Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides

This work was supported in part by grants from the Canadian Institutes of Health Research (FDN_148364) (to C.W.). S.D.L. is a recipient of a Natural Science and Engineering Research Council Alexander Graham Bell Canada Graduate Scholarship and Michael Smith Foreign Study Supplement. C.W. is a Canada Research Chair. J.H.N. is a Wellcome Trust Investigator (100209/Z/12/Z).Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for Oacetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel beta-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S. Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.Publisher PDFPeer reviewe

Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

BACKGROUND: The enzymatic hydrolysis of alpha-mannosides is catalyzed by glycoside hydrolases (GH), termed alpha-mannosidases. These enzymes are found in different GH sequence-based families. Considerable research has probed the role of higher eukaryotic "GH38" alpha-mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 alpha-mannosidase II, which has been shown to be a retaining alpha-mannosidase that targets both alpha-1,3 and alpha-1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)(5)(GlcNAc)(2) hybrid N-glycans to GlcNAc(Man)(3)(GlcNAc)(2). Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 alpha-mannosidases whose activity and specificity is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an alpha-mannosidase with specificity for alpha-1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 A resolution and in complex with the inhibitor swainsonine (K(i) 18 microM) at 2.6 A, reveals a canonical GH38 five-domain structure in which the catalytic "-1" subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn(2+) ion. In contrast, the "leaving group" subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity. CONCLUSIONS/SIGNIFICANCE: Although the in vivo function of this streptococcal GH38 alpha-mannosidase remains unknown, it is shown to be an alpha-mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases

A new isotropic cell for studying the thermo-mechanical behavior of unsaturated expansive clays

This paper presents a new suction-temperature controlled isotropic cell that can be used to study the thermo-mechanical behavior of unsaturated expansive clays. The vapor equilibrium technique is used to control the soil suction; the temperature of the cell is controlled using a thermostat bath. The isotropic pressure is applied using a volume/pressure controller that is also used to monitor the volume change of soil specimen. Preliminary experimental results showed good performance of the cell

Action theory and the value of sport

I present a corrective to the formalist and conventionalist down-playing of physical actions in the understanding of the value of sport. I give a necessarily brief account of the Causal Theory of Action (CTA) and its implications for the normativity of actions. I show that the CTA has limitations, particularly in the case of failed or incomplete actions, and I show that failed or incomplete actions are constitutive of sport. This allows me to open up the space for another model, drawn from Aristotle, for failed or incomplete actions, conceived of as ‘doables’. This avoids some of the problems of the CTA. I explain the importance of difficult but doable actions, at which athletes often fail, and suggest that this establishes pro tanto value. Finally, I claim that this account of the actions that are constitutive of sport deepens our understanding of the value of sport as a whole

Use of a differential pressure transducer for the monitoring of soil volume change in cyclic triaxial test on unsaturated soils

A new experimental set-up using a differential pressure transducer was developed, that enables the monitoring of volume changes in cyclic triaxial tests on unsaturated soils. Calibration tests were performed in order to analyze the performance of the set-up, especially in terms of loading frequencies. Based on calibration results, a low frequency of 0.05 Hz was adopted for the tests carried out on the unsaturated loess from northern France. Five water contents were considered in the tests. The obtained results have confirmed the efficiency of the new system for volume change monitoring under cyclic loading. The effect of water content on the cyclic behavior of loess was clearly evidenced. Finally, some suggestions were made to improve the accuracy of the system

Professionalism, Golf Coaching and a Master of Science Degree: A commentary

As a point of reference I congratulate Simon Jenkins on tackling the issue of professionalism in coaching. As he points out coaching is not a profession, but this does not mean that coaching would not benefit from going through a professionalization process. As things stand I find that the stimulus article unpacks some critically important issues of professionalism, broadly within the context of golf coaching. However, I am not sure enough is made of understanding what professional (golf) coaching actually is nor how the development of a professional golf coach can be facilitated by a Master of Science Degree (M.Sc.). I will focus my commentary on these two issues

Half-metallicity and Slater-Pauling behavior in the ferromagnetic Heusler alloys

Introductory chapter for the book "Halfmetallic Alloys - Fundamentals and Applications" to be published in the series Springer Lecture Notes on Physics, P. H. Dederichs and I. Galanakis (eds). It contains a review of the theoretical work on the half-metallic Heusler alloys.Comment: Introductory chapter for the book "Halfmetallic Alloys - Fundamentals and Applications" to be published in the series Springer Lecture Notes on Physics, P. H. Dederichs and I. Galanakis (eds