The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick-mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.

Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi. The gene for outer surface protein A (OspA) from B. burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli. C3H/HeJ mice actively immunized with live transformed E. coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B. burgdorferi. Recombinant OspA is a candidate for a vaccine for Lyme borreliosis.

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.

Tick saliva has pleiotropic properties that facilitate persistence of the arthropod upon the host. We now describe a feeding-inducible protein in Ixodes scapularis saliva, Salp15, that inhibits CD4(+) T cell activation. The mechanism involves the repression of calcium fluxes triggered by TCR ligation and results in lower production of interleukin-2. Salp15 also inhibits the development of CD4(+) T cell-mediated immune responses in vivo, demonstrating the functional importance of this protein. Salp15 provides a molecular basis for understanding the immunosuppressive activity of I. scapularis saliva and vector-host interactions.

Although recombinant outer surface protein (6), as well as the immune-deficient scid (7,8). Indeed, we previously showed that mice actively immunized with a recombinant OspA were protected from infection and disease when challenged with an intradermal injection of three virulent B. burgdorferi isolates (6). The effectiveness of immunization against the natural mode of transmission of this tick-borne infection, however, was not reported. Tick-mediated transmission may differ from syringe transmission in several crucial ways. Antiinflammatory properties of the saliva of ticks may enhance pathogen transmission (9, 10) as occurs in the case of phlebotomine sandfly saliva and infection by Leishmania braziliensis (11). In addition, vector-borne pathogens may differ from those propagated in vitro in terms of immunogenicity as well as other transmission-related properties. A realistic challenge of a vector-borne agent of disease seems essential in evaluating a vaccine. We now demonstrate that active immunization with recombinant OspA protects mice against tick-borne spirochetal infection by destroying B. burgdorferi in ticks feeding on vaccinated mice. MATERIALS AND METHODSMice. Three-week-old, random sex, virus-antibody-free C3H/HeJ (C3H) mice were obtained from The Jackson Laboratory. They were shipped in filter-equipped crates and housed in micro-isolator cages. Food and water were provided ad libitum. Mice were killed with carbon dioxide gas. Outbred CD-1 mice were obtained from Charles River Breeding Laboratories.B. burgdorferi. Low in vitro passage isolates of B. burgdorferi N40, with previously proven infectivity and pathogenicity in C3H mice, were utilized (12). The spirochetes were grown to logarithmic phase in modified BarbourStoenner-Kelly (BSK II) medium and counted in a hemocytometer under darkfield microscopy.Recombinant OspA Fusion Protein. Recombinant OspA was expressed and purified as a fusion protein with glutathione transferase (GT) (6). In brief, the gene for OspA from N40 was ligated into plasmid pGEX-2T (Pharmacia) in frame with the GT gene. The recombinant plasmid was used to transform Escherichia coli strain DH5a. Production of the recombinant fusion protein was induced with isopropyl -Dthiogalactopyranoside, and the protein was purified from the cell extract by affinity chromatography on a glutathioneSepharose 4B column (Pharmacia) (6). Infection of Tiks with B. bwgdorfer. Ixodes dammini tickswere from a laboratory colony (maintained at the Harvard School of Public Health) derived from an Ipswich, Massachusetts, population, and had been determined to be free of inherited spirochetal infection. Outbred CD-1 mice were infected by means of intradermal inoculation of 103 lowpassage N40 spirochetes 3 weeks prior to serving as hosts. Ticks were infected with B. burgdorferi by allowing larvae to feed to repletion on these mice. Upon repletion, engorged larvae were collected, pooled in groups of 100-200, and permitted to molt to the nymphal stage at 21'C and 95% relative humidity. Prevalence of...