Borrelia burgdorferi ­ morphological structure and motility as adaptation for transmission and survival in the habitat of a tick-vertebrate setup

Lyme borreliosis is a multisystem chronic disease caused by Borrelia burgdorferi sensu lato (s.l.) spirochete transmitted by Ixodes. This bacterium has a remarkable ability to survive in tick-vertebrate setup. Its infection causes diagnostic and clinical difficulties. It was distinguished as a separate disease entity over 30 years ago. Observations made by Steere et al. proved to be a milestone since they found correlation between the occurrence of skin and joint lesions with tick bites. Further studies showed that the disease affects not only joints and skin, but also nervous and circulatory systems. Shortly afterwards, an etiological factor was identified ­ spirochete isolated by W. Burgdorfer (from ticks) as well as Steer and Benach (from blood). Research conducted by other authors confirmed that the spirochete named after its discoverer (Borrelia burgdorferi) is a common etiological factor for disease entities classified as Lyme borreliosis. The high incidence of Lyme borreliosis among the residents of endemic areas, along with diagnostic and therapeutic difficulties, make it a serious academic, clinical and social problem. The present article elaborates on bacterium structure and selected mechanisms facilitating the colonisation of particular hosts. Knowledge of those processes might be useful in understanding complex pathogenesis of lesions occurring in Lyme disease.

Influence of MKP medium stored for prolonged periods on growth and morphology of Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto.

Modified Kelly-Pettenkofer (MKP) medium is one of the several media used for isolation and cultivation of Borrelia. The aim of the study was to assess whether particular Borrelia species (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) have the ability to grow in MKP medium stored at +4 °C for periods for 1 month up to 1 year, and how prolonged storage may influences Borrelia growth and morphology. The growth of Borrelia was evaluated after 5 days of incubation at 33 °C: cell count per mL, morphology, and motility were assessed. The results of this study showed that the duration of storage of MKP medium had statistically significant influence on growth of B. afzelii (p = 0.021) and B. garinii (p = 0.004), but not on growth of B. burgdorferi sensu stricto (p = 0.204), whereas duration of storage of the medium had no impact on Borrelia morphology and motility. The results of the study indicate that medium stored for more than 1 and up to 12 months supports Borrelia growth.

Borrelia Burgdorferi "sensu lato" in Brazil: Occurrence confirmed by immunohistochemistry and focus floating microscopy.

In the present study, we report the occurrence of Lyme's borreliosis in patients from the Brazilian Amazon Region. Borreliosis was investigated by immunohistochemistry and focus floating microscopy for Borrelia burgdorferi in skin biopsy samples from 22 patients with both clinical and histopathology evidences compatible with Erythema Migrans. Spirochetes were detected by specific immunohistochemistry and focus floating microscopy for B. burgdorferi in samples from five patients. Clinical cure of the cutaneous lesions was observed in all the patients after treatment with doxycycline regimen as proposed by the Center Disease Control guidelines. A limitation of our study was the fact that we were not able to isolate and culture these organisms. These are the first known Brazilian cases of borreliosis to have Focus Floating Microscopy confirmation.

Comparative cryo-electron tomography of pathogenic Lyme disease spirochetes.

Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to investigate structural features of three species, B. burgdorferi, B. garinii and B. afzelii, known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii, a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.

Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo.

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Analysis of the cellular localization of Bdr paralogs in Borrelia burgdorferi, a causative agent of lyme disease: evidence for functional diversity.

The bdr (Borrelia direct repeat) gene family of the genus Borrelia encodes a polymorphic group of proteins that carry a central repeat motif region containing putative phosphorylation sites and a hydrophobic carboxyl-terminal domain. It has been postulated that the Bdr proteins may anchor to the inner membrane via the C-terminal domain. In this study, we used cellular fractionation methodologies, salt and detergent treatments, and immunoblot analyses to assess the association of the Bdr proteins with the cellular infrastructure in both Borrelia burgdorferi (a Lyme disease spirochete) and B. turicatae (a relapsing fever spirochete). Triton X-114 extraction and partitioning experiments demonstrated that most Bdr paralogs are associated with the inner membrane-peptidoglycan complex. Analyses of cells treated with the highly chaotropic bile salt detergent deoxycholic acid demonstrated that some Bdr paralogs may also interact with the peptidoglycan, as evidenced by their tight association with the insoluble cellular matrix. In addition, immunoprecipitation (IP) experiments revealed an enhanced IP of all Bdr paralogs when the cell lysates were boiled prior to addition of the precipitating antibody. Furthermore, some Bdr paralogs were accessible to antibody in the IP experiments only in the boiled cell lysates. These observations suggest that different Bdr paralogs may carry out different structural-functional roles. Demonstration of the inner membrane localization of the Bdr proteins and of the differences in nature of the interaction of individual Bdr paralogs with the cell infrastructure is an important step toward defining the functional role of this unique protein family in the genus Borrelia.

The generation of enzymatically active plasmin on the surface of spirochetes.

The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to the host by a feeding Ixodid tick. The spirochete subsequently disseminates through the skin, enters the bloodstream, and becomes systemic. A potential mechanism for this invasiveness was identified with the discovery that B. burgdorferi can bind components of the plasminogen activation system (PAS). The methodology for analyzing the generation of enzymatically active plasmin on the surface of this organism is given, and applied to measure spirochete viability, strain differences, and breakdown of extracellular matrix (ECM) macromolecules. Plasmin acquisition by B. burgdorferi was measured photometrically by a specific chromogenic substrate. The growth of B. burgdorferi in culture was not affected by the presence of active plasmin on the spirochete surface. Plasmin-coated B. burgdorferi degraded the purified (ECM) components fibronectin, laminin, and vitronectin, but not collagen. The addition of B. burgdorferi with surface plasmin to a radiolabeled, native ECM resulted in degradation of noncollagenous protein, as measured by release of solubilized radioactivity. Breakdown of purified ECM components or native ECM did not occur after exposure to untreated spirochetes or spirochetes treated with uPA or PLG alone. These results provide in vitro evidence that enzymatically active plasmin on the surface of B. burgdorferi may be partially responsible for its invasiveness.

Chemotaxis in Borrelia burgdorferi.

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.

Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.

Heterogeneity of Borrelia burgdorferi in the skin.

The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium.

Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.