In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.
Treponema denticola is a proteolytic anaerobic spirochete and key contributor to periodontal disease of microbial etiology. As periodontal disease develops and progresses, T. denticola thrives in the hostile environment of the subgingival crevice by exploiting the negative regulatory activity of the complement protein, factor H (FH). FH bound to the cell surface receptor, FhbB (FH binding protein B), is competent to serve as a cofactor for the Factor I mediated‐cleavage of the opsonin C3b. However, bound FH is ultimately cleaved by the T. denticola protease, dentilisin. As the T. denticola population expands, the rate of FH cleavage may exceed its rate of replenishment leading to local FH depletion and immune dysregulation culminating in tissue and ligament destruction and tooth loss. The goal of this study was to develop a T. denticola FhbB based‐vaccine antigen that can block FH binding and cleavage and kill cells via antibody‐mediated bactericidal activity. Tetra (FhbB‐ch4) and pentavalent fhbB (FhbB‐ch5) chimerics were engineered to have attenuated FH binding ability. The chimerics were immunogenic and elicited high‐titer bactericidal and agglutinating antibody. Anti‐Fhb‐ch4 antisera blocked FH binding and cleavage by the T. denticola protease, dentilisin, in a dose dependent manner. Precedent for the use of FH binding proteins comes from the successful development of two FDA approved vaccines for type B Neiserria meningitidis. This study is the first to extend this approach to the development of a preventive or therapeutic vaccine (or monoclonal Ab) for periodontal disease.
Lyme disease (LD) is a tick-transmitted bacterial infection caused by
Borreliella burgdorferi
and other closely related species collectively referred to as the LD spirochetes. The LD spirochetes encode an uncharacterized family of proteins originally designated
p
rotein
f
amily
t
welve (PF12).
Periodontal disease (PD) is a progressive inflammatory condition characterized by degradation of the gingival epithelium, periodontal ligament, and alveolar bone ultimately resulting in tooth loss. Treponema denticola is a keystone periopathogen that contributes to immune dysregulation and direct tissue destruction. As periodontal disease develops, T. denticola must adapt to environmental, immunological and physiochemical changes in the subgingival crevice. T. denticola produces bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), an important regulatory nucleotide. While T. denticola encodes several putative diguanylate cyclases (DGCs), none have been studied and hence the biological role of c-di-GMP in oral treponemes remains largely unexplored. Here we demonstrate that the T. denticola open reading frame, TDE0125, encodes a functional DGC designated as DgcA (Diguanylate cyclase A). The dgcA gene is universal among T. denticola isolates, highly conserved and is a stand-alone GGEEF protein with a GAF domain. Recombinant DgcA converts GTP to c-di-GMP using either manganese or magnesium under aerobic and anaerobic reaction conditions. Size exclusion chromatography revealed that DgcA exists as a homodimer and in larger oligomers. Site-directed mutagenesis of residues that define the putative inhibitory site of DgcA suggest that c-di-GMP production is allosterically regulated. This report is the first to characterize a DGC of an oral treponeme.
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