Characterization of the Flagellar Collar Reveals Structural Plasticity Essential for Spirochete Motility.

Spirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for this unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi. We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in situ structures reveal that the multiprotein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility. IMPORTANCE Many spirochetes cause serious human diseases. They are well recognized by their distinct morphology and motility. Spirochete motility is driven by a periplasmic flagellum, which possesses a unique collar essential for flagellar assembly and spirochete motility. Here, we discover two novel collar proteins in the Lyme disease spirochete Borrelia burgdorferi. We demonstrate, for the first time, that the collar is a multiprotein complex with a remarkable plasticity that enables the motor to accommodate the highly curved membrane of spirochetes and generate the high torque necessary for spirochete motility.

Molecular mechanism for rotational switching of the bacterial flagellar motor.

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

BB0326 is responsible for the formation of periplasmic flagellar collar and assembly of the stator complex in Borrelia burgdorferi.

Borrelia burgdorferi is a highly motile spirochete due to its periplasmic flagella. Unlike flagella of other bacteria, spirochetes' periplasmic flagella possess a complex structure called the collar, about which little is known in terms of function and composition. Using various approaches, we have identified a novel protein, BB0326, as a key component of the collar. We show that a peripheral portion of the collar is diminished in the Δbb0326 mutant and restored in the complemented bb0326+ cells, leading us to rename BB0326 as periplasmic flagellar collar protein A or FlcA. The ΔflcA mutant cells produced fewer, abnormally tilted and shorter flagella, as well as diminished stators, suggesting that FlcA is crucial for flagellar and stator assemblies. We provide further evidence that FlcA interacts with the stator and that this collar-stator interaction is essential for the high torque needed to power the spirochete's periplasmic flagellar motors. These observations suggest that the collar provides various important functions to the spirochete's periplasmic flagellar assembly and rotation.

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins.

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

Co-culture of human fibroblasts and Borrelia burgdorferi enhances collagen and growth factor mRNA.

Skin fibrosis has been reported in Borrelia burgdorferi infection in Europe, but has been questioned by several authors. The objective of the present study was to examine the interaction of skin fibroblasts with B. burgdorferi sensu stricto B31 (BB) and B. afzelii (BA) in vitro by electron microscopy. We also determined the expression of collagen type I, TGF-ß, FGF-1, calreticulin (CALR), decorin (DCN), and PDGF-α at the mRNA level in Borrelia/fibroblast co-cultures. Intact Borrelia attach to and transmigrate fibroblasts, and undergo cystic transformation outside the fibroblasts. Fibroblasts preserve their vitality and express a prominent granular endoplasmic reticulum, suggesting activated protein synthesis. On two different semi-quantitative real-time PCR assays, BB- and BA/fibroblast co-cultures showed a significant induction of type I collagen mRNA after 2 days compared to fibroblasts (fourfold for BA and 1.8-fold for BB; p < 0.02). In addition, there was a significant upregulation of mRNA expression of TGF-ß, CALR, PDGF-α, and DCN in BA and BB co-cultures compared to control fibroblasts in monolayer cultures after 2 days (p < 0.01). The BA/fibroblast co-culture induced a considerably greater upregulation of collagen and growth factor mRNA compared to BB/fibroblast co-culture. In contrast, a significant down-regulation of FGF-1 (20-fold for BA and 4.5-fold for BB) mRNA expression was detected in co-cultures compared to controls (p < 0.01). The results of the study support the hypothesis that BB sensu lato, and BA in particular, enhances collagen mRNA expression and can stimulate growth factors responsible for increased collagen production.

A high frequency of viral agents yet absence of Borrelia burgdorferi is seen within the myocardium of subjects with normal left ventricular systolic function: an electron microscopy study.

A wide range of viral agents is associated with the development of acute myocarditis and its possible chronic sequela, dilated cardiomyopathy (DCM). There is also increasing evidence that Borrelia burgdorferi (Bb) is associated with DCM in endemic regions for Bb infection. This study sought to use electron microscopy to prospectively analyze the presence of viruses and Bb within the myocardium of 40 subjects with preserved left ventricular (LV) ejection fraction and 40 patients with new-onset unexplained DCM during the same time period. Virus particles were found within the myocardium of 23 subjects (58%) of both cohorts studied, yet there was no statistically significant difference in virus family presence between those with DCM versus those with preserved LV systolic function. In contrast, Bb was detected only in those subjects with DCM (0 versus 5 subjects; p ˂ 0.05). Polymerase chain reaction was performed on samples from patients who were positive for Bb according to electron microscopy, and Bb was confirmed in 4 out of 5 individuals. Our results demonstrate that the prevalence of viral particles does not differ between subjects with preserved LV systolic function versus those with DCM and therefore suggests that the mere presence of a viral agent within the myocardium is not sufficient to establish a clear link with the development of DCM. In contrast, the presence of Bb was found only within myocardial samples of patients with DCM; this finding supports the idea of a causal relationship between Bb infection and DCM development.

Spirochetal motility and chemotaxis in the natural enzootic cycle and development of Lyme disease.

Two-thirds of all bacterial genomes sequenced to-date possess an organelle for locomotion, referred to as flagella, periplasmic flagella or type IV pili. These genomes may also contain a chemotaxis-signaling system which governs flagellar rotation, thus leading a coordinated function for motility. Motility and chemotaxis are often crucial for infection or disease process caused by pathogenic bacteria. Although motility-associated genes are well-characterized in some organisms, the highly orchestrated synthesis, regulation, and assembly of periplasmic flagella in spirochetes are just being delineated. Recent advances were fostered by development of unique genetic manipulations in spirochetes coupled with cutting-edge imaging techniques. These contemporary advances in understanding the role of spirochetal motility and chemotaxis in host persistence and disease development are highlighted in this review.

The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation.

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; relBbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Biofilm formation by Borrelia burgdorferi sensu lato.

Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology.

Motor rotation is essential for the formation of the periplasmic flagellar ribbon, cellular morphology, and Borrelia burgdorferi persistence within Ixodes scapularis tick and murine hosts.

Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts.

Borrelia burgdorferi tissue morphologies and imaging methodologies.

This manuscript offers an image presentation of diverse forms of Borrelia burgdorferi spirochetes which are not spiral or corkscrew shaped. Explanations are offered to justify the legitimacy of tissue forms of Borrelia which may confuse the inexperienced microscopic examiner and which may lead to the misdiagnosis of non-spiral forms as artifacts. Images from the author's personal collection of Borrelia burgdorferi images and a few select images of Borrelia burgdorferi from the peer-reviewed published literature are presented. A commentary justifying each of the image profiles and a survey of the imaging modalities utilized provides the reader with a frame of reference. Regularly spiraled Borrelia are rarely seen in solid tissues. A variety of straightened, undulating, and clipped-off profiles are demonstrated, and the structural basis for each image is explained. Tissue examination is a diagnostic tool and a quality control for judging the eradication or the persistence of borreliosis following attempts to eradicate the infection with antibiotic therapy. The presence or absence of chronic Lyme borreliosis may be objectively adjudicated by tissue examinations which demonstrate or which fail to show pathogenic microbes in patients who have received a full course of antibiotics.

[The use of electron microscopy and virus isolation in the diagnosis of aseptic neuroinfections]. / Vyuzití elektronové mikroskopie a izolace viru v diagnostice aseptických neuroinfekcí.

OBJECTIVE: In aseptic neuroinfections, the etiology is usually known in 50-70% of cases. The aim was to increase the rates using electron microscopy (EM) and virus isolation in cell cultures. MATERIAL AND METHODS: The prospective study included 34 patients with aseptic neuroinfections hospitalized at the Department of Infectious Diseases in Ostrava fromJuly to November 2012. EM examined cerebrospinal fluid of all patients and virus isolation in tissue cultures was performed in all cerebrospinal fluid samples. Cerebrospinal fluid was examined by polymerase chain reaction for enteroviruses in 30 patients and for herpes simplex virus 1 and 2 in 29 patients. Detection of antibodies against Borrelia burgdorferi and tick-borne encephalitis was performed in all 34 patients. RESULTS: Possible etiological agents were discovered in 31 out of 34 patients (91%), with one agent being found in 23 patients (68%) and two agents being detected in 8 patients (24%). EM revealed the agents in 26 patients and virus isolation was successful in 10 patients. EM was the only method to identify 10 agents. A group of 23 patients with a single agent detected included 14 patients with enteroviral meningitis, 4 patients with Lyme borreliosis and 4 patients with tick-borne encephalitis; EM detected an undefined virus in the last patient. An unusual group of 8 patients with two agents detected comprised 5 patients with enteroviruses and spirochetes, 2 patients with tick-borne encephalitis and undefined viruses and 1 patient with a spirochete and an undetermined virus. CONCLUSION: EM can aid in explaining the etiology of aseptic neuroinfections. However, the clinical interpretation of results remains problematic, such as detection of unknown viruses or two possible agents in 8 out of 34 patients.

A single-domain FlgJ contributes to flagellar hook and filament formation in the Lyme disease spirochete Borrelia burgdorferi.

FlgJ plays a very important role in flagellar assembly. In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile. The FlgJ proteins consist of two functional domains. The N-terminal rod-capping domain acts as a scaffold for rod assembly, and the C-terminal domain acts as a peptidoglycan (PG) hydrolase (PGase), which allows the elongating flagellar rod to penetrate through the PG layer. However, the FlgJ homologs in several bacterial phyla (including spirochetes) often lack the PGase domain. The function of these single-domain FlgJ proteins remains elusive. Herein, a single-domain FlgJ homolog (FlgJ(Bb)) was studied in the Lyme disease spirochete Borrelia burgdorferi. Cryo-electron tomography analysis revealed that the flgJ(Bb) mutant still assembled intact flagellar basal bodies but had fewer and disoriented flagellar hooks and filaments. Consistently, Western blots showed that the levels of flagellar hook (FlgE) and filament (FlaB) proteins were substantially decreased in the flgJ(Bb) mutant. Further studies disclosed that the decreases of FlgE and FlaB in the mutant occurred at the posttranscriptional level. Microscopic observation and swarm plate assay showed that the motility of the flgJ(Bb) mutant was partially deficient. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these results indicate that FlgJ(Bb) is involved in the assembly of the flagellar hook and filament but not the flagellar rod in B. burgdorferi. The observed phenotype is different from that of flgJ mutants in the enteric bacteria.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi fliL mutant.

Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, including Borrelia burgdorferi, the causative agent of Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing σ(70). Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defective-motility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.

Metamorphosis of Borrelia burgdorferi organisms--RNA, lipid and protein composition in context with the spirochetes' shape.

Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells--nucleic acids, proteins, lipids--are possibly modified during the process. Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres. The results of our comparisons and the failure to generate mutants lacking the ability to convert to spheres suggest that the metamorphosis of B. burgdorferi results in a conditional reconstruction of the outer membrane. The spheres, which appear to be more resistant to unfavorable conditions and exhibit reduced immune reactivity when compared to spirochetes, might allow the B. burgdorferi to escape complete clearance and possibly ensure long-term survival in the host.

Borrelia-like organism in heart capillaries of patient with Lyme-disease seen by electron microscopy.

A case of a patient who developed an acute myocarditis due to Lyme disease is reported. An increased serum antibody titer to Borrelia burgdorferi suggested a diagnosis and in addition of basic clinical methods, endomyocardial biopsy performed and analyzed by transmission electron microscopy. The lumen of myocardial capillaries was founded mostly filled with detritus and fibrin precipitate, between them several bacterial fragments were identified. The electron-microscopic characteristics of the microorganisms in this specimen, revealing irregularly coiled appearance and consistent thickness of 0.2 µm, correspond to the spiral-like structure of Lyme disease borrelia. The presence of fibrin deposits on the capillary endothelium and necrosis of myocardiocytes, suggests that the cardiopathy in our patient was represent borrelia-mediated damage of the heart microcirculation.

Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline.

Persistence of tissue spirochetes of Borrelia burgdorferi as helices and round bodies (RBs) explains many erythema-Lyme disease symptoms. Spirochete RBs (reproductive propagules also called coccoid bodies, globular bodies, spherical bodies, granules, cysts, L-forms, sphaeroplasts, or vesicles) are induced by environmental conditions unfavorable for growth. Viable, they grow, move and reversibly convert into motile helices. Reversible pleiomorphy was recorded in at least six spirochete genera (>12 species). Penicillin solution is one unfavorable condition that induces RBs. This antibiotic that inhibits bacterial cell wall synthesis cures neither the second "Great Imitator" (Lyme borreliosis) nor the first: syphilis. Molecular-microscopic techniques, in principle, can detect in animals (insects, ticks, and mammals, including patients) helices and RBs of live spirochetes. Genome sequences of B. burgdorferi and Treponema pallidum spirochetes show absence of >75% of genes in comparison with their free-living relatives. Irreversible integration of spirochetes at behavioral, metabolic, gene product and genetic levels into animal tissue has been documented. Irreversible integration of spirochetes may severely impair immunological response such that they persist undetected in tissue. We report in vitro inhibition and destruction of B. burgdorferi (helices, RBs = "cysts") by the antibiotic Tigecycline (TG; Wyeth), a glycylcycline protein-synthesis inhibitor (of both 30S and 70S ribosome subunits). Studies of the pleiomorphic life history stages in response to TG of both B. burgdorferi and Treponema pallidum in vivo and in vitro are strongly encouraged.

Characterization of Borrelia burgdorferi aggregates.

Lyme disease is caused by the tick-borne spirochete, Borrelia burgdorferi. It has been documented that B. burgdorferi form aggregates within ticks and during in vitro culture. However, Borrelia aggregates remain poorly characterized, and their functional significance is unknown. Here we have characterized Borrelia aggregates using microscopy and flow cytometry. Borrelia aggregation was temperature, pH, and growth phase dependent. Environmental conditions (high temperature, low pH, and high cell density) favorable for aggregation were similar to the conditions that increased the expression of B. burgdoferi genes, such as outer surface protein C (ospC), that are regulated by the RpoN/RpoS sigma factors. Experiments were conducted to determine if there is a relationship between aggregation and gene regulation through the RpoN/RpoS pathway. ospC Transcript levels were similar between aggregates and free cells. Moreover, no differences were observed in aggregate formation when null mutants of rpoS, rpoN, or ospC were compared to wild-type spirochetes. These results indicated that, despite the similar external signals that promoted aggregation and the RpoN/RpoS pathway, the two processes were not linked at the molecular level. The methods developed here to study B. burgdorferi aggregates will be useful for further studies on spirochete aggregates.