Characterization of c-di-AMP signaling in the periodontal pathobiont, Treponema denticola.

Pathobionts associated with periodontitis, such as Treponema denticola, must possess numerous sensory transduction systems to adapt to the highly dynamic subgingival environment. To date, the signaling pathways utilized by T. denticola to rapidly sense and respond to environmental stimuli are mainly unknown. Bis-(3'-5') cyclic diadenosine monophosphate (c-di-AMP) is a nucleotide secondary messenger that regulates osmolyte transport, central metabolism, biofilm development, and pathogenicity in many bacteria but is uncharacterized in T. denticola. Here, we studied c-di-AMP signaling in T. denticola to understand how it contributes to T. denticola physiology. We demonstrated that T. denticola produces c-di-AMP and identified enzymes that function in the synthesis (TDE1909) and hydrolysis (TDE0027) of c-di-AMP. To investigate how c-di-AMP may impact T. denticola cellular processes, a screening assay was performed to identify putative c-di-AMP receptor proteins. This approach identified TDE0087, annotated as a potassium uptake protein, as the first T. denticola c-di-AMP binding protein. As potassium homeostasis is critical for maintaining turgor pressure, we demonstrated that T. denticola c-di-AMP concentrations are impacted by osmolarity, suggesting that c-di-AMP negatively regulates potassium uptake in hypoosmotic solutions. Collectively, this study demonstrates T. denticola utilizes c-di-AMP signaling, identifies c-di-AMP metabolism proteins, identifies putative receptor proteins, and correlates c-di-AMP signaling to osmoregulation.

The leptospiral OmpA-like protein (Loa22) is a surface-exposed antigen that elicits bactericidal antibody against heterologous Leptospira.

Leptospirosis is the most widespread zoonosis, affecting over 1 million humans each year, with more than 60,000 deaths worldwide. Leptospirosis poses a significant health threat to dogs, horses, cattle, and wildlife. The disease may be self-limiting or progress to a life-threatening multi-system disorder affecting the kidneys, liver, and lungs. Currently, bacterin vaccine formulations that consist of one or more laboratory-cultivated strains are used for prevention. However, the antibody response elicited by these vaccines is directed primarily at lipopolysaccharide and is generally serovar-specific. The development of broadly protective subunit vaccines for veterinary and human applications would be a significant step forward in efforts to combat this emerging and antigenically variable pathogen. This study assessed the properties and potential utility of the Leptospira Loa22 (Leptospira OmpA-like 22 kDa protein) protein as a vaccine antigen. Loa22 is a virulence factor that is predicted to transverse the outer membrane and present its N-terminal domain on the cell surface. This report demonstrates that diverse Leptospira strains express Loa22 in vitro and that the protein is antigenic during infection in dogs. Immunoblot and size exclusion chromatography revealed that Loa22 exists in monomeric and trimeric forms. Immunization of rats with recombinant Loa22 elicited bactericidal antibodies against diverse Leptospira strains. The immunodominant bactericidal epitopes were localized within the N-terminal domain using protein-blocking bactericidal assays. This study supports the utility of Loa22, or subfragments thereof, in developing a multivalent chimeric subunit vaccine to prevent leptospirosis and sheds new light on the cellular localization of Loa22.

FtlA and FtlB Are Candidates for Inclusion in a Next-Generation Multiantigen Subunit Vaccine for Lyme 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 protein family twelve (PF12). In B. burgdorferi strain B31, PF12 consists of four plasmid-carried genes, encoding BBK01, BBG01, BBH37, and BBJ08. Henceforth, we designate the PF12 proteins family twelve lipoprotein (Ftl) A (FtlA) (BBK01), FtlB (BBG01), FtlC (BBH37), and FtlD (BBJ08). The goal of this study was to assess the potential utility of the Ftl proteins in subunit vaccine development. Immunoblot analyses of LD spirochete cell lysates demonstrated that one or more of the Ftl proteins are produced by most LD isolates during cultivation. The Ftl proteins were verified to be membrane associated, and nondenaturing PAGE revealed that FtlA, FtlB, and FtlD formed dimers, while FtlC formed hexamers. Analysis of serum samples from B. burgdorferi antibody (Ab)-positive client-owned dogs (n = 50) and horses (n = 90) revealed that a majority were anti-Ftl Ab positive. Abs to the Ftl proteins were detected in serum samples from laboratory-infected dogs out to 497 days postinfection. Anti-FtlA and FtlB antisera displayed potent complement-dependent Ab-mediated killing activity, and epitope localization revealed that the bactericidal epitopes reside within the N-terminal domain of the Ftl proteins. This study suggests that FtlA and FtlB are potential candidates for inclusion in a multivalent vaccine for LD.

High-resolution crystal structure of the Borreliella burgdorferi PlzA protein in complex with c-di-GMP: new insights into the interaction of c-di-GMP with the novel xPilZ domain.

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.

The Treponema denticola DgcA protein (TDE0125) is a functional diguanylate cyclase.

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. Treponema 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.

Development of an FhbB based chimeric vaccinogen that elicits antibodies that block Factor H binding and cleavage by the periopathogen Treponema denticola.

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.