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.

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.

Serologic Evidence for the Exposure of Eastern Coyotes (Canis latrans) in Pennsylvania to the Tick-Borne Pathogens Borreliella burgdorferi and Anaplasma phagocytophilum.

Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively.IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.

House finches with high coccidia burdens experience more severe experimental Mycoplasma gallisepticum infections.

Parasites co-infecting hosts can interact directly and indirectly to affect parasite growth and disease manifestation. We examined potential interactions between two common parasites of house finches: the bacterium Mycoplasma gallisepticum that causes conjunctivitis and the intestinal coccidian parasite Isospora sp. We quantified coccidia burdens prior to and following experimental infection with M. gallisepticum, exploiting the birds' range of natural coccidia burdens. Birds with greater baseline coccidia burdens developed higher M. gallisepticum loads and longer lasting conjunctivitis following inoculation. However, experimental inoculation with M. gallisepticum did not appear to alter coccidia shedding. Our study suggests that differences in immunocompetence or condition may predispose some finches to more severe infections with both pathogens.