Research Article: Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission

Date Published: May 29, 2018

Publisher: Public Library of Science

Author(s): Thomas Hart, Ngoc Thien Thu Nguyen, Nancy A. Nowak, Fuming Zhang, Robert J. Linhardt, Maria Diuk-Wasser, Sanjay Ram, Peter Kraiczy, Yi-Pin Lin, D. Scott Samuels.

http://doi.org/10.1371/journal.ppat.1007106

Abstract

Borrelia burgdorferi sensu lato (Bbsl), the causative agent of Lyme disease, establishes an initial infection in the host’s skin following a tick bite, and then disseminates to distant organs, leading to multisystem manifestations. Tick-to-vertebrate host transmission requires that Bbsl survives during blood feeding. Complement is an important innate host defense in blood and interstitial fluid. Bbsl produces a polymorphic surface protein, CspA, that binds to a complement regulator, Factor H (FH) to block complement activation in vitro. However, the role that CspA plays in the Bbsl enzootic cycle remains unclear. In this study, we demonstrated that different CspA variants promote spirochete binding to FH to inactivate complement and promote serum resistance in a host-specific manner. Utilizing a tick-to-mouse transmission model, we observed that a cspA-knockout B. burgdorferi is eliminated from nymphal ticks in the first 24 hours of feeding and is unable to be transmitted to naïve mice. Conversely, ectopically producing CspA derived from B. burgdorferi or B. afzelii, but not B. garinii in a cspA-knockout strain restored spirochete survival in fed nymphs and tick-to-mouse transmission. Furthermore, a CspA point mutant, CspA-L246D that was defective in FH-binding, failed to survive in fed nymphs and at the inoculation site or bloodstream in mice. We also allowed those spirochete-infected nymphs to feed on C3-/- mice that lacked functional complement. The cspA-knockout B. burgdorferi or this mutant strain complemented with cspA variants or cspA-L246D was found at similar levels as wild type B. burgdorferi in the fed nymphs and mouse tissues. These novel findings suggest that the FH-binding activity of CspA protects spirochetes from complement-mediated killing in fed nymphal ticks, which ultimately allows Bbsl transmission to mammalian hosts.

Partial Text

Lyme disease is caused by spirochetes of Bbsl and is transmitted to humans by the bites of infected Ixodes ticks. It is the most common vector-borne disease in North America and Europe [1, 2]. Upon blood feeding, spirochetes migrate from the ticks’ midguts to salivary glands, where they are transmitted to the host’s skin at the tick bite sites [2, 3]. In humans, Lyme borreliae initiate local skin infection often leading to erythema migrans, commonly known as a “bull’s-eye” rash [1, 2]. If left untreated, spirochetes are capable of entering the bloodstream and spreading to multiple tissues and organs, leading to arthritis, carditis, neuroborreliosis, and acrodermatitis chronica atrophicans [4]. The three main Lyme disease causing species, B. afzelii, B. garinii, and B. burgdorferi sensu stricto (hereafter B. burgdorferi), survive not only in humans, but also in other vertebrate animals [5]. These spirochete species tend to be associated with different vertebrate hosts: B. afzelii is typically isolated from small mammals, B. garinii from birds, and B. burgdorferi from both hosts [6, 7]. Specific spirochete-host associations are thought to be caused by the selective ability of these spirochetes to evade innate immune responses of different hosts. One such immune response, complement, is the first-line defense mechanism in humans and other vertebrates [7, 8].

Each of the Lyme borreliae species has been associated with specific vertebrate host(s) [7, 52, 53]. This spirochete-host association has been correlated with the ability of B. burgdorferi to survive in the blood (or serum) from the corresponding hosts [7, 54–56], but the mechanism that drives this association is still unclear. An attractive hypothesis is that the spirochetes exhibit host-specific immune evasion, which leads to the observed spirochete-host association. This could be due to variable spirochete outer surface proteins that interact with components of host complement in a host-specific manner. One such protein is CspA, which displays variant-to-variant differences in binding to human FH [25]. However, the recombinant CspA protein from B. burgdorferi strain B31 is incapable of binding to any other vertebrate animals’ FH, when the FH has been subjected to SDS-PAGE followed by a far-western blot [57]. In contrast, FH from the serum of multiple animals including human and mouse recognizes CspA variants run on a similar blot [58, 59]. This discrepancy may be due to structural alternations of animals’ FH on SDS-PAGE and the following far-western blot [8, 22, 23]. Therefore, we further verified the binding of CspA to FH from mouse, horse, and quail by demonstrating that the CspA of three main Lyme borreliae (B. burgdorferi, B. afzelii, or B. garinii) bind to purified FH in a host-specific manner. Additionally, we observed that the ability of different CspA variants to bind to FH correlates with their ability to inhibit complement activation on the spirochete surface and facilitate spirochete survival in serum in a host-specific manner. We found that these correlations not only apply to human complement (consistent with a previous study [28]) but also to complement of other animals. Further, the high-resolution structure of CspA suggests the recombinant version of this protein forms a dimer [35]. Two FH-binding regions have been localized on the central cleft of the CspA dimer and on the C-termini of this protein including leucine-246 [34, 60]. By ectopically producing a CspA point mutant (CspA-L246D) selectively lacking FH-binding ability on spirochetes, we further demonstrated that the CspA-mediated FH-binding activity contributes to the inactivation of host complement and spirochete survival in sera. Note that similar non-polar features of this position (leucine-246) of CspAB31 and the equivalent location of other CspA variants (phenylalanine-237 of CspAPKo and leucine-252 of CspAZQ1) suggests a possibility that this amino acid is critical for the FH-binding activity of these variants (S1 Fig).

 

Source:

http://doi.org/10.1371/journal.ppat.1007106