Recognition of extracellular DNA by type IV pili promotes biofilm formation by \u3ci\u3eClostridioides difficile\u3c/i\u3e

Clostridioides difficile is a Gram-positive bacillus, which is a frequent cause of gastrointestinal infections triggered by the depletion of the gut microbiome. Because of the frequent recurrence of these infections after antibiotic treatment, mechanisms of C. difficile persistence and recurrence, including biofilm formation, are of increasing interest. Previously, our group and others found that type IV pili, filamentous helical appendages polymerized from protein subunits, promoted microcolony and biofilm formation in C. difficile. In Gram-negative bacteria, the ability of type IV pili to mediate bacterial self-association has been explained through interactions between the pili of adjacent cells, but type IV pili from several Gram-negative species are also required for natural competence through DNA uptake. Here, we report the ability of two C. difficile pilin subunits, PilJ and PilW, to bind to DNA in vitro, as well as the defects in biofilm formation in the pilJ and pilW gene-interruption mutants. Additionally, we have resolved the X-ray crystal structure of PilW, which we use to model possible structural mechanisms for the formation of C. difficile biofilm through interactions between type IV pili and the DNA of the extracellular matrix. Taken together, our results provide further insight into the relationship between type IV pilus function and biofilm formation in C. difficile and, more broadly, suggest that DNA recognition by type IV pili and related structures may have functional importance beyond DNA uptake for natural competence

Structural diversity in the type IV pili of multidrug-resistant Acinetobacter

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating thatA. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter. Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species

Submillimeter Observations of The Isolated Massive Dense Clump IRAS 20126+4104

We used the CSO 10.4 meter telescope to image the 350 micron and 450 micron continuum and CO J=6-5 line emission of the IRAS 20126+4104 clump. The continuum and line observations show that the clump is isolated over a 4 pc region and has a radius of ~ 0.5 pc. Our analysis shows that the clump has a radial density profile propto r ^{-1.2} for r <~ 0.1 pc and has propto r^{-2.3} for r >~ 0.1 pc which suggests the inner region is infalling, while the infall wave has not yet reached the outer region. Assuming temperature gradient of r^{-0.35}, the power law indices become propto r ^{-0.9} for r < ~0.1 pc and propto r^{-2.0} for r >~ 0.1 pc. Based on a map of the flux ratio of 350micron/450micron, we identify three distinct regions: a bipolar feature that coincides with the large scale CO bipolar outflow; a cocoon-like region that encases the bipolar feature and has a warm surface; and a cold layer outside of the cocoon region. The complex patterns of the flux ratio map indicates that the clump is no longer uniform in terms of temperature as well as dust properties. The CO emission near the systemic velocity traces the dense clump and the outer layer of the clump shows narrow line widths (< ~3 km/s). The clump has a velocity gradient of ~ 2 km/s pc^{-1}, which we interpret as due to rotation of the clump, as the equilibrium mass (~ 200 Msun) is comparable to the LTE mass obtained from the CO line. Over a scale of ~ 1 pc, the clump rotates in the opposite sense with respect to the >~ 0.03 pc disk associated with the (proto)star. This is one of four objects in high-mass and low-mass star forming regions for which a discrepancy between the rotation sense of the envelope and the core has been found, suggesting that such a complex kinematics may not be unusual in star forming regions.Comment: 34 pages, 13 figures, Accepted for publication in the Ap

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCE: Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world\u27s population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus\u27s high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus

Exploring morphological correlations among H2CO, 12CO, MSX and continuum mappings

There are relatively few H2CO mappings of large-area giant molecular cloud (GMCs). H2CO absorption lines are good tracers for low-temperature molecular clouds towards star formation regions. Thus, the aim of the study was to identify H2CO distributions in ambient molecular clouds. We investigated morphologic relations among 6-cm continuum brightness temperature (CBT) data and H2CO (111-110; Nanshan 25-m radio telescope), 12CO (1--0; 1.2-m CfA telescope) and midcourse space experiment (MSX) data, and considered the impact of background components on foreground clouds. We report simultaneous 6-cm H2CO absorption lines and H110\alpha radio recombination line observations and give several large-area mappings at 4.8 GHz toward W49 (50'\times50'), W3 (70'\times90'), DR21/W75 (60'\times90') and NGC2024/NGC2023 (50'\times100') GMCs. By superimposing H2CO and 12CO contours onto the MSX color map, we can compare correlations. The resolution for H2CO, 12CO and MSX data was about 10', 8' and 18.3", respectively. Comparison of H2CO and 12CO contours, 8.28-\mu m MSX colorscale and CBT data revealed great morphological correlation in the large area, although there are some discrepancies between 12CO and H2CO peaks in small areas. The NGC2024/NGC2023 GMC is a large area of HII regions with a high CBT, but a H2CO cloud to the north is possible against the cosmic microwave background. A statistical diagram shows that 85.21% of H2CO absorption lines are distributed in the intensity range from -1.0 to 0 Jy and the \Delta V range from 1.206 to 5 km/s.Comment: 18 pages, 22 figures, 5 tables. Accepted to be published in Astrophysics and Space Scienc

Structure of \u3ci\u3eClostridium difficile\u3c/i\u3e PilJ Exhibits Unprecedented Divergence from Known Type IV Pilins

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Grampositive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface

Structural and Evolutionary Analyses Show Unique Stabilization Strategies in the Type IV Pili of Clostridium difficile

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion and horizontal gene transfer. However, many Gram-positive species, including C. difficile, also produce Type IV pili. Here, we identify the major subunit of the Type IV pili of C. difficile, PilA1, and describe multiple three-dimensional structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative Type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the Type IVb pilins from Gram-negative bacteria that suggest that the Type IV pili of C. difficile are involved in microcolony formation

The Cygnus X region XXII. A probable HAeBe star with a giant bipolar outflow in DR 16

From medium-resolution radio images, DR 16 was suspected to be a large cometary nebula. To test this suggestion we obtained a higher resolution (15\arcsec) VLA continuum map. We also analyzed data from the Canadian Galactic Plane Survey in continuum, H I line, and IR. These data were supplemented by published near-infrared (J, H, K) stellar photometric results and MSX 8.28 micrometer data. We suggest that DR 16 is the diffuse H II region of an ongoing star formation site at a distance of about 3 kpc. The complicated radio picture arises from the superposition of diffuse H II with the remains of a giant bipolar outflow. The outflow was generated by a probable Herbig AeBe star, and the lobes are the remnants of its working surfaces. Additional ring-like features are discussed. DR 16 is part of a larger volume of space in the local spiral arm where star formation is an ongoing process.Comment: 15 pages, 14 figures, accepted by A&

First images of 6.7-GHz methanol masers in DR21(OH) and DR21(OH)N

The first images of 6.7-GHz methanol masers in the massive star-forming regions DR21(OH) and DR21(OH)N are presented. By measuring the shapes, radial velocities and polarization properties of these masers it is possible to map out the structure, kinematics and magnetic fields in the molecular gas that surrounds newly-formed massive stars. The intrinsic angular resolution of the observations was 43 mas (~100 AU at the distance of DR21), but structures far smaller than this were revealed by employing a non-standard mapping technique. This technique was used in an attempt to identify the physical structure (e.g. disc, outflow, shock) associated with the methanol masers. Two distinct star-forming centres were identified. In DR21(OH) the masers had a linear morphology, and the individual maser spots each displayed an internal velocity gradient in the same direction as the large-scale structure. They were detected at the same position as the OH 1.7-GHz ground-state masers, close to the centre of an outflow traced by CO and class I methanol masers. The shape and velocity gradients of the masers suggests that they probably delineate a shock. In DR21(OH)N the methanol masers trace an arc with a double-peaked profile and a complex velocity gradient. This velocity gradient closely resembles that of a Keplerian disc. The masers in the arc are 4.5% linearly polarized, with a polarization angle that indicates that the magnetic field direction is roughly perpendicular to the large-scale magnetic field in the region (indicated by lower angular resolution measurements of the CO and dust polarization). The suitability of channel-by-channel centroid mapping is discussed as an improved and viable means to maximise the information gained from the data.Comment: Accepted by MNRA

Generalized eigenfunctions and spectral theory for strongly local Dirichlet forms

We present an introduction to the framework of strongly local Dirichlet forms and discuss connections between the existence of certain generalized eigenfunctions and spectral properties within this framework. The range of applications is illustrated by a list of examples