In Situ Structural Analysis of Leptospira spp. by Electron Cryotomography.

Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is capable of determining protein structures at resolutions high enough to visualize their individual domains and secondary structures in their cellular environment.

Spirochete Flagella and Motility.

Spirochetes can be distinguished from other flagellated bacteria by their long, thin, spiral (or wavy) cell bodies and endoflagella that reside within the periplasmic space, designated as periplasmic flagella (PFs). Some members of the spirochetes are pathogenic, including the causative agents of syphilis, Lyme disease, swine dysentery, and leptospirosis. Furthermore, their unique morphologies have attracted attention of structural biologists; however, the underlying physics of viscoelasticity-dependent spirochetal motility is a longstanding mystery. Elucidating the molecular basis of spirochetal invasion and interaction with hosts, resulting in the appearance of symptoms or the generation of asymptomatic reservoirs, will lead to a deeper understanding of host-pathogen relationships and the development of antimicrobials. Moreover, the mechanism of propulsion in fluids or on surfaces by the rotation of PFs within the narrow periplasmic space could be a designing base for an autonomously driving micro-robot with high efficiency. This review describes diverse morphology and motility observed among the spirochetes and further summarizes the current knowledge on their mechanisms and relations to pathogenicity, mainly from the standpoint of experimental biophysics.

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes.

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

[Morphology and motility of the spirochetes].

Spirochetes have flagella within the cell body and swim by wriggling the spiral cell body. Besides they have been known to be critical agents causing various infectious diseases, their eccentric appearances and motilities have been attracting many scientists in a wide variety of fields other than bacteriologists. Unlike externally flagellated bacteria that swim by using flagella as a screw propeller, spirochetes progress in a liquid by changing their cell shapes. To understand the unique motion mechanism of spirochetes, many experiments and theoretical studies are being carried out. In this review, I will summarize morphological and motile properties of various species of spirochete, such as Borrelia, Treponema and Brachyspira. I will also expound on the motion mechanism of Leptospira with our latest results obtained by high-resolution optical photometry.

Molecular architecture of the bacterial flagellar motor in cells.

The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

Spirochaetes and their twisted ways.

Biological systematists have had a long tradition of encountering organisms that are not quite what they seem to be. Among the microbes, horizontal gene transfer and evolutionary pressures result in organisms that have distinguished themselves from their closest relatives. The recent analyses of several Spirochetes reveal members that are not spiral shaped, and ones that appear to have extensively acquired genetic material from phylogenetically distant, but environmentally proximate, organisms.

Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochaetes.

Free-living bacteria with spherical cells 0.5-2.5 µm in diameter were isolated from freshwater sediment. 16S rRNA gene sequence analysis placed the new isolates within the phylum Spirochaetes ('spirochaetes'). The isolates never displayed a helical morphology or motility. Growth occurred in the presence of 100 mg ampicillin l(-1) in complex and defined mineral salts medium amended with vitamins, yeast extract and monosaccharides, disaccharides or soluble starch as fermentable substrates. Two distinct isolates, designated Buddy(T) and Grapes(T), exhibited doubling times of 21±2 and 15±1 h in glucose-amended medium and grew at 15-37 and 15-30 °C. Optimum growth was observed between 25 and 30 °C and pH 6.5-7.5, with no growth below pH 5 or above pH 10. Hexose and pentose fermentation yielded ethanol, acetate and formate as major end products. Growth was strictly fermentative and anaerobic, but the isolates tolerated brief oxygen exposure. Nitrate, sulfate, thiosulfate and carbon dioxide were not used as electron acceptors, but soluble Fe(III) was reduced to Fe(II) in glucose-amended medium. The DNA G+C base contents of isolates Buddy(T) and Grapes(T) were 45.5-46.4 and 47.0-49.2 mol%, respectively. Phospholipid fatty acid (PLFA) profiles contained large proportions of C(14:0) and C(16:0) straight-chain saturated fatty acids; C(16:1)ω7c and C(16:1)ω9c dominated the mono-unsaturated PLFAs in isolate Grapes(T), whereas isolate Buddy(T) also possessed C(18:1)ω5c, C(18:1)ω7c and C(18:1)ω9c fatty acids. Branched monoenoic acids accounted for up to 12.4 and 30% of the total PLFA in isolates Grapes(T) and Buddy(T), respectively. Based on their unique morphological features and the phylogenetic distance from their closest relatives, we propose the new genus, Sphaerochaeta gen. nov., to accommodate the new isolates within the novel species Sphaerochaeta globosa sp. nov. (type strain Buddy(T) =DSM 22777(T) =ATCC BAA-1886(T)) and Sphaerochaeta pleomorpha sp. nov. (type strain Grapes(T) =DSM 22778(T) =ATCC BAA-1885(T)). Sphaerochaeta globosa is the type species of the genus.

Separation of microscale chiral objects by shear flow.

We show that plane parabolic flow in a microfluidic channel causes nonmotile, helically shaped bacteria to drift perpendicular to the shear plane. Net drift results from the preferential alignment of helices with streamlines, with a direction that depends on the chirality of the helix and the sign of the shear rate. The drift is in good agreement with a model based on resistive force theory, and separation is efficient (>80%) and fast (<2 s). We estimate the effect of Brownian rotational diffusion on chiral separation and show how this method can be extended to separate chiral molecules.

Electron cryotomography reveals novel structures of a recently cultured termite gut spirochete.

Electron cryotromography, a relatively new methodology in the field of microbiology, has been exploited by Murphy et al. (in this issue of Molecular Microbiology) in their analysis of the recently isolated termite gut spirochete Treponema primitia. Unique structures (bowls, arcades of hooks, cones at the cell ends, two layers of wall material) were evident from the analysis of its surface and internal constituents. These results, coupled to video microscopy analysis of swimming cells, allowed the authors to propose a model of cell motility. This highly significant paper highlights the importance of electron cryotomography to the field of microbiology. It also illustrates that newly cultured recalcitrant bacteria from complex environments are likely to possess novel structures not previously seen in other species.

The last eukaryotic common ancestor (LECA): acquisition of cytoskeletal motility from aerotolerant spirochetes in the Proterozoic Eon.

We develop a symbiogenetic concept of the origin of eukaryotic intracellular motility systems from anaerobic but aerotolerant spirochetes in sulfide-rich environments. The last eukaryotic common ancestors (LECAs) have extant archaeprotist descendants: motile nucleated cells with Embden-Meyerhof glycolysis and substrate-level phosphorylation that lack the alpha-proteobacterial symbiont that became the mitochondrion. Swimming and regulated O(2)-tolerance via sulfide oxidation already had been acquired by sulfidogenic wall-less archaebacteria (thermoplasmas) after aerotolerant cytoplasmic-tubule-containing spirochetes (eubacteria) attached to them. Increasing stability of sulfide-oxidizing/sulfur-reducing consortia analogous to extant sulfur syntrophies (Thiodendron) led to fusion. The eubacteria-archaebacteria symbiosis became permanent as the nucleus evolved by prokaryotic recombination with membrane hypertrophy, analogous to Gemmata obscuriglobus and other delta-proteobacteria with membrane-bounded nucleoids. Histone-coated DNA, protein-synthetic RNAs, amino-acylating, and other enzymes were contributed by the sulfidogen whereas most intracellular motility derives from the spirochete. From this redox syntrophy in anoxic and microoxic Proterozoic habitats LECA evolved. The nucleus originated by recombination of eu- and archaebacterial DNA that remained attached to eubacterial motility structures and became the microtubular cytoskeleton, including the mitotic apparatus. Direct LECA descendants include free-living archaeprotists in anoxic environments: archamoebae, metamonads, parabasalids, and some mammalian symbionts with mitosomes. LECA later acquired the fully aerobic Krebs cycle-oxidative phosphorylation-mitochondrial metabolism by integration of the protomitochondrion, a third alpha-proteobacterial symbiont from which the ancestors to most protoctists, all fungi, plants, and animals evolved. Secondarily anaerobic eukaryotes descended from LECA after integration of this oxygen-respiring eubacterium. Explanatory power and experimental predictions for molecular biology of the LECA concept are stated.

Morphological properties of a human intestinal spirochete first isolated from a patient with diarrhea in Japan.

A human intestinal spirochete isolated from a rectal biopsy specimen was morphologically characterized. The isolate was comma-shaped, 3-6 microm in length, 0.2 micro m in diameter and had tapered ends. The surface layer, external to the outer envelope, was amorphous. Four string-like periplasmic flagella with a diameter of 20 nm were presented at each end of the SDS-treated cells. Thin sections of the bacterial cell revealed a high-density cytoplasmic membrane and flagella in the periplasmic space between the cytoplasmic membrane and the outer envelope. Three segments of equal length were observed in some of the cells, while other cells were bi-segmented and more frequently observed.

Papillomatous pastern dermatitis with spirochetes and Pelodera strongyloides in a Tennessee Walking Horse.

Papillomatous digital dermatitis is a common disease in cattle. The pastern dermatitis observed in a horse shared many of the gross characteristics of papillomatous digital dermatitis in cattle. Lesions included a mixture of proliferative and erosive changes, with a verrucose appearance in some areas. Microscopic similarities included pseudoepitheliomatous and papillomatous epidermal hyperplasia with hyperkeratosis, spongiosis of the epidermis, and intraepidermal spirochetes. The horse was also concurrently infected with Pelodera strongyloides. Papillomatous digital dermatitis in cattle is associated with poor husbandry practices. The environment of the affected horse was heavily contaminated with urine, manure, and other organic debris. Verrucous pododermatitis of horses may be the same as or similar to bovine papillomatous digital dermatitis, and these conditions have similar etiologies.

Ultrastructure of plaque associated with polytetrafluoroethylene (PTFE) membranes used for guided tissue regeneration.

The purpose of the study was to examine the ultrastructure of plaque contaminating polytetrafluoroethylene (PTFE) membranes used for guided periodontal tissue regeneration. 8 patients treated with Gore-Tex membranes received daily antibiotics (650 mg x 2 Femepen) and rinsed with 10 ml 0.2% chlorhexidine during a healing period of 30 days. Following retrieval, the membranes were processed for electron microscopy. External aspects of 12 portions from 4 partially exposed membranes were selected for detailed ultrastructural examination. The plaque-membrane interface was characterized by the presence of fibrin or discontinuous accumulation of intermicrobial matrix. Adjacent plaque-free areas of membrane surface exhibited no detectable electron-dense material. 3 structurally different groups of bacterial aggregations were observed on the strips: (i) dense layers of gram-positive cocci and rods dominated the external aspect of the open microstructure portion; (ii) cocci, rods and filamentous microorganisms embedded in fibrin filled the spaces of the open microstructure; (iii) a loosely arranged mixed microbiota consisting of gram-positive cocci and rods as well as of gram-negative microorganisms and spirochetes were present on the occlusive portion. Areas with morphologically intact bacteria alternated with areas with empty bacterial cell walls. One specimen also displayed degenerated Candida-like blastospores. This study shows that oral micro-organisms may colonize and extensively invade the open microstructure of PTFE material and that adhesion of plaque to the membrane surface is mediated either by fibrin or a discontinuous layer of intermicrobial matrix.

Human intestinal spirochetes are distinct from Serpulina hyodysenteriae.

Twenty-nine intestinal spirochetes isolated from Australian aboriginal children and six strains from Italian adults (HRM1, -2, -4, -5, -7, and -14) were genetically examined at 15 enzyme loci by using multilocus enzyme electrophoresis. Results were compared with those previously obtained for 188 porcine intestinal spirochetes. DNA from human strain HRM7 and porcine strain Serpulina hyodysenteriae P18A were also radioactively labeled and hybridized with DNA from 12 other human and porcine intestinal spirochetes. Both the multilocus enzyme electrophoresis and hybridization techniques demonstrated that the human spirochetes were not S. hyodysenteriae. They belonged to another distinct genetic group of spirochetes that included P43/6/78, the bacterium recovered from the first recorded case of porcine intestinal spirochetosis. Bacteria in this distinct group also differed from Serpulina spp. in possessing only four, five, or occasionally six axial filaments, being slightly thinner, and having more pointed ends. These findings add further weight to the possibility that human intestinal spirochetes may act as enteric pathogens.

[Intestinal spirochetosis in HIV infection: prevalence, isolation and morphology of spirochetes]. / Intestinale Spirochätose bei HIV-Infektion. Vorkommen, Isolierung und Morphologie der Spirochäten.

The prevalence of intestinal spirochaetosis was investigated in 39 HIV-positive homosexual males (mean age 39 [24-65] years) in different stages of HIV infection (3 with the lymphadenopathy syndrome, 8 with AIDS-related complex and 28 with AIDS). Biopsies for cultural and histological demonstration of spirochaetes were obtained during routine ileoscopies. At the time of examination 35 of the 39 patients had intestinal symptoms. 27 patients had had no previous antimicrobial treatment. In 12 of the 27 previously untreated patients with intestinal symptoms spirochaetes were demonstrated, to different extent, from the terminal ileum to the rectum, while the treated group of eight and the control group of four were negative. There were no significant inflammatory changes histologically. Treatment with metronidazole in most cases improved symptoms. In their ultrastructure the microorganisms showed several complete convolutions, cone-shaped cell endings, cell length of 4-18 microns, cell diameter of 0.21-0.35 microns and five subterminal flagella. Morphological considerations favour the inclusion of these microorganisms in the genus Treponema rather than Brachyspira.

Morphology as a basis for taxonomy of large spirochetes symbiotic in wood-eating cockroaches and termites: Pillotina gen. nov., nom. rev.; Pillotina calotermitidis sp. nov., nom. rev.; Diplocalyx gen. nov., nom. rev.; Diplocalyx calotermitidis sp. nov., nom. rev.; Hollandina gen. nov., nom.[TRUNCATED].

The purposes of this paper are (i) to present a framework for the morphometric analysis of large uncultivable spirochetes that are symbiotic in wood-eating cockroaches and termites; (ii) to revive, in accordance with the rules of the International Code of Nomenclature of Bacteria, the names of three genera (Pillotina, Diplocalyx, and Hollandina) and three species (Pillotina calotermitidis, Diplocalyx calotermitidis, and Hollandina pterotermitidis) for the same organisms to which the names were originally applied, because these names were not included on the 1980 Approved Lists of Bacterial Names; and (iii) to formally propose the name Clevelandina reticulitermitidis for a new genus and species of spirochetes from the termite Reticulitermes tibialis. None of these genera and species has been cultivated either axenically or in mixed culture; hence, all are based on type-descriptive material.

Isolation of spirochetes from the skin of patients with erythema chronicum migrans in Austria.

Spirochetal organisms were isolated from biopsies (skin punches) of the erythematous anular skin lesion of three patients with erythema chronicum migrans. The organisms were cultivated in modified Kelly's medium and - after an incubation period of 2 weeks - discovered by dark field microscopy (X 200). Compared with the Lyme disease agent Borrelia burgdorferi the organisms did not differ in shape, motility and reaction with polyclonal antiserum. Differences to B. burgdorferi were found in (i) very slow growth of the isolated organisms in modified Kelly's medium and in (ii) non reactivity with monoclonal antibodies against the outer membrane protein of B. burgdorferi. It is concluded that the Austrian isolates are closely related to but not identical with the Lyme disease agent.