Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Microbial motility frequently depends on flagella or type?IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with type?IV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type?II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP [2'-/3'-O-(N'- methylanthraniloyl)adenosine-5'-O-triphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75?°C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.     

Ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential

Pilin proteins assemble into Type IV pili (T4P), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. However, challenges in crystallizing full-length fiber-forming and membrane protein pilins leave unanswered questions regarding pilin structures, assembly, functions, and vaccine potential. Here we report pilin structures of full-length DnFimA from the sheep pathogen Dichelobacter nodosus and FtPilE from the human pathogen Francisella tularensis at 2.3 and 1 ? resolution, respectively. The DnFimA structure reveals an extended kinked N-terminal α-helix, an unusual centrally located disulfide, conserved subdomains, and assembled epitopes informing serogroup vaccines. An interaction between the conserved Glu-5 carboxyl oxygen and the N-terminal amine of an adjacent subunit in the crystallographic dimer is consistent with the hypothesis of a salt bridge between these groups driving T4P assembly. The FtPilE structure identifies an authentic Type IV pilin and provides a framework for understanding the role of T4P in F. tularensis virulence. Combined results define a unified pilin architecture, specialized subdomain roles in pilus assembly and function, and potential therapeutic targets.     

Interferon-β-related tumefactive brain lesion in a Caucasian patient with neuromyelitis optica and clinical stabilization with tocilizumab

Background

Neuromyelitis optica (NMO) is a severely disabling inflammatory disorder of the central nervous system and is often misdiagnosed as multiple sclerosis (MS). There is increasing evidence that treatment options shown to be beneficial in MS, including interferon-β (IFN-β), are detrimental in NMO.

Case presentation

We here report the first Caucasian patient with aquaporin 4 (AQP4) antibody (NMO-IgG)-seropositive NMO presenting with a tumefactive brain lesion on treatment with IFN-β. Disease started with relapsing optic neuritis and an episode of longitudinally extensive transverse myelitis (LETM) in the absence of any brain MRI lesions or cerebrospinal fluid-restricted oligoclonal bands. After initial misdiagnosis of multiple sclerosis (MS) the patient received subcutaneous IFN-β1b and, subsequently, subcutaneous IFN-β1a therapy for several years. Under this treatment, the patient showed persisting relapse activity and finally presented with a severe episode of subacute aphasia and right-sided hemiparesis due to a large T2 hyperintensive tumefactive lesion of the left brain hemisphere and a smaller T2 lesion on the right side. Despite rituximab therapy two further LETM episodes occurred, resulting in severe neurological deficits. Therapeutic blockade of the interleukin (IL)-6 signalling pathway by tocilizumab was initiated, followed by clinical and radiological stabilization.

Conclusion

Our case (i) illustrates the relevance of correctly distinguishing NMO and MS since these disorders differ markedly in their responsiveness to immunomodulatory and -suppressive therapies; (ii) confirms and extends a previous report describing the development of tumefactive brain lesions under IFN-β therapy in two Asian NMO patients; and (iii) suggests tocilizumab as a promising therapeutic alternative in highly active NMO disease courses.

     

Voxel-Based MRI Intensitometry Reveals Extent of Cerebral White Matter Pathology in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons. Advanced MRI techniques such as diffusion tensor imaging have shown great potential in capturing a common white matter pathology. However the sensitivity is variable and diffusion tensor imaging is not yet applicable to the routine clinical environment. Voxel-based morphometry (VBM) has revealed grey matter changes in ALS, but the bias-reducing algorithms inherent to traditional VBM are not optimized for the assessment of the white matter changes. We have developed a novel approach to white matter analysis, namely voxel-based intensitometry (VBI). High resolution T1-weighted MRI was acquired at 1.5 Tesla in 30 ALS patients and 37 age-matched healthy controls. VBI analysis at the group level revealed widespread white matter intensity increases in the corticospinal tracts, corpus callosum, sub-central, frontal and occipital white matter tracts and cerebellum. VBI results correlated with disease severity (ALSFRS-R) and patterns of cerebral involvement differed between bulbar- and limb-onset. VBI would be easily translatable to the routine clinical environment, and once optimized for individual analysis offers significant biomarker potential in ALS.     

FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.     

Urea: a nitrogen source for the aquatic resurrection plant Chamaegigas intrepidus Dinter

Chamaegigas intrepidus Dinter is a poikilohydric aquatic plant that lives in rock pools on granite outcrops in central Namibia. The pools are filled with water only intermittently during the wet season, and the plants may pass through up to 20 rehydration/dehydration cycles during the summer rains. The potential nitrogen sources for the rehydrated plants are ammonium, which is only present at 10–20 μm, amino acids, particularly glycine, and urea, which is generally present at 20–30 μm. We show that urea can be utilised by plants in the field through the presence of urease in the sediments of the rock pools. Urease activity is higher in non-submerged than in submerged sediments, and it can survive 6 months of complete dryness at temperatures up to 60°C. Experiments with [¹⁴C]urea under laboratory conditions show that the roots of C. intrepidus are unable to take up urea; while ¹⁵N-nuclear magnetic resonance experiments show that [¹⁵N]urea is only metabolised to labelled glutamine and glutamate after ammonium has been released by the action of urease. Thus urease plays a vital role in allowing urea to be utilised as a major N source in this nutrient-limited aquatic ecosystem. Received: 23 April 1999 / Accepted: 8 November 1999     

Finite element analysis of an additional implant for intramedullary nailing]

In the surgical treatment of fractured femurs, the fracture is bridged by a medullary nail fixed in the bone with interlocking screws. Failure of bone substance in the region of the interlocking screws is the most common complication in the treatment of osteoporotic bone. With the aim of preventing this complication, an additional implant was developed. A finite element analysis of an ideal bone/implant system was carried out to investigate the role of the additional implant. Three defined finite element models were generated, and the associated stress situations compared. The first model is a standard fixation without the additional implant. In the second model, the additional implant is integrated within the bone/implant system. The third model uses a modified form of the additional implant. The results show that both additional implants reduce the stresses occurring, both in the bone substance and at the screws. The modified form of the additional implant proved to be the most favorable version. In the case of the original additional implant, the negative effect of the sharp edges of the thread was demonstrable.     

Strain Diversity of CTX-M-Producing Enterobacteriaceae in Individual Pigs: Insights into the Dynamics of Shedding during the Production Cycle

The aim of this study was to evaluate the population dynamics of CTX-M-producing Enterobacteriaceae in individual pigs on a farm positive for CTX-M-14-producing Escherichia coli. Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and tested for bla_CTX-M. CTX-M-positive isolates (n = 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboring bla_CTX-M were characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producing E. coli strains during the course of life. Concomitant shedding of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination of bla_CTX-M-14 within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producing Enterobacteriaceae in pig farms.