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

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 OspEBBN38, OspEBBL39, and OspEBBP38. 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.

A single immunization of Borreliella burgdorferi-infected mice with Vanguard crLyme elicits robust antibody responses to diverse strains and variants of outer surface protein C.

Lyme disease, caused by Borreliella burgdorferi and related species, is a growing health threat to companion animals across North America and Europe. Vaccination is an important preventive tool used widely in dogs living in, or near, endemic regions. In this report, we assessed anti-outer surface protein (Osp) A and anti-OspC antibody responses in B. burgdorferi-infected and -naïve mice (C3H/HeN) after immunization with a murine-optimized single dose of the Lyme disease subunit vaccine, Vanguard crLyme. crLyme is comprised of OspA and an OspC chimeritope-based immunogen designated as CH14. Mice that were infected and immunized developed higher levels of anti-OspC antibodies (Abs) than those infected only or that received one vaccine dose. The anti-OspC Abs that developed in the infected/immunized mice bound to all OspC variants tested (n = 22), whereas OspC Abs in serum from infected mice bound predominantly to the OspC variant (type A) produced by the infecting B. burgdorferi strain. Consistent with the absence of OspA expression in infected mammals, none of the infected mice developed Abs to OspA and did not develop anti-OspA Abs after single dose immunization. Lastly, serum from infected/immunized mice displayed significantly higher and broader killing activity than serum from non-immunized infected mice. The results of this study demonstrate that a single vaccination of actively infected mice results in strong anti-OspC Ab responses. This study contributes to our understanding of Ab responses to vaccination in actively infected mammals.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

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.

Human and Veterinary Vaccines for Lyme Disease.

Lyme disease (LD) is an emerging zoonotic infection that is increasing in incidence in North America, Europe, and Asia. With the development of safe and efficacious vaccines, LD can potentially be prevented. Vaccination offers a cost-effective and safe approach for decreasing the risk of infection. While LD vaccines have been widely used in veterinary medicine, they are not available as a preventive tool for humans. Central to the development of effective vaccines is an understanding of the enzootic cycle of LD, differential gene expression of Borrelia burgdorferi in response to environmental variables, and the genetic and antigenic diversity of the unique bacteria that cause this debilitating disease. Here we review these areas as they pertain to past and present efforts to develop human, veterinary, and reservoir targeting LD vaccines. In addition, we offer a brief overview of additional preventative measures that should employed in conjunction with vaccination.

Immunization against Anaplasma phagocytophilum Adhesin Binding Domains Confers Protection against Infection in the Mouse Model.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola.

The oral microbiome consists of a remarkably diverse group of 500-700 bacterial species. The microbial etiology of periodontal disease is similarly complex. Of the ~400 bacterial species identified in subgingival plaque, at least 50 belong to the genus Treponema. As periodontal disease develops and progresses, T. denticola transitions from a low to high abundance species in the subgingival crevice. Changes in the overall composition of the bacterial population trigger significant changes in the local physical, immunological and physiochemical conditions. For T. denticola to thrive in periodontal pockets, it must be nimble and adapt to rapidly changing environmental conditions. The purpose of this chapter is to review the current understanding of the molecular basis of these essential adaptive responses, with a focus on the role of two component regulatory systems with global regulatory potential.

The Treponema denticola PAS Domain-Containing Histidine Kinase Hpk2 Is a Heme Binding Sensor of Oxygen Levels.

Periodontal disease (PD) results from a shift in the composition of the microbial community of the subgingival crevice. As the bacterial population transitions from Gram-positive bacteria to predominantly Gram-negative anaerobes and spirochetes, dramatic changes occur in the physiological and immunological environment at diseased sites. Treponema denticola thrives in periodontal pockets, indicating that it has a unique ability to adapt to changing environmental conditions. Hpk2 (tde1970), a Per-Arnt-Sim motif (PAS) domain-containing histidine kinase (HK), is part of the T. denticola Hpk2-Rrp2 (tde1969) two-component regulatory (TCR) system. This TCR system is growth phase regulated and has been postulated to play a key role in adaptive responses. In this study, we employ predictive structural analyses and site-directed mutagenesis to investigate the functional role of specific amino acid residues located within the Hpk2 PAS domain. Specific substitutions impacted autophosphorylation (AP), phosphotransfer (PT), oligomerization, and hemin binding. The AP, PT, hemin binding, and oligomerization potential of some mutated Hpk2 proteins differed under aerobic versus anaerobic reaction conditions. The data presented here suggest that the regulatory activity of Hpk2 is linked to diatomic gas levels. In a broader sense, this study highlights the importance of studying proteins produced by anaerobes under conditions that approximate the environment in which they thrive.IMPORTANCE Periodontal disease affects nearly 60% of the global adult population. Its costs to individuals, and to society as a whole, are enormous. As periodontal disease develops, there is a shift in the composition of the oral microbial community. The bacteria that become dominant are able to cause significant damage to the tissues that support the teeth, leading to tooth loss. Treponema denticola is one of the keystone pathogens associated with periodontal disease. An earlier study demonstrated that the Hpk2 and Rrp2 proteins play an important role in adaptive responses. Here, we explore the role of specific Hpk2 amino acids in environmental sensing and function, using structural analyses and site-directed mutagenesis.

Orientia tsutsugamushi Ank9 is a multifunctional effector that utilizes a novel GRIP-like Golgi localization domain for Golgi-to-endoplasmic reticulum trafficking and interacts with host COPB2.

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that afflicts 1 million people annually. This obligate intracellular bacterium boasts one of the largest microbial arsenals of ankyrin repeat-containing protein (Ank) effectors, most of which target the endoplasmic reticulum (ER) by undefined mechanisms. Ank9 is the only one proven to function during infection. Here, we demonstrate that Ank9 bears a motif that mimics the GRIP domain of eukaryotic golgins and is necessary and sufficient for its Golgi localization. Ank9 reaches the ER exclusively by retrograde trafficking from the Golgi. Consistent with this observation, it binds COPB2, a host protein that mediates Golgi-to-ER transport. Ank9 destabilizes the Golgi and ER in a Golgi localization domain-dependent manner and induces the activating transcription factor 4-dependent unfolded protein response. The Golgi is also destabilized in cells infected with O. tsutsugamushi or treated with COPB2 small interfering RNA. COPB2 reduction and/or the cellular events that it invokes, such as Golgi destabilization, benefit Orientia replication. Thus, Ank9 or bacterial negative modulation of COPB2 might contribute to the bacterium's intracellular replication. This report identifies a novel microbial Golgi localization domain, links Ank9 to the ability of O. tsutsugamushi to perturb Golgi structure, and describes the first mechanism by which any Orientia effector targets the secretory pathway.

Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLex). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLex fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

Essential domains of Anaplasma phagocytophilum invasins utilized to infect mammalian host cells.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLe(x)-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLe(x) sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLe(x) but express the structurally similar glycan, 6-sulfo-sLe(x), required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLe(x) antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLe(x) antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.

The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin.

The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation.

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs.

UNLABELLED: A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCE: Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets. Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates that O. tsutsugamushi strain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that an Orientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fide Orientia effector and the first example of an endogenous F-box-containing Ank-mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

Anaplasma phagocytophilum surface protein AipA mediates invasion of mammalian host cells.

Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA1-87 ) were found to be essential for host cell invasion. Recombinant AipA1-87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1-87 , but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1-87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis.

Comparison of effectiveness of cefovecin, doxycycline, and amoxicillin for the treatment of experimentally induced early Lyme borreliosis in dogs.

BACKGROUND: While Koch's postulates have been fulfilled for Lyme disease; causing transient fever, anorexia and arthritis in young dogs; treatment of sero-positive dogs, especially asymptomatic animals, remains a topic of debate. To complicate this matter the currently recommended antibiotic treatments of Lyme Disease in dogs caused by Borrelia burgdorferi require daily oral administrations for 31 days or longer, which makes non-compliance a concern. Additionally, there is no approved veterinary antimicrobial for the treatment of Lyme Disease in dogs in the USA and few recommended treatments have been robustly tested. In vitro testing of cefovecin, a novel extended-spectrum cephalosporin, demonstrated inhibition of spirochete growth. A small pilot study in dogs indicated that two cefovecin injections two weeks apart would be as efficacious against B. burgdorferi sensu stricto as the recommended treatments using doxycycline or amoxicillin daily for 31 days. This hypothesis was tested in 17-18 week old Beagle dogs, experimentally infected with B. burgdorferi sensu stricto, using wild caught ticks, 75 days prior to antimicrobial administration. RESULTS: Clinical observations for lameness were performed daily but were inconclusive as this characteristic sign of Lyme Disease rarely develops in the standard laboratory models of experimentally induced infection. However, each antibiotic tested was efficacious against B. burgdorferi as measured by a rapid elimination of spirochetes from the skin and reduced levels of circulating antibodies to B. burgdorferi. In addition, significantly less cefovecin treated animals had Lyme Disease associated histopathological changes compared to untreated dogs. CONCLUSIONS: Convenia was efficacious against B. burgdorferi sensu stricto infection in dogs as determined by serological testing, PCR and histopathology results. Convenia provides an additional and effective treatment option for Lyme Disease in dogs.

The Borrelia hermsii factor H binding protein FhbA is not required for infectivity in mice or for resistance to human complement in vitro.

The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii. It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro, B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen for B. hermsii infection in humans. The ability to test the hypothesis that FhbA is a critical virulence factor in vivo has been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated a B. hermsii fhbA deletion mutant (the B. hermsii YORΔfhbA strain) through allelic exchange mutagenesis. Deletion of fhbA abolished FH binding by the YORΔfhbA strain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbA strain remained infectious in mice and retained resistance to killing in vitro by human complement. Collectively, these results indicate that B. hermsii employs an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

Development of novel multi-protein chimeric immunogens that protect against infection with the Lyme disease agent, Borreliella burgdorferi.

Lyme disease is the most common tick-borne disease in North America. A vaccine for use in humans is not available. Here, we detail the development of two chimeric vaccine antigens, BAF and Chv2M. BAF elicits Abs that target proteins and protein variants produced by Borreliella species in ticks (OspB and OspA) and mammals (FtlA/B). OspB serves as the backbone structure for the BAF chimeric. Two OspA221-240 epitope-containing domain (ECD) variants (#A1 and #A15) were inserted into a loop in OspB. The N-terminal region of the FtlA protein was joined to the C-terminus of the chimeric. The second chimeric, Chv2M, consists of L5 (loop 5) and H5 (helix 5) ECDs derived from diverse OspC proteins. Borreliella species produce OspC upon exposure to the bloodmeal and during early infection in mammals. Here, we demonstrate that BAF and Chv2M are potent immunogens that elicit Abs that bind to each component protein (FtlA, FtlB, OspB, and multiple OspA and OspC variants). Anti-BAF and anti-Chv2M Abs kill Borreliella burgdorferi strains through Ab-mediated complement-dependent and complement-independent mechanisms. Eighty percent (32/40) of mice that received a three-dose vaccine regimen were protected from infection with B. burgdorferi B31. Efficacy increased to 90% (18/20) when the amount of Chv2M was increased in the third vaccine dose. Readouts for infection were flaB PCR and seroconversion to VlsE. This study establishes proof of principle for a chimeric immunogen vaccine formulation that elicits Abs to multiple targets on the B. burgdorferi cell surface produced during tick and mammalian stages of the enzootic cycle.IMPORTANCELyme disease is a growing public health threat across parts of the Northern Hemisphere. Regions that can support sustained tick populations are expanding, and the incidence of tick-borne diseases is increasing. In light of the increasing risk of Lyme disease, effective preventive strategies are needed. Most vaccine development efforts have focused on outer surface protein A, a Borreliella burgdorferi protein produced only in ticks. Herein, we describe the development of a novel vaccine formulation consisting of two multivalent chimeric proteins that are immunogenic and elicit antibodies with bactericidal activity that target several cell surface proteins produced by the Lyme disease spirochetes in feeding ticks and mammals. In a broader sense, this study advances efforts to develop custom-designed vaccinogens comprised of epitope-containing domains from multiple proteins.

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.