| 1. |
- Frost, Stefan, et al.
(författare)
-
Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscU CC
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:11
-
Tidskriftsartikel (refereegranskat)abstract
- Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.
|
|
| 2. |
|
|
| 3. |
- Lundgren, E, et al.
(författare)
-
Invasin of Yersinia pseudotuberculosis activates human peripheral B cells.
- 1996
-
Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 64:3, s. 829-35
-
Tidskriftsartikel (refereegranskat)abstract
- The Yersinia pseudotuberculosis cell surface-located protein invasin was found to promote binding between the pathogen and resting peripheral B cells via beta 1 integrin receptors (CD29). B cells responded by expressing several activation markers and by growing, In contrast, T cells did not react, although these cells express CD29. An isogenic invA mutant failed to activate B cells. The mutation could be complemented by providing the invA+ gene in trans. Purified invasin alone did not activate B cells, although it was able to block the binding of bacteria to the cells.
|
|
| 4. |
- Weise, Christoph F, et al.
(författare)
-
Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein
- 2014
-
Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 107:8, s. 1950-1961
-
Tidskriftsartikel (refereegranskat)abstract
- The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.
|
|
| 5. |
- Arencibia, I, et al.
(författare)
-
Yersinia invasin, a bacterial beta1-integrin ligand, is a potent inducer of lymphocyte motility and migration to collagen type IV and fibronectin.
- 1997
-
Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 159:4, s. 1853-9
-
Tidskriftsartikel (refereegranskat)abstract
- The Yersinia pseudotuberculosis invasin protein was found to be a potent inducer of pseudopodia formation and chemotactic and haptotactic migration in human T lymphocytes. Checkerboard analysis confirmed that migration was directional. The Yersinia invasin triggered migration of otherwise poorly migratory normal T cells on fibronectin and in particular on collagen type IV, and augmented the migration of leukemic T cell lines on these components. Invasin-induced lymphocyte migration was inhibited by staurosporin that selectively prevented pseudopodia formation but, noteworthy, augmented adhesion. The motogenic and attractant properties of invasin (Inv) were mediated via beta1-integrins, as shown by lack of effect of Inv on the motility of a beta1-integrin-negative lymphoid cell line and inhibition of invasin-induced lymphocyte motility by anti-beta1 Abs. Inv was markedly more effective than the extracellular matrix components fibronectin, collagen type IV, and laminin, which also interact with lymphocyte beta1-integrins, with respect to induction of pseudopodia, chemotaxis, and haptotaxis. Thus, Yersinia invasin is a model ligand for induction of lymphocyte motility via beta1-integrins. The extraordinary capacity of Inv to trigger and guide T lymphocyte motility and potentiate lymphocyte migration to extracellular matrix components may be of pathogenetic significance for the movement of lymphocytes to extraintestinal sites secondary to Yersinia infection.
|
|
| 6. |
- Andersson, K, et al.
(författare)
-
YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis.
- 1996
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 20:5, s. 1057-69
-
Tidskriftsartikel (refereegranskat)abstract
- The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.
|
|
| 7. |
- Beery, T. H., et al.
(författare)
-
Nature to place : Rethinking the environmental connectedness perspective
- 2014
-
Ingår i: Journal of Environmental Psychology. - Amsterdam : Elsevier BV. - 0272-4944 .- 1522-9610. ; 40, s. 198-205
-
Tidskriftsartikel (refereegranskat)abstract
- The environmental connectedness perspective posits that direct encounter with generalized, or non-specific "nature," leads to environmental connectedness and subsequent pro-environmental behavior. This article examines this perspective and proposes a place-based application of the nature encounter-environmental behavior relation. An empirical study using data from a national survey on outdoor recreation and nature-based tourism is presented. Results show a minimal relationship between measures of environmental connectedness and self-reports of environmental behavior. The following examination of the environmental connectedness perspective reveals that environmental connectedness is rooted in a material/objective perspective, neglecting the human domain of perceptions, values, and representations. The environment as "nature" is portrayed as a geographically undefined agent with the inherent power to change human attitudes and behavior. Based on this, the article concludes with a proposed replacement of the elusive concept of nature for the relational concept of place. © 2014 Elsevier Ltd.
|
|
| 8. |
- Björnfot, Ann-Catrin, 1981-, et al.
(författare)
-
Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3S
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to secrete and deliver effectors called Yops into target cells. These processes are highly regulated and the pathogen senses cell contact and respond accordingly by inducing Yop-effector expression. A key component of the T3SS is the YscF needle present on the surface of the pathogen. It has been suggested that the bacterium can switch from needle export to Yop expression and secretion and that this substrate switch is important for proper regulation during infection. YscU is an essential protein regulating the substrate switch and autoproteolysis of YscU is essential for accurate T3SS regulation. To study regulation of Yop translocation in more detail, we generated mutants defective for expression of the heat shock proteins (HSPs) DnaJ and DnaK, since earlier studies had indicated a role of these proteins in regulation of effector translocation in Salmonella. The dnaJ mutant and the double dnaK/J mutant showed significant defects in Yop translocation, but surprisingly both mutants were able to secrete Yops in vitro much like the wild type. However, both mutants showed a changed export pattern of the YscF needle with a pronounced increased export of the YscF needle protein after incubation in calcium containing media. This phenotype was linked to defects in YscU autoproteolysis and in this respect the hsp-mutants were identical to earlier identified autoprocessing defective mutants in YscU (Single amino acid exchange mutants N263A and P264A). The hsp-mutants and the processing mutants accumulated full-length YscU, which surprisingly was associated with the outer membrane, while the processed form of YscU was found in the inner membrane fraction. The dnaJ and dnaK/J mutants were strongly affected in YscU autoproteolysis, which indicates a possible direct role for DnaJ in this process. Indeed a specific interaction between YscU and DnaJ could be found suggesting a direct role of the HSPs in regulation of the substrate switch in the T3SS.
|
|
| 9. |
- Bohr, J, et al.
(författare)
-
Yersinia species in collagenous colitis.
- 2002
-
Ingår i: Scandinavian Journal of Gastroenterology. - 0036-5521 .- 1502-7708. ; 37, s. 711-714
-
Tidskriftsartikel (refereegranskat)
|
|
| 10. |
- Engström, Patrik, et al.
(författare)
-
A 2-Pyridone-Amide Inhibitor Targets the Glucose Metabolism Pathway of Chlamydia trachomatis
- 2015
-
Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 6:1
-
Tidskriftsartikel (refereegranskat)abstract
- In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.
|
|
| 11. |
- Engström, Patrik, 1982-, et al.
(författare)
-
Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity
- 2013
-
Ingår i: Journal of Bacteriology. - Washington DC, USA : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 195:18, s. 4221-4230
-
Tidskriftsartikel (refereegranskat)abstract
- Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.
|
|
| 12. |
- Eriksson, Jonas, et al.
(författare)
-
Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis
- 2012
-
Ingår i: Advances in Yersinia Research. - New York : Springer. - 9781461435600 - 9781461435617 ; , s. 357-363
-
Bokkapitel (refereegranskat)abstract
- Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.
|
|
| 13. |
- Kovermann, Michael, et al.
(författare)
-
Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- 2015
-
Ingår i: Nature Communications. - : Macmillan Publishers Ltd.. - 2041-1723. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
|
|
| 14. |
- Kovermann, Michael, et al.
(författare)
-
Structural basis for ligand binding to an enzyme by a conformational selection pathway
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:24, s. 6298-6303
-
Tidskriftsartikel (refereegranskat)abstract
- Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
|
|
| 15. |
- Login, Frederic H., et al.
(författare)
-
YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal
- 2015
-
Ingår i: Journal of Biological Chemistry. - Umea Univ, Dept Mol Biol, Umea Ctr Microbial Res, SE-90187 Umea, Sweden. Umea Univ, Lab Mol Infect Med Sweden MIMS, Umea Ctr Microbial Res, SE-90187 Umea, Sweden.. - 0021-9258 .- 1083-351X. ; 290:43, s. 26282-26291
-
Tidskriftsartikel (refereegranskat)abstract
- All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscU(CC), the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+ -depleted medium at 37 degrees C. Here, we investigated the secretion of this early T3S-substrate and showed that YscU(CC) encompasses a specific C-terminal T3S signal within the 15 last residues (U-15). U-15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U-15 interacted with the ATPase YscN. Although U-15 is critical for YscU(CC) secretion, deletion of the C-terminal secretion signal of YscU(CC) did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion.
|
|
| 16. |
|
|
| 17. |
- Najdenski, H, et al.
(författare)
-
Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model
- 2009
-
Ingår i: Zoonoses and Public Health. - : John Wiley & Sons. - 1863-1959 .- 1863-2378. ; 56:4, s. 157-168
-
Tidskriftsartikel (refereegranskat)abstract
- Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.
|
|
| 18. |
- Nam, Kwangho, et al.
(författare)
-
Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release
- 2024
-
Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 64:1, s. 150-163
-
Tidskriftsartikel (refereegranskat)abstract
- This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.
|
|
| 19. |
- Nordfelth, R., et al.
(författare)
-
Small-molecule inhibitors specifically targeting type III secretion
- 2005
-
Ingår i: Infection and Immunity. - Washington : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 73:5, s. 3104-3114
-
Tidskriftsartikel (refereegranskat)abstract
- The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. A number of small organic molecules having this potential have recently been identified (A. M. Kauppi, R. Nordfelth, H. Uvell, H. Wolf-Watz, and M. Elofsson, Chem. Biol. 10:241-249, 2003). Using different reporter gene constructs, we showed that compounds that belong to a class of acylated hydrazones of different salicylaldehydes target the TTS system of Yersinia pseudotuberculosis. One of these compounds, compound 1, was studied in detail and was found to specifically block Yop effector secretion under in vitro conditions by targeting the TTS system. In this respect the drug mimics the well-known effect of calcium on Yop secretion. In addition, compound I inhibits Yop effector translocation after infection of HeLa cells without affecting the eukaryotic cells or the bacteria. A HeLa cell model that mimics in vivo conditions showed that compound 1 chemically attenuates the pathogen to the advantage of the eukaryotic cell. Thus, our results show proof of concept, i.e., that small compounds targeting the TTS system can be identified, and they point to the possible use of TTS inhibitors as a novel class of antibacterial agents.
|
|
| 20. |
- Norquist, A., et al.
(författare)
-
Protection against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.
- 1989
-
Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 55:6, s. 1400-1405
-
Tidskriftsartikel (refereegranskat)abstract
- The fish pathogen Vibrio anguillarum causes a lethal infectionin rainbow trout (Salmo gairdneri). Three different avirulentmutants, constructed by transposon insertion mutagenesis (VAN20and VAN70) or as antibiotic-resistant mutants (VAN1000), wereisolated by screening 200 individual isolated mutants for avirulence.When used as live vaccines, all three avirulent mutants wereable to induce protective immunity against the homologous aswell as a heterologous strain of V. anguillarum. When VAN1000was used, protective immunity could be recorded 1 week afterbath vaccination with 10(7) bacteria per ml of water for 30min. A single-dose immunization was effective for at least 12weeks. Western immunoblotting showed that strains of V. anguillarumhave antigenic determinants in common with Aeromonas strains.Therefore, we tested and confirmed that VAN1000 also was ableto induce protective immunity against challenge with Aeromonassalmonicida.
|
|
| 21. |
- Ojeda-May, Pedro, et al.
(författare)
-
Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions
- 2021
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 60:28, s. 2246-2258
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.
|
|
| 22. |
|
|
| 23. |
- O'Toole, R, et al.
(författare)
-
Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum.
- 1996
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 19:3, s. 625-637
-
Tidskriftsartikel (refereegranskat)abstract
- The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intra-peritoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum.
|
|
| 24. |
- O'Toole, R, et al.
(författare)
-
RpoN of the fish pathogen Vibrio (Listonella) anguillarum is essential for flagellum production and virulence by the water-borne but not intraperitoneal route of inoculation.
- 1997
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 143 ( Pt 12)
-
Tidskriftsartikel (refereegranskat)abstract
- To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative sigma 54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.
|
|
| 25. |
- Rehnstam, A.-S., et al.
(författare)
-
Identification of Vibrio anguillarum in fish using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.
- 1989
-
Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 55:8, s. 1907-1910
-
Tidskriftsartikel (refereegranskat)abstract
- 16S rRNA from seven different Vibrio anguillarum strains waspartially sequenced and compared. From this sequence informationwe could design a 25-base-long oligonucleotide and use it asa specific probe for identification of V. anguillarum. Thiswas determined by RNA-DNA colony hybridization and slot-blothybridization. Strong, specific hybridization to the probe wasobserved for all V. anguillarum strains tested. Furthermore,no cross-hybridization could be seen against five other bacterialspecies. The detection limit was 5 x 10(3) bacteria per ml.It was even possible to detect V. anguillarum, by slot-blothybridization, directly in a homogenized kidney from a fishthat had died of vibriosis. The partial sequence informationrevealed small but significant differences between strains ofthe same species. These sequence differences are sufficientlysignificant to allow serotyping on the RNA level. Comparingstrains of different serotypes revealed a 10-base and an 11-basedifference in V. anguillarum serotypes O8 and O9, respectively,in a 122-base partial sequence.
|
|
| 26. |
- Rogne, Per, et al.
(författare)
-
Molecular mechanism of ATP versus GTP selectivity of adenylate kinase
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. 3012-3017
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
|
|
| 27. |
|
|
| 28. |
|
|
| 29. |
- Verma, Apoorv, et al.
(författare)
-
Insights into the evolution of enzymatic specificity and catalysis : from Asgard archaea to human adenylate kinases
- 2022
-
Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:44
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.
|
|
| 30. |
|
|
