species. The second specific aim was to use Borrelia specific primers to screen 344 individual ticks from Missouri, Texas and the Northeast to determine the prevalence of Borrelia species in ticks. To screen for Borrelia species, two housekeeping genes, uvrA and recG, were selected as well as the 16S-23S rDNA intergenic spacer. Ticks from Missouri, Texas and New York were screened. None of the Missouri or Texas ticks tested positive for Borrelia spp. The rate of I. scapularis infection by B.burgdorferi is dependent on tick feeding activity as well as reservoir availability. B. burgdorferi is endemic in the Northeast, sometimes reported as highly present in over 50% of all I. scapularis ticks. 11.6% of all New York ticks were positive for a species of Borrelia, however only 6.9% of all New York ticks were positive for B. burgdorferi. Despite being significantly lower than 50%, the results still fall in line with previous reports of about the prevalence of B. burgdorferi. 1.5% of all Texas ticks were positive for a Borrelia species, specifically B. lonestari. While this study was unable to identify the causative agent for STARI, 454 sequencing was able to provide a tremendous insight into the bacterial flora and possible pathogenic species of both the I. scapularis and the A. americanum tick.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^