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RESEARCH ARTICLES
M. tuberculosis Gene Expression during Transition to the "Non-Culturable" State
E.G. Salina1, H.J. Mollenkopf2*, S.H.E. Kaufmann2, A.S. Kaprelyants1
1A.N. Bach Institute of Biochemistry, RAS, 119071, Moscow, Leninsky pr., 33, fax (495) 954-27-32, elenasalina@mail.ru
2Max Planck Institute for Infection Biology, 10117, Berlin, Chariteplatz 1, fax 49-30-284 605 01, *E-mail: mollenkopf@mpiib-berlin.mpg.de
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.
Keywords: M. tuberculosis, latent tuberculosis, “non-culturable” cells, global gene expression profile
INTRODUCTION
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.
METHODS
M. tuberculosis total RNA 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 1^g RNA using random hex-amers and reverse transcriptase (Superscript III, Invitro-gen, 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 KREApure 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 ^m (Microarray Scanner BA, Agilent, Technologies, Waldbronn, Germany). Image analysis was carried out with Agilent’s feature extraction software version (Agilent, Technologies, Waldbronn, Germany). The extracted MAGE-ML files were further analyzed with the Rosetta Resolver Biosoftware, Build 7.1 (Rosetta Biosoftware, Seattle, USA). Ratio profiles comprising color-swap hybridizations were combined in an error-weighted fashion to create ratio experiments. Anticorrelation of dye-reversals was determined by the compare function of Resolver. Next we applied a Student's t-test. Finally, by combining a 1.5-fold change cutoff to ratio experiments and the anticorrelation criterion together with the signatures from the Student's t-test, all valid data points had a P-value < 0.01, rendering the analysis highly robust and reproducible.
RESULTS AND DISCUSSION
We found earlier that M. tuberculosis cultivation in the modified Sauton medium without K+ supplemented by dextrose, BSA, and sodium chloride led to a decrease in colony forming units (CFU) on the solid medium in the stationary phase [4]. After 60 days of cultivation, the CFU count dropped to 105 per ml (Fig. 1), which meant a transition of 99.9% of cells to the NC state. During further cultivation of cells, spontaneous recovery of NC cells was observed. It is important that the NC state was reversible, and that cells with a minimum CFu count could be reactivated after regrowing them in fresh medium.
Comparison of the gene expression profile at different time points from the stationary to the logarithmic phase (5-day cultivation) revealed a different expression (at least 1.5-fold) for a significant number of genes (566), which corresponds to 14% of the M. tuberculosis genome. Some 238 genes are upregulated and 237 downregulated over all time points during the entire culture period. Table 1 shows the functional category of differentially regulated genes during the transition of cells to the NC state.
Besides the significant amount of conserved hypotheticals/unknown genes, many genes involved in the intermediary metabolism and respiration, virulence, detoxification and adaptation, lipid metabolism, information pathways, cell wall and cell processes were downregulated.
A considerable amount of genes coding hypothetical proteins were also found to be upregulated in the NC state: remarkably, genes encoding insertion sequences and phages represented about a quarter of the genes upregulated in the NC state, whereas their proportion in the genome was smaller - only 3.7%. This fact is a possible illustration of the high intensity of genome modification processes during the transition of cells to the NC state.
A significant proportion of upregulated genes belonged to the intermediary metabolism and respiration category, in par-
cultivation time, days
Fig. 1. Formation of NC M. tuberculosis cells in the stationary phase.
Time points where RNA was isolated are marked by arrows
ticular, gcvB and ald, coding, respectively, glycine dehydrogenase and L-alanine dehydrogenase, proteases pepR and clpC2. icl1 - one of the genes coding isocitrate lyase, anaplerotic enzyme, existing in the M. tuberculosis cells in two isoforms icl 1 and icl 2 - was found upregulated. Isocitrate lyase is the key enzyme of the glyoxylate cycle - a metabolic pathway, which is an alternative for the tricarboxylic acid cycle and allows the synthesis of carbohydrates from simple precursors. In particular, it plays an important role in seed germination, where fatty acids are used as the main storage of carbon and energy. The induction of some genes involved in lipid degradation, such as fadD9, fadE24, fadE26, and fatty acid degradation, scoA, is indicative of the active role of the glyoxylate cycle in NC cells already found for the Wayne persistence model [2].
Table 1. Functional categories of M. tuberculosis genes with changed expression level during transition to the NC state
Functional categories Genes induced during transition to the NC state Genes repressed during transition to the NC state Percent (%) in the genome
Number of genes % Number of genes %
Virulence, detoxification, adaptation 5 2.1 7 2.9 2.6
Lipid metabolism 6 2.5 20 8.4 5.9
Information pathways 13 5.5 23 9.7 5.8
Cell wall and cell processes 24 10.1 59 24.8 18.8
Insertion sequences and phages 58 24.4 1 0.4 3.7
PE/PPE 7 2.9 11 4.6 4.2
Intermediary metabolism and respiration 42 17.7 50 21.1 22.4
Regulatory proteins 16 6.7 4 1.7 4.8
Unknown/hypothetical 67 28.1 63 26.5 31.9
Total number of genes 238 - 237 - 3924/100
Table 2. Significantly upregulated genes during transition to the NC state in the stationary phase
ORF Gene Gene product Change of gene expression level
5 days 21 days 30 days 41 days 62 days
Rv0186 bglS Beta-glucosidase 1 4.20459 8.33686 6.51867 5.24295
Rv0840c pip Proline iminopeptidase 1 6.33559 11.0004 4.58881 3.86572
Rv0841c Transmembrane protein 1 31.11093 52.56174 13.79488 11.85425
Rv0989c grcC2 Polyprenil-diphosphate synthase 1 7.60797 6.29748 7.58723 3.94285
Rv0990c Hypothetical protein 1 7.12899 6.60915 6.652 3.57343
Rv0991c Conserved hypothetical protein 1 3.31598 3.87521 5.44297 3.70462
Rv1369c Transposase 1 3.17178 3.9213 4.22925 3.86883
Rv1394c cyp132 Cytochrome P450 132 1 8.89047 7.50161 3.72981 3.12534
Rv1395 Transcriptional regulatory protein 1 3.22394 11.65875 7.03908 4.27327
Rv1397c Conserved hypothetical protein 1 6.95276 11.79184 5.97336 5.77752
Rv1460 Transcriptional regulatory protein 1 3.87617 5.50637 6.90405 3.78332
Rv1575 phiRVl phage protein 1 17.29509 37.08693 51.7473 20.53329
Rv1576c phiRVl phage protein 1 28.17817 33.97652 10.11378 12.88182
Rv1577c phiRVl phage protein 1 26.27261 39.87495 19.41512 11.49041
Rv1584c phiRVl phage protein 1 3.27674 5.68552 3.3055 3.02886
Rv1831 Hypothetical protein 1 3.1468 5.74692 5.14019 4.04747
Rv1991c Conserved hypothetical protein 1 4.04696 4.12618 4.06786 4.65579
Rv1992c ctpG Metal cation transporter ATPase 1 5.2883 7.31348 4.7442 4.22806
Rv2106 Transposase 1 3.01418 5.61324 4.77882 5.09925
Rv2254c Integral membrane protein 1 7.09534 6.53956 3.33899 4.63885
Rv2278 Transposase 1 3.28663 6.78129 6.28036 4.13102
Rv2354 Transposase 1 3.1594 6.15299 5.21098 3.13151
Rv2497c pdhA Pyruvate dehydrogenase alpha subunit 1 3.73133 4.52197 5.04976 4.00306
Rv2642 ArsR family transcriptional regulator 1 3.76985 5.16757 4.39006 3.93426
Rv2644c Hypothetical protein 1 3.36059 7.58921 5.36796 3.51825
Rv2645 Hypothetical protein 1 3.45006 8.21393 6.70101 3.25709
Rv2646 Integrase 1 5.04391 12.16535 7.82435 9.96087
Rv2647 Hypothetical protein 1 5.32983 13.40623 9.43796 7.2163
Rv2649 Transposase IS6110 1 3.2505 5.3557 5.59089 3.74714
Rv2650c phiRv2 prophage protein 1 21.46669 29.74372 16.65359 20.66349
Rv2651c phiRv2 prophage protease 1 20.04086 34.29153 20.61728 13.41666
Rv2660c Hypothetical protein 1 13.43717 41.25793 67.29882 19.6699
Rv2661c Hypothetical protein 1 9.23174 28.30861 52.34967 11.04351
Rv2662 Hypothetical protein 1 20.62942 18.83647 14.72059 12.88898
Rv2663 Hypothetical protein 1 7.61461 9.43216 8.19525 7.3034
Rv2664 Hypothetical protein 1 6.24636 8.49102 7.10191 5.60291
Rv2666 Truncated transposase IS1081 1 6.91867 13.89339 7.89331 5.86579
Rv2667 clpC2 ATP-dependent protease 1 9.44815 17.89662 9.64508 6.46149
Rv2707 Conserved transmembrane protein 1 3.35002 5.09024 14.83903 4.53239
Rv2711 ideR Transcriptional regulatory protein 1 3.48877 4.30099 6.06795 3.83858
Rv2713 sthA Soluble pyridine nucleotide transhydrogenase 1 4.43327 6.35516 6.80833 3.83838
Rv2780 ald Secreted L-alanine dehydrogenase ALD 1 5.2891 4.65988 4.52694 4.92656
Rv2814c Transposase 1 3.3279 5.52338 4.86873 4.60102
Rv2815c Transposase 1 3.13667 6.24306 5.87423 4.84337
Rv3185 Transposase 1 3.58899 6.43621 5.67335 5.82686
Rv3186 Transposase 1 3.2903 6.21375 6.14822 5.77427
Rv3290c lat L-lysine aminotransferase 1 4.32023 5.06387 3.54801 3.9704
Rv3474 Transposase IS6110 1 3.04947 6.19754 6.19869 3.22266
Rv3475 Transposase IS6110 1 3.73966 5.79892 5.63617 6.23465
Rv3580c cysS Cysteinyl-tRNA synthetase 1 3.87797 6.67899 3.14124 3.40852
Rv3582c ispD 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase 1 3.50012 4.07861 3.78626 3.51221
During transition to the NC state, some genes used as markers of stress conditions were induced: the heat-shock protein hsp, the chaperones Rv0440 and Rv3417c, as well as sigma-factors: sigG — regulating genes which are necessary for survival inside the macrophages and sigB, which can control stationary phase regulons and general resistance to stress. Induction of ccsA, whose product takes part in the cytochrome biosynthesis at the step of heme attachment, and cyp132, coding one of the cytochrome’s P450 oxidizing different xenobiotics, could evidently reflect accumulation of toxic components in cultures during transition. Enzymes of the non-mevalonate pathway of isoprenoid biosynthesis ispF and ispD were also induced in the NC cells. There are data indicating that some of the metabolites of this pathway can affect the immune response of the host [6]. A number of induced genes are involved in the information pathways and those encoding regulatory proteins; in particular, the transcriptional regulator furA, which acts as a global negative control element, employing Fe2+ as a cofactor to bind the operator of the repressed genes. It seems to regulate the transcription of katG, which is induced in the NC state. katG encodes a multifunctional enzyme, exhibiting both catalase, a broad-spec-trum peroxidase and peroxynitritase activities and is believed to play a role in the intracellular survival of mycobacteria within macrophages, protecting them against the aggressive components produced by phagocytic cells. Some genes taking part in the cell wall and cell processes, in particular the transporters ctpG and ctpC encoding ATPases-transporting metal cations and the transporter Rv2688c involved in antibiotic resistance and export of antibiotics across the membrane, are activated.
To identify the genes that were significantly upregulated during transition to the NC state, we used stringent criteria: the expression level during the whole time course in the stationary phase was upregulated at least 3-fold in comparison to the expression in the logarithmic phase. Fifty-one genes met this criterion (Table 2).
Among the genes with a substantially high level of expression, genes encoding insertion sequences and phages -20 genes out of the 51- are prime candidates, while 13 genes encode hypothetical proteins with unknown function. It is remarkable that the significantly upregulated genes belonged to intermediary metabolism and the respiration category. Moreover, these genes mainly encode proteins involved in degradation processes; namely bglS - beta-glycosidase (hydrolyzes the terminal, non-reducing beta-D-glucose residue); pip - proline iminopeptidase (specifically catalyses the removal of N-terminal proline residues from small peptides); clpC2 ATP-dependent protease; and ald - L-alanine dehydrogenase (catalyses alanine hydrolyze - an important constituent of the peptidoglycan layer). In addition, the pdhA coding the alpha subunit of pyruvate dehydrogenase and taking part in the energetic metabolism and catalyzing the conversion of pyruvate to acetyl-CoA was highly expressed. Significant upregulation of sthA, a soluble pyridine nucleotide transhydrogenase that catalyses the conversion of NADPH generated by catabolic pathways to NADH, which is oxidized by the respiratory chain for energy generation, is a sign of the prevalence of catabolic reactions in cell metabolism in the NC state.
Analysis of the global gene expression profile has been published for several M. tuberculosis persistence models, in particular the Wayne model of the non-replicating state during hypoxia [5,7,8], the gradual depletion of the carbon source under decreased oxygen tension [9], the adaptation of M. tuberculosis within macrophages [10], and in vivo within artificial granulomas in mice [11]. Considering the results of these studies, the gene expression profile in our model of “non-culturability” in the stationary phase has, evidently, some overlaps with the above-mentioned models of persistence (Table 3).
Little in common was found between the genes induced in our model of “non-culturability” and the Wayne dormancy model during hypoxia (Table 3). The Wayne model is characterized by the induction of genes of the dormancy survival regulon (Dos-regulon), a group of 49 genes under the control of devR which codes the regulatory part of the two-component system. Upregulation of the Dos-regulon was found not only for dormant cells under hypoxia in vitro, but also for M. tuberculosis cells within macrophages [10], and in the artificial granulomas in mice [11]. In our model of M. tuberculosis transition to the NC state in the stationary phase, only two genes from Dos-regulon - Rv0571c and Rv2631 - were found upregulated. Dos-regulon induction was not found in the persisting cells during starvation [12], and only two genes of Dos-regulon were activated during persistence at gradual depletion of the carbon source [9].
A recently published paper [13] demonstrated that the role of Dos-regulon is apparently overestimated not only as a universal regulator of the dormant state of mycobacteria, but also as a general response on hypoxia. Genes of the Dos-regulon were shown to be activated only 2 hours after hypoxia.
Table 3. Comparison of genes upregulated during transition to the NC state in the stationary phase (at least 1.5-fold) to the genes activated in other models of persistence
Models of M. tuberculosis persistence Overlapping to 238 genes activated in the stationary phase during transition to the NC state.
Number of genes %
Wayne non-replicating state (Voskuil et al., 2004) 23 9.7
Persistence at gradual depletion of carbon source at 50% oxygen tension (Hampshire et al., 2004) 82 34.5
Persistence within macrophages (Schnappinger et al., 2003) 77 32.4
Artificial granuloma in mice (Karakousis et al., 2004) 32 13.4
Enduring hypoxia response (Rustad et al., 2008) 40 16.8
Table 4. Shared genes of M. tuberculosis persistence state. Genes of EHR regulon are in bold
ORF Gene Non-replicating state of Wayne (Voskuil et al., 2004) Gradual depletion of carbon source (Hampshire et al., 2004) Persistence within macrophages (Schnappinger et al., 2003) Artificial granuloma in mice (Karakousis et al., 2004). NC state in the stationary phase (this study)
Rv0188 0.8 67.2 2.8 2.7 2.5
Rv0211 pckA - 1.7 3.6 2.6 1.64
Rv0251c hsp 4.5 18.6 25.6 3.9 4.5
Rv1894c 2.0 5.1 1.8 - 2.8
Rv1909c furA - 5.4 2.2 2.8 2.7
Rv2011c 2.1 9.5 2.5 - 2.8
Rv2497c pdhA 3.4 8.4 2.1 2.0 4.0
Rv2660c 1.5 4.3 2.1 3.3 19.7
Rv2662 1.5 1.5 2.0 - 12. 9
Rv2710 sigB - 34.6 3.8 4.7 4.6
Rv2780 ald 6.1 2.6 2.4 2.4 4.9
Rv3139 fadE24 - 2.2 2.0 5.8 2.4
Rv3290c lat 3.6 25.9 7.5 5.6 4.0
Thereafter expression of at least half of these returned to the baseline [13]. The authors observed a significant induction of another 230 genes after further cultivation during hypoxia, and hereafter their expression level was stable. Thus, the authors refer to this group of genes as enduring hypoxia response (EHR) genes. Considering the gene expression profile for our model of transition to the NC state, we found significant overlap with this group of genes (Table 3), which was rather unexpected because the conditions for NC cell formation developed in our laboratory did not imply any oxygen limitation. Some overlap with EHR [13] was found for the persistence model of gradual depletion of the carbon source [9] and the transcriptional response to multiple stresses [14]. Therefore, it is possible to conclude that EHR genes may not only play a role as hypoxia markers, but may also be a general regulon of the dormant state of M. tuberculosis, independent of its induction.
Thus, the data presented here indicate that cell transition to dormant state is an active process and that numerous genes are involved in it. The future task is to investigate this process in detail in order to understand the molecular mechanisms in the cells during the transition to the dormant state.
Based on the results of the transcriptome analysis of the NC cells obtained in our model and those obtained in several models of persistence, it is possible to pinpoint some shared genes that are upregulated in these models (Table 4). The genes presented in Table 4 and their products are believed to be important for further study, because some of them could represent new targets for anti-tuberculosis drug candidates directed mainly against latent tuberculosis.
This work was supported by the Program of the Presidium of the RAS “Molecular and Cellular Biology"
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