Mechanism of Borrelia immune evasion by FhbA-related proteins.

Immune evasion facilitates survival of Borrelia, leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 Å crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19-20, combined with extensive mutagenesis, we identified the structural mechanism by which B. hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia. Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B. duttonii. By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia.

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology.

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

Fibronectin-binding protein of Borrelia hermsii expressed in the blood of mice with relapsing fever.

To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp(-) cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins.

Dual binding specificity of a Borrelia hermsii-associated complement regulator-acquiring surface protein for factor H and plasminogen discloses a putative virulence factor of relapsing fever spirochetes.

Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.

High production of CXCL13 in blood and brain during persistent infection with the relapsing fever spirochete Borrelia turicatae.

Relapsing fever (RF) is a multisystemic borrelial infection with frequent neurologic involvement referred to as neuroborreliosis. The absence of an effective antibody response results in persistent infection. To study the consequences to the brain of persistent infection with the RF spirochete Borrelia turicatae, we studied B cell (Igh6-/-) and B and T (Rag1-/-) cell-deficient mice inoculated with isogenic serotypes 1 (Bt1) or 2 (Bt2). We found that Bt1 was more tissue tropic than Bt2, not only for brain but also for heart. Igh6-/- mice developed more severe clinical disease than Rag1-/- mice. Bt1-infected brains had widespread microgliosis/brain macrophage activation despite localization of spirochetes in the leptomeninges rather than the brain parenchyma itself. Oligoarray analysis revealed that CXCL13 was the most upregulated gene in the brain of Bt1-infected Igh6-/- mice. CXCL13 was also the most abundant of the chemokines we measured in infected blood. Persistent infection did not result in injury to the brain. Treatment with exogenous interleukin-10 reduced microgliosis in the brain and production of CXCL13 in the blood. We concluded that brain involvement in B cell-deficient mice persistently infected with B. turicatae is characterized by prominent microgliosis and production of CXCL13 without detectable injury.

Relapsing fever spirochetes Borrelia recurrentis and B. duttonii acquire complement regulators C4b-binding protein and factor H.

Relapsing fever is a rapidly progressive and severe septic disease caused by certain Borrelia spirochetes. The disease is divided into two forms, i.e., epidemic relapsing fever, caused by Borrelia recurrentis and transmitted by lice, and the endemic form, caused by several Borrelia species, such as B. duttonii, and transmitted by soft-bodied ticks. The spirochetes enter the bloodstream by the vector bite and live persistently in plasma even after the development of specific antibodies. This leads to fever relapses and high mortality and clearly indicates that the Borrelia organisms utilize effective immune evasion strategies. In this study, we show that the epidemic relapsing fever pathogen B. recurrentis and an endemic relapsing fever pathogen, B. duttonii, are serum resistant, i.e., resistant to complement in vitro. They acquire the host alternative complement pathway regulator factor H on their surfaces in a similar way to that of the less serum-resistant Lyme disease pathogen, B. burgdorferi sensu stricto. More importantly, the relapsing fever spirochetes specifically bind host C4b-binding protein, a major regulator of the antibody-mediated classical complement pathway. Both complement regulators retained their functional activities when bound to the surfaces of the spirochetes. In conclusion, this is the first report of complement evasion by Borrelia recurrentis and B. duttonii and the first report showing capture of C4b-binding protein by spirochetes.

Horizontally acquired genes for purine salvage in Borrelia spp. causing relapsing fever.

Unlike Borrelia burgdorferi, the relapsing fever agent Borrelia hermsii and the related Borrelia miyamotoi had purA and purB genes of the purine salvage pathway. These were located among the rRNA genes. Phylogenetic analysis indicated that these genes had a different evolutionary history than those of orthologs in other spirochetes.

Vector interactions and molecular adaptations of lyme disease and relapsing fever spirochetes associated with transmission by ticks.

Pathogenic spirochetes in the genus Borrelia are transmitted primarily by two families of ticks. The Lyme disease spirochete, Borrelia burgdorferi, is transmitted by the slow-feeding ixodid tick Ixodes scapularis, whereas the relapsing fever spirochete, B. hermsii, is transmitted by Ornithodoros hermsi, a fast-feeding argasid tick. Lyme disease spirochetes are generally restricted to the midgut in unfed I. scapularis. When nymphal ticks feed, the bacteria pass through the hemocoel to the salivary glands and are transmitted to a new host in the saliva after 2 days. Relapsing fever spirochetes infect the midgut in unfed O. hermsi but persist in other sites including the salivary glands. Thus, relapsing fever spirochetes are efficiently transmitted in saliva by these fast-feeding ticks within minutes of their attachment to a mammalian host. We describe how B. burgdorferi and B. hermsii change their outer surface during their alternating infections in ticks and mammals, which in turn suggests biological functions for a few surface-exposed lipoproteins.

Variable major lipoprotein is a principal TNF-inducing factor of louse-borne relapsing fever.

Massive release of tumor necrosis factor is responsible for the potentially fatal larisch-Herxheimer reaction that follows antibiotic treatment of relapsing fever due to Borrelia recurrentis. We have undertaken the quantitative purification of the components of B. recurrentis that stimulate human monocytes to produce tumor necrosis factor. We show that the predominant factor inducing tumor necrosis factor is a variable lipoprotein homologous to the variable major protein of B. hermsii. We found antibodies to different forms of variable major protein in two patients with louse-borne relapsing fever. The three purified variable major proteins studied here differ in their ability to induce tumor necrosis factor production, which may partly explain the variable clinical severity of borrelial infection. These results may be of considerable relevance for the pathogenesis of Lyme disease and other forms of human borreliosis.