| 1. |
- Garbom, Sara, 1975-
(författare)
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A strategy to identify novel antimicrobial compounds : a bioinformatics and HTS approach
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets.We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant.Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells.We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.
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| 2. |
- Garbom, Sara, et al.
(författare)
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Identification of novel virulence-associated genes via genome analysis of hypothetical genes.
- 2004
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 72:3, s. 1333-1340
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Tidskriftsartikel (refereegranskat)abstract
- The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (vag genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans: Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi, Streptococcus pneumoniae, and Treponema pallidum. This comparison was limited to genes annotated as hypothetical in the T. pallidum genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with Yersinia pseudotuberculosis in-frame deletion mutants. In addition, these five vag genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding vag gene products may constitute novel targets for antimicrobial therapy and that some vag mutants could serve as carrier strains for live vaccines.
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| 3. |
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| 4. |
- Garbom, Sara, et al.
(författare)
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Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system.
- 2007
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Ingår i: Microbiology. - : Society for General Microbiology. - 1350-0872 .- 1465-2080. ; 153:Pt 5, s. 1464-73
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Tidskriftsartikel (refereegranskat)abstract
- Recently, a number of attenuated mutants of Yersinia pseudotuberculosis have been identified using a bioinformatics approach. One of the target genes identified in that study was vagH, which the authors now characterized further. VagH shows homology to HemK of Escherichia coli, possessing methyltransferase activity similar to that of HemK, and targeting release factors 1 and 2. Microarray studies comparing the wild-type and the vagH mutant revealed that the mRNA levels of only a few genes were altered in the mutant. By proteome analysis, expression of the virulence determinant YopD was found to be increased, indicating a possible connection between VagH and the virulence plasmid-encoded type III secretion system (T3SS). Further analysis showed that Yop expression and secretion were repressed in a vagH mutant. This phenotype could be suppressed by trans-complementation with the wild-type vagH gene or by deletion of the negative regulator yopD. Also, in a similar manner to a T3SS-negative mutant, the avirulent vagH mutant was rapidly cleared from Peyer's patches and could not reach the spleen after oral infection of mice. In a manner analogous to that of T3SS mutants, the vagH mutant could not block phagocytosis by macrophages. However, a vagH mutant showed no defects in the T3SS-independent ability to proliferate intracellularly and replicated to levels similar to those of the wild-type in macrophages. In conclusion, the vagH mutant exhibits a virulence phenotype similar to that of a T3SS-negative mutant, indicating a tight link between VagH and type III secretion in Y. pseudotuberculosis.
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| 7. |
- Liu, Yanjie, et al.
(författare)
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The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes
- 2008
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory Press (CSHL). - 0890-9369 .- 1549-5477. ; 22:8, s. 1051-1068
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Tidskriftsartikel (refereegranskat)abstract
- The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion.
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| 8. |
- Najdenski, H, et al.
(författare)
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Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model
- 2009
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Ingår i: Zoonoses and Public Health. - : John Wiley & Sons. - 1863-1959 .- 1863-2378. ; 56:4, s. 157-168
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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.
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| 9. |
- Rüssmann, Holger, et al.
(författare)
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Alternative Endogenous Protein Processing via an Autophagy-Dependent Pathway Compensates for Yersinia-Mediated Inhibition of Endosomal Major Histocompatibility Complex Class II Antigen Presentation
- 2010
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 78:12, s. 5138-5150
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins, Yops) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and MHC class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by Yersinias T3SS. Our data demonstrate Yersinias ability to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it neither required the function of the proteasome nor of the TAP. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.
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| 10. |
- Wiedig, Carolin A, et al.
(författare)
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Induction of CD8+ T cell responses by Yersinia vaccine carrier strains.
- 2005
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Ingår i: Vaccine. - 0264-410X. ; 23:42, s. 4984-98
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Tidskriftsartikel (refereegranskat)abstract
- Yersinia enterocolitica employs a type III secretion system (TTSS) to target virulence factors (e.g. YopE) into the cytosol of the host cells. We utilized the TTSS to introduce a recombinant antigen directly into the cytosol of host cells and to investigate the potential of Y. enterocolitica and Y. pseudotuberculosis as live carrier for vaccines. The model antigen ovalbumin (Ova) was fused to defined secretion or translocation domains of the Yersinia effector protein YopE and introduced into attenuated mutant strains of Y. enterocolitica and Y. pseudotuberculosis. In vitro experiments showed secretion and translocation of YopE-Ova hybrid proteins into host cells. To investigate the resulting immune responses, mice expressing transgenic Ova-specific T cell receptors were used. Both Y. enterocolitica and Y. pseudotuberculosis mutants induced efficaciously Ova-specific CD8+ T cell responses. The translocation domain of YopE was required for induction of CD8+ T cell responses in vivo, but not for T cell responses induced in vitro. The in vivo frequency of Ova-specific splenic T cells was up to six-fold higher in mice immunized with YopE-Ova-translocating Y. enterocolitica/Y. pseudotuberculosis mutants than in control mice. The Ova-specific T cells were shown to produce high amounts of IFN-gamma. We did not observe significant Ova-specific CD4+ T cell or antibody responses upon vaccination with either of the strains. In conclusion, Yersinia live carrier vaccine strains are suitable to target antigens into the MHC class I pathway and stimulate CD8+ T cell responses and thus, might be useful in vaccine approaches against intracellular pathogens.
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