Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response. Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne pathogen that successfully colonizes both ticks and a variety of vertebrate hosts. In mammals, B. burgdorferi is frequently found associated with connective tissues (1-13), and the bacterium is often detected in cartilaginous or collagen-rich tissues, such as skin and joints (6,7,9,(11)(12)(13)(14)(15)(16)(17)(18). Adhesins expressed by B. burgdorferi facilitate interactions with these tissues. B. burgdorferi expresses a plethora of outer surface proteins that interact with numerous components of the extracellular matrix (ECM), such as fibronectin, decorin, and integrins (19-23). The attachment of B. burgdorferi to the host ECM is likely critical for pathogenesis and persistence in mammals. Indeed, for many of these adhesins, deletion mutants have significant defects in infectivity, fail to disseminate widely in the host, or have other effects on disease, such as alterations in the severity of arthritis (24-27).The interactions of B. burgdorferi with fibronectin are facilitated by several distinct bacterial surface proteins, including BBK32, RevA, BB0347, and CspA (BbCRASP-1) (28-31). The redundancy in adhesins makes it difficult to dissect the role of each B. burgdorferi fibronectin binding protein in the pathogenesis of Lyme disease. For example, real-time microscopic imaging in live mice revealed a significant role for BBK32 in interactions with host vasculature, yet bbk32 mutants are still able to infect mammals (32).RevA is a 19-kDa surface protein encoded on the circular plasmid 32 (cp32) family of plasmids (33). RevA expression is elevated during mammalian infection over its expression in the tick vector, suggesting a role in pathogenesis (30,(34)(35)(36). RevA binds to fibronectin, and anti-RevA antibodies block the binding of whole B. burgdorferi bacteria to fibronectin (30). RevA is antigenic, as evidenced by the fact that both mice and human Lyme disease patients produce antibodies...

Borrelia burgdorferi SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete

“…The pCR5 constructs were reisolated from the spoVG-ON strains and were found to have sequences that were identical to that of the initial plasmid, indicating that no mutations had occurred within B. burgdorferi. A control plasmid that produced elevated levels of revA mRNA yielded elevated levels of the RevA protein, indicating that transcripts could be translated from such constructs (22).…”

“…Strains of B. burgdorferi that constitutively transcribe elevated levels of spoVG were generated as follows. The spoVG gene was cloned into the previously described plasmid pBLS715 (22), directly 3= of the gentamicin resistance gene, to create pCRS5 (Fig. 7).…”

“…The plasmid insert was sequenced to ensure that spoVG was cloned in-frame without mutations. The spoVG ribosomal binding site was modified to AGGAGG to ensure efficient translation of SpoVG protein (22). B. burgdorferi B31-A3 was transformed with pCRS5 on three separate occasions.…”

“…Relative spoVG expression levels during tick colonization and murine infection were determined by qRT-PCR using the oligonucle-FIG 7 pCRS5 (spoVG-ON) plasmid construction. spoVG was cloned into the BamHI and KpnI sites of our previously described pBLS715 plasmid (22). The parental construct was created to contain a consensus ribosome-binding site (RBS) appropriately placed 5= of the insert.…”

The Lyme disease spirochete's BpuR DNA/RNA‐binding protein is differentially expressed during the mammal–tick infectious cycle, which affects translation of the SodA superoxide dismutase

“…The initial studies on the impacts of elevated BpuR concentrations were performed using a plasmid construct that puts bpuR under control of a constitutive promoter. Plasmid pBLS715 was derived from cloning vector pBSV2‐G by removing the multiple cloning sequence region, then inserting an RBS and restriction endonuclease cleavage sites immediately 3′ of aac1 , the gentamicin resistance‐encoding gene (Elias et al , ; Byram et al , ). Open reading frames cloned into those sites are transcribed by RNA polymerase initiating from the constitutive P flgB promoter that is 5′ of aac1 .…”