M. tuberculosis gene Expression during Transition to the “Non-Culturable” State

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Abstract


We analyzed the gene expression profile under specific conditions during reversible transition of M. tuberculosis cells to the “non-culturable” (NC ) state in a prolonged stationary phase. More than 500 genes were differentially regulated, while 238 genes were upregulated over all time points during NC cell formation. Approximately a quarter of these upregulated genes belong to insertion and phage sequences indicating a possible high intensity of genome modification processes taking place under transition to the NC state. Besides the high proportion of hypothetical/conserved hypothetical genes in the cohort of upregulated genes, there was a significant number of genes belonging to intermediary metabolism, respiration, information pathways, cell wall and cell processes, and genes encoding regulatory proteins. We conclude that NC cell formation is an active process involved in the regulation of many genes of different pathways. A more detailed analysis of the experimental data will help to understand the precise molecular mechanisms of dormancy/latency/persistence of M. tuberculosis in the future. The list of upregulated genes obtained in this study includes many genes found to be upregulated in other models of M. tuberculosis persistence. Thirteen upregulated genes, which are common for different models, can be considered as potential targets for the development of new anti-tuberculosis drugs directed mainly against latent tuberculosis.

Mycobacterium tuberculosis – the causative agent of tuberculosis – can persist in the human host for decades after infection. Such a latent M. tuberculosis state is traditionally connected with its transition to the dormant state, accompanied by loss of culturability [1]. This makes it practically impossible to reveal latent infection by traditional biochemical and microbiological means and attempt to cure it by antibiotic therapy. To study latent infection in live organisms, several modifications of the experimental model of dormancy during hypoxia in vitro are used [2, 3]. However, none of them imitates such an important state of bacteria as their “non-culturability” in the dormant state. We have established an experimental model where dormant M. tuberculosis cells are “non-culturable” (NC ) and can reactivate under special conditions [4]. To reveal the biochemical processes accompanying the transition of bacteria to the NC state and to understand the mechanisms of this phenomenon, we analyzed M. tuberculosis gene expression profile during the formation of NC cells. Met hods M. tuberculosis total RN A samples were extracted from cells in the late logarithmic phase (5 days of cultivation) and during the transition of cells to the NC state under incubation in the stationary phase at different time points (21, 30, 41 and 62 days of cultivation) as described previously [5]. cDNA was generated from 1µg RN A using random hexamers and reverse transcriptase (Superscript III, Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Reverse transcribed samples were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and labeled with Cy3- and Cy5-ULS according the suppliers' recommendations (Kreatech Diagnostics, Amsterdam, The Netherlands). Finally, labeled samples were purified with KRE Apure spin columns. Microarray experiments were performed as dual-color hybridizations. In order to compensate for the specific effects of the dyes and to ensure statistically relevant data, a color-swap dye-reversal analysis was performed. Cy3-labeled cDNA (250ng) corresponding to cells from different time points in the stationary phase was competitively hybridized with the same amount of Cy5-labeled cDNA of the control sample as color-swap technical replicates onto self-printed microarrays comprising a collection of 4,325 M. tuberculosis-specific “Array-Ready” 70mer DNA oligonucleotide capture probes and 25 control sequences (Operon Biotechnologies, Koeln, Germany) at 42°C for 20 h. Arrays were washed 3 times using a SSC wash protocol followed by scanning at 10 µm (Microarray Scanner BA, Agilent, Technologies, Waldbronn, Germany).

E G Salina

A.N. Bach Institute of Biochemistry, RAS

Email: elenasalina@mail.ru
119071, Moscow, Leninsky pr., 33

H J Mollenkopf

Max Planck Institute for Infection Biology

Email: mollenkopf@mpiib-berlin.mpg.de
10117, Berlin, Chariteplatz 1

S HE Kaufmann

Max Planck Institute for Infection Biology

10117, Berlin, Chariteplatz 1

A S Kaprelyants

A.N. Bach Institute of Biochemistry, RAS

119071, Moscow, Leninsky pr., 33

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Copyright (c) 2009 Salina E.G., Mollenkopf H.J., Kaufmann S.H., Kaprelyants A.S.

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