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. ^

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. ^

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. ^