DNA Uptake by Type IV Filaments

Bacterial uptake of DNA through type IV filaments is an essential component of natural competence in numerous gram-positive and gram-negative species. Recent advances in the field have broadened our understanding of the structures used to take up extracellular DNA. Here, we review seminal experiments in the literature describing DNA binding by type IV pili, competence pili and the flp pili of Micrococcus luteus; collectively referred to here as type IV filaments. We compare the current state of the field on mechanisms of DNA uptake for these three appendage systems and describe the current mechanistic understanding of both DNA-binding and DNA-uptake by these versatile molecular machines

DNA Uptake by Type IV Filaments

Bacterial uptake of DNA through type IV filaments is an essential component of natural competence in numerous gram-positive and gram-negative species. Recent advances in the field have broadened our understanding of the structures used to take up extracellular DNA. Here, we review seminal experiments in the literature describing DNA binding by type IV pili, competence pili and the flp pili of Micrococcus luteus; collectively referred to here as type IV filaments. We compare the current state of the field on mechanisms of DNA uptake for these three appendage systems and describe the current mechanistic understanding of both DNA-binding and DNA-uptake by these versatile molecular machines

Type IV Pilus-Mediated Inhibition of \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e Biofilm Formation by Phenothiazine Compounds

Infections by pathogenic Acinetobacter species represent a significant burden on the health care system, despite their relative rarity, due to the difficulty of treating infections through oral antibiotics. Multidrug resistance is commonly observed in clinical Acinetobacter infections and multiple molecular mechanisms have been identified for this resistance, including multidrug efflux pumps, carbapenemase enzymes, and the formation of bacterial biofilm in persistent infections. Phenothiazine compounds have been identified as a potential inhibitor of type IV pilus production in multiple Gram-negative bacterial species. Here, we report the ability of two phenothiazines to inhibit type IV pilus-dependent surface (twitching) motility and biofilm formation in multiple Acinetobacter species. Biofilm formation was inhibited in both static and continuous flow models at micromolar concentrations without significant cytotoxicity, suggesting that type IV pilus biogenesis was the primary molecular target for these compounds. These results suggest that phenothiazines may be useful lead compounds for the development of biofilm dispersal agents against Gram-negative bacterial infections

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

The basis for limited specificity and MHC restriction in a T cell receptor interface

αβ Tcell receptors (TCRs) recognize peptides presented by major histocompatibility complex (MHC) proteins using multiple complementarity-determining region (CDR) loops. TCRs display an array of poorly understood recognition properties, including specificity, crossreactivity and MHC restriction. Here we report a comprehensive thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A*0201, uncovering the physical basis for the receptor’s recognition properties. Broadly, our findings are in conflict with widely held generalities regarding TCR recognition, such as the relative contributions of central and peripheral peptide residues and the roles of the hypervariable and germline CDR loops in engaging peptide and MHC. Instead, we find that the recognition properties of the receptor emerge from the need to engage the composite peptide/MHC surface, with the receptor utilizing its CDR loops in a cooperative fashion such that specificity, crossreactivity and MHC restriction are inextricably linked

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

The JCMT 12CO(3-2) Survey of the Cygnus X Region: I. A Pathfinder

Cygnus X is one of the most complex areas in the sky. This complicates interpretation, but also creates the opportunity to investigate accretion into molecular clouds and many subsequent stages of star formation, all within one small field of view. Understanding large complexes like Cygnus X is the key to understanding the dominant role that massive star complexes play in galaxies across the Universe. The main goal of this study is to establish feasibility of a high-resolution CO survey of the entire Cygnus X region by observing part of it as a Pathfinder, and to evaluate the survey as a tool for investigating the star-formation process. A 2x4 degree area of the Cygnus X region has been mapped in the 12CO(3-2) line at an angular resolution of 15" and a velocity resolution of ~0.4km/s using HARP-B and ACSIS on the James Clerk Maxwell Telescope. The star formation process is heavily connected to the life-cycle of the molecular material in the interstellar medium. The high critical density of the 12CO(3-2) transition reveals clouds in key stages of molecule formation, and shows processes that turn a molecular cloud into a star. We observed ~15% of Cygnus X, and demonstrated that a full survey would be feasible and rewarding. We detected three distinct layers of 12CO(3-2) emission, related to the Cygnus Rift (500-800 pc), to W75N (1-1.8 kpc), and to DR21 (1.5-2.5 kpc). Within the Cygnus Rift, HI self-absorption features are tightly correlated with faint diffuse CO emission, while HISA features in the DR21 layer are mostly unrelated to any CO emission. 47 molecular outflows were detected in the Pathfinder, 27 of them previously unknown. Sequentially triggered star formation is a widespread phenomenon.Comment: 18 pages, 13 figures, accepted for publication in Astronomy & Astrophysic

Fluorine substitutions in an antigenic peptide selectively modulate T cell receptor binding in a minimally perturbing manner

T cell receptor (TCR) recognition of antigenic peptides bound and presented by major histocompatibility complex (MHC) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide/MHC molecules weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Further, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. Here, we studied how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the HTLV-1 Tax11-19 peptide (Tax) enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with A6, independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, in probing the mechanism of fluorine’s effect on TCR binding, our data were most consistent with fluorine’s unique “polar hydrophobicity,” a finding which should impact other attempts to alter molecular recognition with fluorine