The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

Iñaki de Diego et al.In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, UMO-2011/01/D/NZ1/01169, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; BIO2013-49320-EXP; MDM-2014-0435; 1306/MOB/IV/2015/0 (“Mobilność Plus” MK) and 2014SGR9). The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRFPeer Reviewe

Trypanosoma cruzi macrophage infectivity potentiator has a rotamase core and a highly exposed α-helix

The macrophage infectivity potentiator protein from Trypanosoma cruzi (TcMIP) is a major virulence factor secreted by the etiological agent of Chagas' disease. It is functionally involved in host cell invasion. We have determined the three-dimensional crystal structure of TcMIP at 1.7 Å resolution. The monomeric protein displays a peptidyl-prolyl cis-trans isomerase (PPlase) core, encompassing the characteristic rotamase hydrophobic active site, thus explaining the strong inhibition of TcMIP by the immunosuppressant FK506 and related drugs. In TcMIP, the twisted β-sheet of the core is extended by an extra β-strand, preceded by a long, exposed N-terminal α-helix, which might be a target recognition element. An invasion assay shows that the MIP protein from Legionella pneumophila (LpMIP), which has an equivalent N-terminal α-helix, can substitute for TcMIP. An additional exposed α-helix, this one unique to TcMIP, is located in the C-terminus of the protein. The high-resolution structure reported here opens the possibility for the design of new inhibitory drugs that might be useful for the clinical treatment of American trypanosomiasis.This work was supported by grants from Ministerio de Educación y Cultura (PB98-1631 and 2FD97-0518), CSIC and Generalitat de Catalunya (CERBA and 1999SGR188) to M.C., grant PB98-0479 to A.G. and by grant BIO2000-1659 to F.X.G.-R. P.J.B.P. and S.M.-R. acknowledge postdoctoral fellowships from FCT (Portugal). Data collection at DESY was supported by EC grants ERBFMGECT980134 and HPRI-CT-1999-00017 to EMBL-HamburgPeer Reviewe

Crystallization and preliminary X-ray diffraction analysis of eukaryotic α2-macroglobulin family members modified by methylamine, proteases and glycosidases

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Summary: α2-Macroglobulin (α2M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2M (hα2M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.European, Spanish, and Catalan Agencies. Grant Number: FP7-HEALTH-2010-261460; Gums&Joints. Grant Number: FP7-PEOPLE-2011-ITN-290246; RAPID. Grant Number: FP7-HEALTH-2012-306029-2; TRIGGER. Grant Numbers: BFU2012-32862, CSD2006-00015; La Marató de TV3. Grant Numbers: 2009-100732, 2009SGR1036; ESRFPeer Reviewe

Efficacy of liposomal amphotericin B and anidulafungin using an antifungal lock technique (ALT) for catheter-related Candida albicans and Candida glabrata infections in an experimental model

Funding: This study was co-financed by the European Development Regional Fund "A way to achieve Europe" ERDF and partially by Pfizer, Inc.Objective: The aims of this study were as follows. First, we sought to compare the in vitro susceptibility of liposomal amphotericin B (LAmB) and anidulafungin on Candida albicans and Candida glabrata biofilms growing on silicone discs. Second, we sought to compare the activity of LAmB versus anidulafungin for the treatment of experimental catheter-related C. albicans and C. glabrata infections with the antifungal lock technique in a rabbit model. Methods: Two C. albicans and two C. glabrata clinical strains were used. The minimum biofilm eradication concentration for 90% eradication (MBEC ) values were determined after 48h of treatment with LAmB and anidulafungin. Confocal microscopy was used to visualize the morphology and viability of yeasts growing in biofilms. Central venous catheters were inserted into New Zealand rabbits, which were inoculated of each strain of C. albicans and C. glabrata. Then, catheters were treated for 48h with saline or with antifungal lock technique using either LAmB (5mg/mL) or anidulafungin (3.33mg/mL). Results: In vitro: anidulafungin showed greater activity than LAmB against C. albicans and C. glabrata strains. For C. albicans: MBEC of anidulafungin versus LAmB: CA176, 0.03 vs. 128 mg/L; CA180, 0.5 vs. 64 mg/L. For C. glabrata: MBEC of anidulafungin versus LAmB: CG171, 0.5 vs. 64 mg/L; CG334, 2 vs. 32 mg/L. In vivo: for C. albicans species, LAmB and anidulafungin achieved significant reductions relative to growth control of log cfu recovered from the catheter tips (CA176: 3.6±0.3 log CFU, p0.0001; CA180: 3.8±0.1 log CFU, p0.01). For C. glabrata, anidulafungin lock therapy achieved significant reductions relative to the other treatments (CG171: 4.8 log CFU, p0.0001; CG334: 5.1 log CFU, p0.0001) Conclusions: For the C. albicans strains, both LAmB and anidulafungin may be promising antifungal lock technique for long-term catheter-related infections; however, anidulafungin showed significantly higher activity than LAmB against the C. glabrata strains

Determination of hemihedral twinning and initial structural analysis of crystals of the procarboxypeptidase A ternary complex

The initial structural analysis of the ternary complex of procarboxypeptidase A from hemihedrally twinned crystals diffracting up to 2.8 Å is described. Detection of twinning by different techniques is presented, including biochemical and intensity statistics approaches. The structure was initially solved using Patterson-search techniques, and the three positioned search models were used to effectively deconvolute the twinned data

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

Theodoros Goulas et al.Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a >Michaelis loop> that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteinehistidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, 2137/7.PR-EU/2011/2, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; FP7-HEALTH-2010-261460 “Gums&Joints”; BFU2012-32862; BFU2012-33516; BFU2012-35367; BIO2013-49320-EXP; MDM-2014-0435; 2014SGR9 and 2014SGR997). IGF acknowledges an FPU-fellowship (AP2010-3799) from the former Spanish Ministry for Education, Culture and Sport. TG acknowledges a “Juan de la Cierva” research contract (JCI-2012-13573) from the Spanish Ministry for Economy and Competitiveness. The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRFPeer Reviewe

Optical Photometric Monitoring of LS i +61 303

Three gamma-ray binaries, namely PSR B1259 63, HESS J0632+057 and LS I +61 303, contain compact objects orbiting around massive Be stars. Around periastron passage the compact objects should produce significant changes in the structure of the Be disks due to gravitational forces and eventually by ram pressure from the putative pulsar wind. Indeed, variability in the Hα emission line has been detected in all these systems, and optical periodic variability has been detected in one of them. However, there is lack of a systematic monitoring with accurate photometry, which could be used to constrain the shape of the disk during the periastron passage. This information is important to build accurate physical models to explain the broadband spectral energy distribution of these sources. Here we present an ongoing program to monitor the optical photometry of gamma-ray binaries and show preliminary results for the case of LS I +61 303

The 2009 multiwavelength campaign on Mrk 421: Variability and correlation studies

Aims: We perform an extensive characterization of the broadband emission of Mrk 421, as well as its temporal evolution, during the non-flaring (low) state. The high brightness and nearby location (z = 0.031) of Mrk 421 make it an excellent laboratory to study blazar emission. The goal is to learn about the physical processes responsible for the typical emission of Mrk 421, which might also be extended to other blazars that are located farther away and hence are more difficult to study. Methods: We performed a 4.5-month multi-instrument campaign on Mrk 421 between January 2009 and June 2009, which included VLBA, F-GAMMA, GASP-WEBT, Swift, RXTE, Fermi-LAT, MAGIC, and Whipple, among other instruments and collaborations. This extensive radio to very-high-energy (VHE; E> 100 GeV) γ-ray dataset provides excellent temporal and energy coverage, which allows detailed studies of the evolution of the broadband spectral energy distribution. Results: Mrk421 was found in its typical (non-flaring) activity state, with a VHE flux of about half that of the Crab Nebula, yet the light curves show significant variability at all wavelengths, the highest variability being in the X-rays. We determined the power spectral densities (PSD) at most wavelengths and found that all PSDs can be described by power-laws without a break, and with indices consistent with pink/red-noise behavior. We observed a harder-when-brighter behavior in the X-ray spectra and measured a positive correlation between VHE and X-ray fluxes with zero time lag. Such characteristics have been reported many times during flaring activity, but here they are reported for the first time in the non-flaring state. We also observed an overall anti-correlation between optical/UV and X-rays extending over the duration of the campaign. Conclusions: The harder-when-brighter behavior in the X-ray spectra and the measured positive X-ray/VHE correlation during the 2009 multi-wavelength campaign suggests that the physical processes dominating the emission during non-flaring states have similarities with those occurring during flaring activity. In particular, this observation supports leptonic scenarios as being responsible for the emission of Mrk 421 during non-flaring activity. Such a temporally extended X-ray/VHE correlation is not driven by any single flaring event, and hence is difficult to explain within the standard hadronic scenarios. The highest variability is observed in the X-ray band, which, within the one-zone synchrotron self-Compton scenario, indicates that the electron energy distribution is most variable at the highest energies. Appendix A is available in electronic form at http://www.aanda.orgThe complete data set shown in Fig. 1 is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/576/A12