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A strategy to identify novel antimicrobial compounds: a bioinformatics and HTS approach
Umeå University, Faculty of Medicine, Molecular Biology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2006. , 57 p.
Keyword [en]
Antibiotic resistance, Yersinia, T3SS, virulence associated genes, VagH, HemK/PrmC, Release factors, small molecular inhibitors, HTS
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-900ISBN: 91-7264-190-8 (print)OAI: oai:DiVA.org:umu-900DiVA: diva2:144978
Public defence
2006-11-17, Major Groove, 6L NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-10-24 Created: 2006-10-24 Last updated: 2009-10-05Bibliographically approved
List of papers
1. Identification of novel virulence-associated genes via genome analysis of hypothetical genes.
Open this publication in new window or tab >>Identification of novel virulence-associated genes via genome analysis of hypothetical genes.
2004 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 72, no 3, 1333-1340 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Animals, Anti-Bacterial Agents/therapeutic use, Bacteria/*genetics/growth & development/*pathogenicity, Bacterial Infections/drug therapy/microbiology, Bacterial Proteins/genetics/physiology, Computational Biology, Drug Resistance; Multiple; Bacterial, Female, Genes; Bacterial, Genome; Bacterial, Hela Cells, Humans, Mice, Mice; Inbred C57BL, Mutagenesis, Phenotype, Virulence/genetics
Identifiers
urn:nbn:se:umu:diva-16697 (URN)10.1128/IAI.72.3.1333-1340.2004 (DOI)14977936 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved
2. Phenotypic characterization of a virulence associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the T3SS
Open this publication in new window or tab >>Phenotypic characterization of a virulence associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the T3SS
Show others...
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5458 (URN)
Available from: 2006-10-24 Created: 2006-10-24 Last updated: 2011-04-12Bibliographically approved
3. The negative regulator YopD regulates LcrF expression on the translational level as studied by a vagH mutant of Yersinia
Open this publication in new window or tab >>The negative regulator YopD regulates LcrF expression on the translational level as studied by a vagH mutant of Yersinia
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5459 (URN)
Available from: 2006-10-24 Created: 2006-10-24 Last updated: 2010-01-13Bibliographically approved
4. Small molecule inhibitors of the methyltransferase VagH resuce cells from T3SS-dependent toxicity
Open this publication in new window or tab >>Small molecule inhibitors of the methyltransferase VagH resuce cells from T3SS-dependent toxicity
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5460 (URN)
Available from: 2006-10-24 Created: 2006-10-24 Last updated: 2010-01-13Bibliographically approved

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