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A comparative study of RNA and DNA as internal gene expression controls early in the developmental cycle of Chlamydia pneumoniae
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). (Bergström)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). (Bergström)
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2010 (Engelska)Ingår i: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 58, nr 2, s. 244-253Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Many microbial pathogens invade and proliferate within host cells and the molecular mechanism underlying this behavior is currently being revealed for several bacterial species. Testing clinically relevant antibacterial compounds and elucidating their effects on gene expression requires adequate controls, especially when studying genetically intractable organisms such as Chlamydia spp., for which various gene fusions cannot be constructed. Until now, relative mRNA levels in Chlamydia have been measured using different internal gene expression controls, including 16S rRNA, mRNAs, and DNA. Here, we compared the advantages and disadvantages of various internal expression controls during the early phase of Chlamydia pneumoniae development. The relative abundance of target mRNAs varied using the different internal control RNAs. This was partly due to variation in the transcript stability of the RNA species. Also, seven out of nine of the analyzed RNAs increased fivefold or more between 2 and 14 h postinfection, while the amount of DNA and number of cells remained essentially unaltered. Our results suggest that RNA should not be used as a gene expression control during the early phase of Chlamydia development, and that intrinsic bacterial DNA is preferable for that purpose because it is stable, abundant, and its relative amount is generally correlated with bacterial numbers.

Ort, förlag, år, upplaga, sidor
2010. Vol. 58, nr 2, s. 244-253
Nyckelord [en]
Chlamydia pneumoniae, gene expression, internal controls, mRNA stabilit
Nationell ämneskategori
Infektionsmedicin Mikrobiologi inom det medicinska området
Identifikatorer
URN: urn:nbn:se:umu:diva-32837DOI: 10.1111/j.1574-695X.2009.00631.xISI: 000274311700011PubMedID: 20002746OAI: oai:DiVA.org:umu-32837DiVA, id: diva2:306266
Tillgänglig från: 2010-03-29 Skapad: 2010-03-29 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. Chemical genetics discloses the importance of heme and glucose metabolism in Chlamydia trachomatis pathogenesis
Öppna denna publikation i ny flik eller fönster >>Chemical genetics discloses the importance of heme and glucose metabolism in Chlamydia trachomatis pathogenesis
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Chlamydiae are important human bacterial pathogens with an intracellular life cycle that consists of two distinct bacterial forms, an infectious form (EB) that infects the eukaryotic host cell, and a non-infectious form (RB) that allows intracellular proliferation. To be successful, chlamydiae need to alternate between EB and RB to generate infectious EB’s which are competent to infect new host cells.

Chemical genetics is an attractive approach to study bacterial pathogenesis; in principal this approach relies on an inhibitory compound that specifically inhibits a protein of interest. An obstacle in using this approach is target identification, however whole genome sequencing (WGS) of spontaneous mutants resistant to novel inhibitory compounds has significantly extended the utility of chemical genetic approaches by allowing the identification of their target proteins and/or biological pathways.

In this thesis, a chemical genetics approach is used, I have found that heme and glucose metabolism of C. trachomatis is specifically important for the transition from the RB form to the infectious EB form. Heme and glucose metabolism are both coupled to energy metabolism, which suggests a common link between the RB-to-EB transitions. In connection with the above findings I have developed strategies that enable the isolation of isogenic C. trachomatis mutant strains. These strategies are based on WGS of spontaneous mutant populations and subsequent genotyping of clonal strains isolated from these mutant populations. Experiments with the mutant strains suggest that the uptake of glucose-6-phosphate (G-6-P) regulates the RB-to-EB transition, representing one of the first examples where genetics has been used to study C. trachomatis pathogenesis. Additional experiments with the mutant strains indicate that G-6-P promotes bacterial growth during metabolic stress.

In concert with other findings presented in this thesis, I have fine-tuned methods that could be employed to reveal how novel inhibitory chemical compounds affect chlamydiae. In a broader context, I suggest that C. trachomatis could be used as a model organism to understand how new inhibitory drugs affect other bacterial pathogens.

In addition, I observed that C. pneumoniae infections resulted in generalized bone loss in mice and that these mice display a cytokine profile similar to infected bone cells in vitro. Thus, this study indicates that C. pneumoniae potentially can infect bone cells in vivo, resulting in bone loss, alternatively, the inflammatory responses seen in vivo could be the causative factor of the bone loss observed.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet, 2013. s. 50
Nyckelord
Chlamydiae, heme metabolism, glucose metabolism, glucose-6-phosphate, RB-to-EB transition
Nationell ämneskategori
Naturvetenskap
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-70304 (URN)978-91-7459-673-1 (ISBN)978-91-7459-674-8 (ISBN)
Disputation
2013-06-04, Biomedicinhuset, Byggnad 6A, NUS, sal A5, Plan 0, Umeå universitet, Umeå, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-05-14 Skapad: 2013-05-13 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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Engström, PatrikBergström, SvenJohansson, Jörgen
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Institutionen för molekylärbiologi (Medicinska fakulteten)Molekylär Infektionsmedicin, Sverige (MIMS)Institutionen för matematik och matematisk statistik
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FEMS Immunology and Medical Microbiology
InfektionsmedicinMikrobiologi inom det medicinska området

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