Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals

Abstract

The causative agent of Lyme disease (LD), Borreliella burgdorferi, binds factor H (FH) and other complement regulatory proteins to its surface. B. burgdorferi B31 (type strain) encodes five FH-binding proteins (FHBPs): CspZ, CspA, and the OspE paralogs OspE_BBN38, OspE_BBL39, and OspE_BBP38. This study assessed potential correlations between the production of individual FHBPs, FH-binding ability, and serum resistance using a panel of infectious B. burgdorferi clonal populations recovered from dogs. FHBP production was assessed in cultivated spirochetes and by antibody responses in naturally infected humans, dogs, and eastern coyotes (wild canids). FH binding specificity and sensitivity to dog and human serum were also assessed and compared. No correlation was observed between the production of individual FHBPs and FH binding with serum resistance, and CspA was determined to not be produced in animals. Notably, one or more clones isolated from dogs lacked CspZ or the OspE proteins (a finding confirmed by genome sequence determination) and did not bind FH derived from canines. The data presented do not support a correlation between FH binding and the production of individual FHBPs with serum resistance and infectivity. In addition, the limited number and polymorphic nature of cp32s in B. burgdorferi clone DRI85A that were identified through genome sequencing suggest no strict requirement for a defined set of these replicons for infectivity. This study reveals that the immune evasion mechanisms employed by B. burgdorferi are diverse, complex, and yet to be fully defined.

Keywords: CspA; CspZ; Lyme disease; OspE; complement evasion; cp32; dogs; factor H.

Fig 1

FHBP production by B. burgdorferi clones derived from infected dogs. Cell lysates of B. burgdorferi clonal populations were fractionated by SDS-PAGE using AnyKda gels. Clone designations are indicated along the top of the figure. B. burgdorferi B31 served as a positive control. One gel was stained with Coomassie brilliant blue to demonstrate similar loading (top panel). Immunoblots were generated and screened with antisera to the FHBPs (indicated to the right). The molecular weight (MW) of OspE_BBN38 and OspE_BBL39 are 20.675 and 19.560 kDa, respectively. Variation in the MW of the OspE paralogs in some clones rendered definitive identification at the paralog level difficult. Hence, the proteins are labeled simply as OspE. Note that anti-OspE_BBN38 also reacts with OspF (indicated), which is not an FHBP. The migration position of MW standards is indicated to the left. The antisera were used at a 1:1,000 dilution. All methods were as described in the text, and the images were cropped for presentation purposes.

Fig 2

Comparative analysis of the FHBP profiles of B. burgdorferi clonal populations. Immunoblots of cell lysates were generated as described in the Materials and Methods section and in Fig. 1. The membranes were incubated with the dog, eastern coyote, or human sera as indicated. The sera, which served as the species-specific FH source, were confirmed to be antibody-negative for B. burgdorferi before use. Bound FH was detected using anti-FH antiserum, as detailed in the text. Clone designations are indicated along the top of the figure. The identities of the proteins that bound FH are indicated to the right.

Fig 3

Immunoblot analysis and ELISA of B. burgdorferi clones DRI85A and B31 using serum from infected mice. Mice were inoculated with B. burgdorferi clone DRI85A or B. burgdorferi B31. Seventeen days post-inoculation, blood was collected, and the serum was harvested. To assess seroconversion, the sera were screened by ELISA against recombinant DbpB and VlsE (B31 sequence) and the VlsE protein derived from clone DRI85A. OspA, a Borreliella protein that is not expressed during infection, and BSA, served as negative controls. Significance was assessed as detailed in the text (* indicates P < 0.05).