scholarly journals Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Vojtech Kuban ◽  
Pavel Macek ◽  
Jozef Hritz ◽  
Katerina Nechvatalova ◽  
Katerina Nedbalcova ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Peptide Bond ◽  
Actinobacillus Pleuropneumoniae ◽  
Lysine Residue ◽  
Structural Basis ◽  
Gram Negative ◽  
Content Type ◽  
Isopeptide Bond ◽  
Autocatalytic Cleavage ◽  
Processing Module

ABSTRACT The posttranslational Ca2+-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface. IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

scholarly journals Draft Genome Sequence of Actinobacillus pleuropneumoniae Serotype 7 Strain S-8

2012 ◽  
Vol 194 (23) ◽  
pp. 6606-6607 ◽  
Author(s):  
Gang Li ◽  
Fang Xie ◽  
Yanhe Zhang ◽  
Chunlai Wang
Keyword(s):  
Genome Sequence ◽  
Draft Genome ◽  
Actinobacillus Pleuropneumoniae ◽  
Draft Genome Sequence ◽  
Economic Losses ◽  
Negative Bacterium ◽  
Swine Industry ◽  
Gram Negative ◽  
Content Type ◽  
Porcine Pleuropneumonia

ABSTRACTThe Gram-negative bacteriumActinobacillus pleuropneumoniaeis the etiological agent of porcine pleuropneumonia, a respiratory disease that leads to severe economic losses in the swine industry. For years, scientists working with it have lacked a reliable genome sequence for comparison with otherActinobacillusspecies. Here, we report the draft genome sequence ofA. pleuropneumoniaeserotype 7 (strain S-8), isolated from swine lung in China in 1992.

scholarly journals Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Christa Litschko ◽  
Insa Budde ◽  
Monika Berger ◽  
Andrea Bethe ◽  
Julia Schulze ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Enzymatic Synthesis ◽  
Actinobacillus Pleuropneumoniae ◽  
Glycerol Phosphate ◽  
Gram Positive ◽  
Content Type ◽  
Teichoic Acids ◽  
Bibersteinia Trehalosi ◽  
Wall Teichoic Acids ◽  
Mix And Match

ABSTRACT Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

scholarly journals Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

2015 ◽  
Vol 197 (22) ◽  
pp. 3583-3591 ◽  
Author(s):  
John R. Brannon ◽  
Jenny-Lee Thomassin ◽  
Samantha Gruenheid ◽  
Hervé Le Moual
Keyword(s):  
Substrate Specificity ◽  
Antimicrobial Peptide ◽  
Outer Membrane ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Structural Basis ◽  
Gram Negative ◽  
Content Type ◽  
Substrate Specificities ◽  
Structural Determinant

ABSTRACTBacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagicEscherichia coli(EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogenCitrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast,C. rodentiumCroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins ofC. rodentiumandE. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments.IMPORTANCEOmptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.

scholarly journals Draft Genome Sequence of Urease-Producing Pseudorhodobacter sp. Strain E13, Isolated from the Yellow Sea in Gunsan, South Korea

2019 ◽  
Vol 8 (23) ◽  
Author(s):  
Si Chul Kim ◽  
Hyo Jung Lee
Keyword(s):  
South Korea ◽  
Genome Sequence ◽  
Yellow Sea ◽  
Draft Genome ◽  
The Yellow Sea ◽  
Draft Genome Sequence ◽  
Protein Coding ◽  
Coding Sequences ◽  
Gram Negative ◽  
Content Type

Here, we report the draft genome sequence of Pseudorhodobacter sp. strain E13, a Gram-negative, aerobic, nonflagellated, and rod-shaped bacterium which was isolated from the Yellow Sea in South Korea. The assembled genome sequence is 3,878,578 bp long with 3,646 protein-coding sequences in 159 contigs.

scholarly journals Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Diseased Ayu (Plecoglossus altivelis altivelis) Caught at Three Sites in the Kagami River in Kochi, Japan

2019 ◽  
Vol 8 (34) ◽  
Author(s):  
Hazuki Yamashita ◽  
Takayuki Wada ◽  
Yusuke Kato ◽  
Takuji Ikeda ◽  
Masayuki Imajoh
Keyword(s):  
Draft Genome ◽  
Psychrophilic Bacterium ◽  
Genome Sequences ◽  
Plecoglossus Altivelis ◽  
Gram Negative ◽  
Content Type ◽  
Flavobacterium Psychrophilum ◽  
Skin Ulcers ◽  
The Family

Flavobacterium psychrophilum is a Gram-negative, psychrophilic bacterium within the family Flavobacteriaceae. Here, we report the draft genome sequences of three F. psychrophilum strains isolated from skin ulcers of diseased ayu caught by tomozuri angling at three sites in the Kagami River in Japan.

scholarly journals Retrospective Molecular Analysis of DIM-1 Metallo-β-Lactamase Discovered in Pseudomonas stutzeri from India in 2000

2013 ◽  
Vol 58 (1) ◽  
pp. 596-598 ◽  
Author(s):  
Lalitagauri M. Deshpande ◽  
Ronald N. Jones ◽  
Leah N. Woosley ◽  
Mariana Castanheira
Keyword(s):  
Molecular Analysis ◽  
Pseudomonas Stutzeri ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Environmental Source ◽  
Lactamase Gene ◽  
Encoding Genes

ABSTRACTAmong 220 clinical isolates of Gram-negative bacilli collected in India during 2000, 22 strains showing elevated imipenem MICs were evaluated for carbapenemase production. One DIM-1-producingPseudomonas stutzeriisolate was detected, and no other carbapenemase-encoding genes were identified. This detection of a DIM-1-producingP. stutzeriisolate from India predating the finding of this gene in the index Dutch strain and the very recent detection of DIM-1 in Africa suggest an unidentified environmental source of this metallo-β-lactamase gene.

scholarly journals Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Joel D. Ernst ◽  
Amber Cornelius ◽  
Miriam Bolz
Keyword(s):  
Protein Secretion ◽  
Immunosorbent Assay ◽  
Bacterial Population ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Protein ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

scholarly journals Structural basis of the interaction between the putative adhesion-involved and iron-regulated FrpD and FrpC proteins of Neisseria meningitidis

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Ekaterina Sviridova ◽  
Pavlina Rezacova ◽  
Alexey Bondar ◽  
Vaclav Veverka ◽  
Petr Novak ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Structural Basis
Sign in / Sign up

Export Citation Format

Share Document