Expression of type I pili is abolished in verotoxin-producing Escherichia coli O157

Verotoxin-producing Escherichia coli (VTEC) were examined for production of type I pili. None of 34 strains of VTEC serogroup O157 examined expressed any pili, whereas 26 strains of 27 VTEC serogroup O26 and seven strains of nine non-VTEC O157 produced type I pili. These VTEC strains were collected from sporadic human cases and cattle in Okinawa in 1997. The genes encoding the major structural component (FimA) and the adhesin (FimH) of type I pili were detected in all 70 strains examined. The inability to express type I pili could be a unique character of VTEC O157 and this trait could be a new candidate to identify the organisms.     

Morphological alterations of Pseudomonas aeruginosa and Escherichia coli by nocardicin A

Abstract Pseudomonas aeruginosa and Escherichia coli were exposed to nocardicin A, and were subsequently observed with transmission and scanning electron microscopes. Although the nocardicin A-induced morphological alterations such as bulges and spheroplast formations were observed both in P. aeruginosa and E. coli , their positions on the cell surface were different in the two species.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon

Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells. In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca. 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili. An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes. These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.     

使用电子断层技术对IgG1抗体逐个分子的三维重构与结构分析

抗体(antibody)又称免疫球蛋白(immunoglobulin,Ig),是人体免疫反应的重要参与者.了解抗体的结构和结构动态特征,是理解人体免疫作用机理、修复或提高免疫能力、定向设计抗体以治疗各种疾病的基础.本文以人体IgG1抗体为对象,综述了使用透射电子显微学方法研究IgG1抗体结构方向的最新进展.详细介绍了使用逐个分子的电子断层三维重构技术(individual-particle electron tomography,IPET)对抗体进行结构研究的方法,包括样品制备、图像处理和数据分析等.并描述了利用该技术,在研究抗体结合肽分子后的结构形变和通过收集不同构象来研究抗体动态结构特征方面所取得的阶段性成果.最后,对尚待解决的关键问题与该技术未来的发展方向进行了讨论与展望.     

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol . The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.     

Structure, folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli strains

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol^− 1) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol^− 1) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.     

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.     

Structural and functional insights into the assembly of type 1 pili from Escherichia coli

Type 1 pili are filamentous protein complexes that are anchored to the outer membrane of uropathogenic Escherichia coli and mediate bacterial adhesion to the surface of urinary epithelium cells. We review here the current status of structural and functional studies on the assembly of type 1 pili.     

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.     

3D structure of the skeletal muscle dihydropyridine receptor

The dihydropyridine receptors (DHPR) are L-type voltage-gated calcium channels that regulate the flux of calcium ions across the cell membrane. Here we present the three-dimensional (3D) structure at approximately 27A resolution of purified skeletal muscle DHPR, as determined by electron microscopy and single particle analysis. Here both biochemical and 3D structural data indicate that DHPR is dimeric. DHPR dimers are composed of two arch-shaped monomers approximately 210A across and approximately 75A thick, that interact very tightly at each end of the arch. The roughly toroidal structure of the two monomers encloses a cylindrical space of approximately 80A diameter, which is then closed on each side by two dome-shaped protein densities reaching over from each monomer arch. The dome-shaped domains have a length of approximately 80-90A and a maximum height of approximately 45A. Small orifices punctuate their exterior surface. The 3D structure disclosed here may have important implications for the understanding of DHPR Ca(2+) channel function. We also propose a model for its in vivo interactions with the calcium release channel at the junctional sarcoplasmic recticulum.     

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.     

Cytopathology, Mode of Aphid Transmission and Search for the Casual Agent of Cotton Bunchy Top Disease

The cytopathological effects of cotton bunchy top (CBT) disease and its mode of transmission by Aphis gossypii Glover (cotton aphid), were studied. CBT infection affected the leaf epidermal layer producing a loose, ruptured and rough surface morphology with many stomata closed and misshapen. Roots of CBT‐infected plants showed reduced growth, small knots and a dark brown appearance. A single aphid per plant was capable of transmitting CBT at 5%, whereas three aphids per plant transmitted CBT to 50% of the cotton seedlings and 20 aphids per plant transmitted the disease agent to 80% of the cotton seedlings. Aphis gossypii acquired CBT after a minimum acquisition access period of 5 min and transmitted the agent after a minimum inoculation access period of 1 h. Both alate and apterous aphids and nymph instars 2, 3 and 4 of A. gossypii transmitted CBT. This preliminary data suggest that A. gossypii transmits CBT in a semi‐persistent manner. Myzus persicae Sulz (green peach aphid) was unable to transmit CBT. A comprehensive attempt to isolate the CBT agent, using a range of virological techniques including double‐stranded RNA extraction, two‐dimensional gel electrophoresis for viroid, circular DNA test, nanovirus polymerase chain reaction (PCR), luteovirus PCR and enzyme‐linked immunosorbent assay, phytoplasma test, nucleoprotein purification and electron microscopy, was unsuccessful, raising the possibility that CBT may be caused by a unique new pathogen.     

3D representation and analysis of enthesis morphology

This comparison of methods for assessing the development of muscle insertion sites, or entheses, suggests that three‐dimensional (3D) quantification of enthesis morphology can produce a picture of habitual muscle use patterns in a past population that is similar to one produced by ordinal scores for describing enthesis morphology. Upper limb skeletal elements (humeri, radii, and ulnae) from a sample of 24 middle‐aged adult males from the Pottery Mound site in New Mexico were analyzed for both fibrous and fibrocartilaginous enthesis development with three different methods: ordinal scores, two‐dimensional (2D) area measurements, and 3D surface areas. The methods were compared using tests for asymmetry and correlations among variables in each quantitative data set. 2D representations of enthesis area did not agree as closely as ordinal scores and 3D surface areas did regarding which entheses were significantly asymmetrical. There was significant correlation between 3D and 2D data, but correlation coefficients were not consistently high. Intraobserver error was also assessed for the 3D method. Cronbach's alpha values fell between 0.68 and 0.73, and error rates for all entheses fell between 10% and 15%. Marginally acceptable intraobserver error and the analytic versatility of 3D images encourage further investigation of using 3D scanning technology for quantifying enthesis development. Am J Phys Anthropol 152:417–424, 2013. © 2013 Wiley Periodicals, Inc.     

The Structure and Morphogenesis of the Ventral Ciliature in Paraurostyla hymenophora*

SYNOPSIS. The structure and morphogenesis of the ventral ciliature of Paraurostyla hymenophora (Stokes) are described. The oral primordium apparently originates in association with transverse cirrus #6, from which it migrates anteriorly simultaneous with kinetosomal proliferation. The primordium eventually forms an elongate ciliary field from which the future opisthe's fronto-ventro-transverse (FVT) and undulating membrane primordial fields arise. Concomitantly, the future proter's FVT primordial field is initiated by the disaggregation of frontal cirri #4, #5, and #6. Primordia then develop simultaneously within marginal and ventral cirral rows by a disaggregation of cirri within the respective rows, and do not give rise to new cirri until the FVT fields complete segregation into discrete cirri. Near the completion of cirral production from the FVT primordia, each ventral cirral primordium (VCP) forms the 2 rightmost transverse cirri. Segregation of new cirri within the marginal cirral primordia and VCP then occurs, eventually replacing all old cirri within their respective marginal and ventral cirral rows. At the end of cortical morphogenesis, all old ciliary organelles, with the exception of the adoral zone of membranelles, are either reorganized or replaced. These results suggest an evolutionary affinity between the ventral and marginal cirral rows and raise questions about the control of the developmental competence of individual primordia.     

Comparative study of the corneal epithelium in some reptiles inhabiting different environments

El‐Bakry, A.M. 2011. Comparative study of the corneal epithelium in some reptiles inhabiting different environments. —Acta Zoologica (Stockholm) 92 : 54–61. The vertebrate cornea functions in either aquatic or aerial environments and in some cases in both. In terrestrial and aerial vertebrates, the cornea contributes most of the refractive powers of the eye because of the large variation in refractive index between the air and the cornea. The present study aimed to examine and compare the main features of the corneal epithelial surface of three reptilian species related to three different families (Caretta caretta, Varanus griseus and Mabuya quinquetaeniata) and inhabiting different environment, by light, scanning (SEM) and transmission electron microscopy. The mean epithelial cell densities of the species of the study were 8.670 ± 3.134, 5.945 ± 2.144 and 2.124 ± 713 respectively. The corneal epithelium of the three species observed by SEM showed a similarity to one another indicating that the apical cell surfaces possess regular polygonal cells with varieties of microprocesses. These microprocesses were represented by microplicae, numerous microvilli and some long microridges in C. caretta, microplicae and minute microholes in V. griseus and microplicae intermingled with short microvilli in M. quinquetaeniata. According to the densities of these microprocesses, three polymorphic cell types (light, medium and dark) appeared in C. caretta, light and medium cell types were observed in V. griseus and medium and dark cell types were noticed in M. quinquetaeniata. Different types of tight adhesions were observed by transmission electron microscopy between the cell borders of the epithelial cells which differ according to environment where the species occupy. In conclusion, variation in the structure of the corneal epithelial cells appears to be related to the living environment, such as aerial, terrestrial and aquatic ones, which is occupied by every species.     

Adhesion of Plasmodium gallinaceum Ookinetes to the Aedes aegypti Midgut: Sites of Parasite Attachment and Morphological Changes in the Ookinete

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered lo and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut. ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.     

COMBO-FISH for focussed fluorescence labelling of gene domains: 3D-analysis of the genome architecture of abl and bcr in human blood cells

Structural analysis and nanosizing of gene domains requires not only high-resolution microscopy but also improved techniques of fluorescence labelling strongly focussed on the gene domains. To investigate the architecture of abl and bcr in blood cell nuclei forming the Philadelphia chromosome in CML, we applied COMBO-FISH using specifically colocalising combinations of triple strand forming oligonucleotide probes for abl on chromosome 9 and bcr on chromosome 22. Each probe set consisting of 31 homopyrimidine oligonucleotides was computer selected from the human genome database. Measurements by 3D microscopy were compared to results obtained after standard FISH using commercially available abl/bcr BAC probes. The relative radial fluorescence distributions in lymphocyte cell nuclei of healthy donors in comparison to cell nuclei of blood cells of CML patients showed a strong correlation in the location of abl and bcr for both labelling techniques. The absolute distances of the homologous bcr domains and the abl domain-nuclear center-abl domain angles in cell nuclei of CML donors differed significantly from those of healthy donors only when COMBO-FISH was applied. These results indicate that COMBO-FISH may be more sensitive than standard FISH in case of slight modifications in the genome architecture.