Alignment and use of microbeam with full-field x-ray microscopes.

Demonstration tests of the alignment of Fresnel zone plate focusing optics using a full-field x-ray microscope and microbeam x-ray diffraction measurements combined with the full-field x-ray microscope were performed. It was confirmed that the full-field x-ray microscope enables direct two-dimensional observation of a microbeam with sub-micrometer spatial resolution. This allowed visualization of the misalignment of the focusing optics, resulting in accurate alignment of the optics within a short time. In addition, the microscope could be used to observe the sample as well as the microbeam, which enabled clarification of the position and two-dimensional shape of the microbeam on the sample. This realized a measurement procedure that a 100-µm-size sample was imaged with sub-micrometer spatial resolution, and then, microbeam-use measurements were performed for only the region of interest determined by the microscope, which has been difficult with conventional microbeam applications. The combination of observations by a full-field x-ray microscope and measurements using a microbeam is expected to open a new style of measurement.

X-ray zooming optics for analyzer-based multi-contrast computed tomography.

An X-ray analyzer-based optics with a zoom function is proposed for observing various samples with apparent-absorption contrast, phase contrast and scattering contrast. The proposed X-ray optics consists of a collimator crystal and an analyzer crystal arranged in a nondispersive (+, -) geometry with a sample placed between them. For the implementation of the zoom function, an asymmetrically cut crystal in the rotated-inclined geometry was used for the analyzer. Proof-of-principle experiments were performed at the vertical wiggler beamline BL-14B of the Photon Factory. First, the magnification was set to 1×, and then it was zoomed into the optimal magnification (10×). At these magnifications, tri-modal contrast cross-sectional images of a sample were obtained by computed tomography. It was confirmed that the image quality at 10× was superior to that at 1×. This achievement opens up new possibilities for observing an entire sample or regions of interest within a sample at optimal magnification, and is expected to be useful for materials science, condensed matter physics, archeology and biomedical science.

X-ray zooming microscopy with two Fresnel zone plates.

We propose a variable-magnification full-field x-ray microscope using two Fresnel zone plates (FZPs). By moving the positions of the two FZPs, the magnification can be continuously changed even if the sample and camera positions are fixed. It was demonstrated that the magnification can be changed in the range of 25-150× using a hard x-ray beam at 14.4 keV. Using the first FZP as a convex lens and the second FZP as a concave lens, high magnification can be achieved at a short camera length. Even under the condition of a camera length of about 7 m, a magnification higher than 300× was achieved, and a line and space pattern with a pitch of 40 nm was observed at 10 keV. By inserting a knife edge at an appropriate position in the optical system, a phase-contrast image can be easily obtained, which is useful for soft-tissue observation of biological samples.

Bordetella bronchiseptica Bcr4 antagonizes the negative regulatory function of BspR via its role in type III secretion.

Bordetella species, including B. pertussis, have a type III secretion system that is highly conserved among gram-negative pathogenic bacteria. Genes encoding the component proteins of the type III secretion system are localized at the bsc locus in the Bordetella genome. Here, the function of a hypothetical protein Bcr4 encoded at the bsc locus in the B. bronchiseptica genome was investigated. A Bcr4-deficient mutant was created and the amounts of type III secreted proteins (e.g., BopB, BopN and Bsp22) in both the supernatant fraction and whole-cell lysates of the Bcr4-deficient mutant were determined. It was found that the amounts of these proteins were significantly lower than in the wild-type strain. The amounts of type III secreted proteins in the supernatant fraction and whole-cell lysates were much greater in a Bcr4-overproducing strain than in the wild-type strain. The type III secreted protein BspR reportedly negatively regulates the type III secretion system. Here, it was observed that a Bcr4 + BspR double-knockout mutant did not secrete type III secreted proteins, whereas the amounts of these proteins in whole-cell lysates of this mutant were nearly equal to those in whole-cell lysates of the BspR-deficient mutant. Bcr4 thus appears to play an essential role in the extracellular secretion of type III secreted proteins. Our data also suggest that Bcr4 antagonizes the negative regulatory function of BspR.

Bordetella effector BopN is translocated into host cells via its N-terminal residues.

Bordetella bronchiseptica infects a wide variety of mammals, the type III secretion system (T3SS) being involved in long-term colonization by Bordetella of the trachea and lung. T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alterations in the host's physiological function. The Bordetella effectors BopN and BteA are known to have roles in up-regulation of IL-10 and cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of translocation of BopN into host cells, truncated derivatives of BopN were built and the derivatives' ability to translocate into host cells evaluated by adenylate cyclase-mediated translocation assay. It was found that N-terminal amino acid (aa) residues 1-200 of BopN are sufficient for its translocation into host cells. Interestingly, BopN translocation was completely blocked by deletion of the N-terminal aa residues 6-50, indicating that the N-terminal region is critical for BopN translocation. Furthermore, BopN appears to play an auxiliary role in BteA-mediated cytotoxicity. Thus, BopN can apparently translocate into host cells and may facilitate activity of BteA.

The Bordetella Secreted Regulator BspR Is Translocated into the Nucleus of Host Cells via Its N-Terminal Moiety: Evaluation of Bacterial Effector Translocation by the Escherichia coli Type III Secretion System.

Bordetella bronchiseptica is genetically related to B. pertussis and B. parapertussis, which cause respiratory tract infections in humans. These pathogens possess a large number of virulence factors, including the type III secretion system (T3SS), which is required for the delivery of effectors into the host cells. In a previous study, we identified a transcriptional regulator, BspR, that is involved in the regulation of the T3SS-related genes in response to iron-starved conditions. A unique feature of BspR is that this regulator is secreted into the extracellular milieu via the T3SS. To further characterize the role of BspR in extracellular localization, we constructed various truncated derivatives of BspR and investigated their translocation into the host cells using conventional translocation assays. In this study, the effector translocation was evaluated by the T3SS of enteropathogenic E. coli (EPEC), since the exogenous expression of BspR triggers severe repression of the Bordetella T3SS expression. The results of the translocation assays using the EPEC T3SS showed that the N-terminal 150 amino acid (aa) residues of BspR are sufficient for translocation into the host cells in a T3SS-dependent manner. In addition, exogenous expression of BspR in HeLa cells demonstrated that the N-terminal 100 aa residues are involved in the nuclear localization. In contrast, the N-terminal 54 aa residues are sufficient for the extracellular secretion into the bacterial culture supernatant via the EPEC T3SS. Thus, BspR is not only a transcriptional regulator in bacteria cytosol, but also functions as an effector that translocates into the nuclei of infected host cells.