Emerging Tick-Borne Diseases.

Increases in tick-borne disease prevalence and transmission are important public health issues. Efforts to control these emerging diseases are frustrated by the struggle to control tick populations and to detect and treat infections caused by the pathogens that they transmit. This review covers tick-borne infectious diseases of nonrickettsial bacterial, parasitic, and viral origins. While tick surveillance and tracking inform our understanding of the importance of the spread and ecology of ticks and help identify areas of risk for disease transmission, the vectors are not the focus of this document. Here, we emphasize the most significant pathogens that infect humans as well as the epidemiology, clinical features, diagnosis, and treatment of diseases that they cause. Although detection via molecular or immunological methods has improved, tick-borne diseases continue to remain underdiagnosed, making the scope of the problem difficult to assess. Our current understanding of the incidence of tick-borne diseases is discussed in this review. An awareness of the diseases that can be transmitted by ticks in specific locations is key to detection and selection of appropriate treatment. As tick-transmitted pathogens are discovered and emerge in new geographic regions, our ability to detect, describe, and understand the growing public health threat must also grow to meet the challenge.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis.

Flavodiiron proteins (FDPs) contain a unique active site consisting of a non-heme diiron carboxylate site proximal to a flavin mononucleotide (FMN). FDPs serve as the terminal components for reductive scavenging of dioxygen (to water) or nitric oxide (to nitrous oxide), which combats oxidative or nitrosative stress in many bacteria. Characterizations of FDPs from spirochetes or from any oral microbes have not been previously reported. Here, we report characterization of an FDP from the anaerobic spirochete, Treponema (T.) denticola, which is associated with chronic periodontitis. The isolated T. denticola FDP exhibited efficient four-electron dioxygen reductase activity and lower but significant anaerobic nitric oxide reductase activity. A mutant T. denticola strain containing the inactivated FDP-encoding gene was significantly more air-sensitive than the wild-type strain. Single turnover reactions of the four-electron-reduced FDP (FMNH2-Fe(II)Fe(II)) (FDPred) with O2 monitored on the milliseconds to seconds time scale indicated initial rapid formation of a spectral feature consistent with a cis-µ-1,2-peroxo-diferric intermediate, which triggered two-electron oxidation of FMNH2. Reaction of FDPred with NO showed apparent cooperativity between binding of the first and second NO to the diferrous site. The resulting diferrous dinitrosyl complex triggered two-electron oxidation of the FMNH2. Our cumulative results on this and other FDPs indicate that smooth two-electron FMNH2 oxidation triggered by the FDPred/substrate complex and overall four-electron oxidation of FDPred to FDPox constitutes a mechanistic paradigm for both dioxygen and nitric oxide reductase activities of FDPs. Four-electron reductive O2 scavenging by FDPs could contribute to oxidative stress protection in many other oral bacteria.

Real-time PCR and pyrosequencing for differentiation of medically relevant Bartonella species.

Multiple Bartonella species cause disease in humans. Although fast and accurate species differentiation could inform effective treatment interventions, species-level diagnosis of Bartonella infections is not typical. Here we describe a real-time PCR and pyrosequencing based algorithm for rapid differentiation of at least 11 medically relevant Bartonella spp.

Treponema denticola superoxide reductase: in vivo role, in vitro reactivities, and a novel [Fe(Cys)(4)] site.

In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, Treponema denticola (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe(His)(4)Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe(Cys)(4)] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe(Cys)(4)] sites of "canonical" 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe(Cys)(4)] protein. Structural modeling is consistent with iron ligation of the "noncanonical" Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe(Cys)(4)] sites. However, quantitative formation of stable [Fe(Cys)(4)] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe(Cys)(4)] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress.

Cryo-electron tomography elucidates the molecular architecture of Treponema pallidum, the syphilis spirochete.

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

The flat-ribbon configuration of the periplasmic flagella of Borrelia burgdorferi and its relationship to motility and morphology.

Electron cryotomography was used to analyze the structure of the Lyme disease spirochete, Borrelia burgdorferi. This methodology offers a new means for studying the native architecture of bacteria by eliminating the chemical fixing, dehydration, and staining steps of conventional electron microscopy. Using electron cryotomography, we noted that membrane blebs formed at the ends of the cells. These blebs may be precursors to vesicles that are released from cells grown in vivo and in vitro. We found that the periplasmic space of B. burgdorferi was quite narrow (16.0 nm) compared to those of Escherichia coli and Pseudomonas aeruginosa. However, in the vicinity of the periplasmic flagella, this space was considerably wider (42.3 nm). In contrast to previous results, the periplasmic flagella did not form a bundle but rather formed a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense. We show how the ribbon configuration of the assembled periplasmic flagella is more advantageous than a bundle for both swimming and forming the flat-wave morphology. Previous results indicate that B. burgdorferi motility is dependent on the rotation of the periplasmic flagella in generating backward-moving waves along the length of the cell. This swimming requires that the rotation of the flagella exerts force on the cell cylinder. Accordingly, a ribbon is more beneficial than a bundle, as this configuration allows each periplasmic flagellum to have direct contact with the cell cylinder in order to exert that force, and it minimizes interference between the rotating filaments.

Development of a novel chloramphenicol resistance expression plasmid used for genetic complementation of a fliG deletion mutant in Treponema denticola.

A new expression plasmid containing the fla operon promoter and a staphylococcal chloramphenicol resistance gene, was constructed to help assess the role of fliG in Treponema denticola motility. Deletion of fliG resulted in a nonmotile mutant with a markedly decreased number of flagellar filaments. Wild-type fliG genes from T. denticola and from Treponema pallidum were cloned into this expression plasmid. In both cases, the gene restored the ability of the mutant to gyrate its cell ends and enabled colony spreading in agarose. This shuttle plasmid enables high-level expression of genes in T. denticola and possesses an efficient selectable marker that provides a new tool for treponemal genetics.