RESEARCH LETTER
Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival Wareed Ahmed1, Shruti Menon1, Adwait Anand Godbole1, Pullela V.D.N.B. Karthik1 & Valakunja Nagaraja1,2 1
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India; and 2Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
Correspondence: Valakunja Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Tel.: +91 080 2360 0668; fax: +91 080 2360 2697; e-mail: [email protected] Received 13 January 2014; revised 25 February 2014; accepted 27 February 2014. DOI: 10.1111/1574-6968.12412
MICROBIOLOGY LETTERS
Editor: Roger Buxton Keywords Mycobacterium tuberculosis; conditional silencing; essential genes; cell growth; topoisomerase I.
Abstract Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M. tuberculosis topoisomerase I (TopoIMt) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M. tuberculosis growth and open up new avenues for targeting the enzyme.
Introduction Tuberculosis (TB) is a life-threatening infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb; Dye et al., 2005). About one-third of the human population is estimated to be latently infected with this etiologic agent, and c. 8 million new cases of active TB and 2 million deaths are documented annually (Corbett et al., 2003; Dye & Williams, 2010). The success of the organism is due to its ability to evade the human immune response and then manipulate the host machinery to persist in the host (Gomez & McKinney, 2004). The elimination of the bacilli existing in the latent form has been largely unsuccessful by the currently used antimycobacterials. The emergence of multidrug resistance (MDR) and extremely drug-resistant TB (XDR) strains of the organism has compounded the problem further (LoBue, 2009; Shenoi et al., 2009). The success rate for the treatment of XDR-TB strain is meager, usually between 30% and 50%, and the mortality can be 100% in patients co-infected with HIV (Gandhi et al., 2006; LoBue, 2009). With this FEMS Microbiol Lett && (2014) 1–8
current scenario, new TB drugs have to be discovered urgently to combat the pathogen. The advent of new TB drugs would need better understanding of the in vivo function(s) of the target gene candidates, which are essential for the growth of the pathogen and can be battered by the chemical inhibitors. Topoisomerases are an important class of enzymes, which maintain topological homeostasis during different DNA transaction processes such as DNA replication, transcription, chromosome segregation, etc. (Wang, 2002; Vos et al., 2011). Based on their structure and mechanism of action, topoisomerases have been classified as type I and type II (Champoux, 2001; Corbett & Berger, 2004). Type I and type II enzymes follow a different mechanism of action but function synchronously to maintain the topological homeostasis inside the cell (Viard & de la Tour, 2007; Laponogov et al., 2010). Because of their essential role in cellular functions, topoisomerases from diverse organisms have been explored as drug targets. Indeed, both the type I and type II topoisomerases from human are being exploited extensively for anticancer therapy (Topcu, 2001; Azarova et al., 2007). Similarly, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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DNA gyrase – the bacterial type II enzyme – has been successfully targeted by various antibacterial agents (Maxwell, 1997). The Mtb genome encodes only one copy of type I and one copy of type II topoisomerase (Cole et al., 1998). The Mtb DNA gyrase has drawn a lot of attention for its chemotherapeutic potential, and newer fluoroquinolones which target the DNA gyrase, such as moxifloxacin and gatifloxacin, exhibit potent activity against Mtb and show promise to shorten the duration for TB treatment (Mdluli & Ma, 2007). In contrast, topoisomerase I which belongs to the type I class has not been explored to its potential as a candidate drug target. The high-throughput saturation mutagenesis studies have predicted the essentiality of TopoIMt (Rv3646c) for Mtb growth and survival (Sassetti et al., 2001, 2003). Validation of its role in Mtb growth and in vivo function is yet to be carried out, although TopoIMt is characterized biochemically (Annamalai et al., 2009; Narula et al., 2010; Godbole et al., 2012). To decipher the importance of TopoI for the cellular processes and Mtb growth, we have constructed an Mtb mutant, where the topoI expression can be conditionally regulated by the addition of anhydrotetracycline (ATc; Boldrin et al., 2010). Using this conditional knockdown mutant, we have examined the consequences of topoI down-regulation on the growth and survival of Mtb.
Materials and methods Bacterial strains, growth media, and transformation conditions
The following bacterial strains were used: Escherichia coli DH10B (Gibco BRL, Rockville, MD); Mycobacterium tuberculosis H37Ra (laboratory stock); Escherichia coli strains were grown at 37 °C in Luria–Bertani (LB) broth or on LB agar plates; Mycobacterium tuberculosis strains were grown at 37 °C in Middlebrook 7H9-broth (Difco) or 7H10-agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80. For growth of M. tuberculosis, 10% ADC (Albumin, Dextrose, Catalase, NaCl) and 10% OADC (Oleic acid, Albumin, Dextrose, Catalase, NaCl) were added in liquid media and 7H10-agar, respectively (Jacobs et al., 1991). Appropriate antibiotics were added to the media, at the following concentrations: streptomycin (Sm): 20 lg mL1; hygromycin (Hyg): 150 lg mL1 (E. coli) or 50 lg mL1 (M. tuberculosis). ATc (Sigma-Aldrich) was added at final concentrations of 200 ng mL1. Construction of conditional mutant of topoI in M. tuberculosis
From the 50 end, the first 750 bp of the TopoIMt gene was cloned downstream of the ATc repressible ptr ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
promoter in the suicide plasmid pFRA50 (Boldrin et al., 2010) to obtain pFRA50-TopoIMt. To replace the native promoter of topoI with the Pip controlled promoter (Pptr), M. tuberculosis H37Ra cells were electroporated with 5 lg of plasmid (pFRA50-TopoIMt). Recombinant colonies were selected on 7H10-agar plates, containing Hyg (50 lg mL1). Integration of the plasmid by singlesite integration was confirmed by PCR. The resulting recombinant strain was transformed with the integrative plasmid pFRA42B (containing the TetR/Pip system) to acquire M. tuberculosis topoI conditional mutant (MtbPptrtopoI). RNA extraction and quantitative PCR
The M. tuberculosis H37Ra (WT) and MtbPptrtopoI cells were grown to exponential phase (OD595 nm = 0.4–0.6). The cultures were treated with ATc (200 ng mL1) for the indicated period of time. Untreated cultures were used as a control. The cells were pelleted down, disrupted by bead beating using zirconia–silica beads, and processed by the SV RNA isolation kit (Promega) according to the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically. Purified total RNA (1 lg) was incubated with random hexamer primers at 70 °C for 5 min. After cooling on ice, 1 mM dNTPs, 40U RNase inhibitor (Ribolock; Fermentas), and 50 U MMLV reverse transcriptase (Fermentas) were added in a final volume of 20 lL. The reactions were incubated at 42 °C for 1 h and then terminated by incubation at 70 °C for 5 min. Further to carry out quantitative PCR, primers were designed using PRIMER3 software (http://frodo.wi.mit.edu/primer3/). The 20 lL PCR reactions consisted of 19 Master Mix (Applied Biosystems), 1 mM each primer, and 1 lL of cDNA. Reactions were carried out in duplicates in an Applied Biosystems Viia7 Real-Time PCR system with the following protocol: denaturation at 95 °C for 5 min and 95 °C for 15 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s, final extension at 72 °C for 5 min, 25 cycles of denaturation at 95 °C for 30 s, and annealing at 57 °C for 30 s with data collection. sigA was used as a reference gene. Immunoblot analysis
Proteins were separated on an 8% SDS PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated in PBS blocking buffer [10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 0.05% Tween-20] with 2% (w/v) BSA for 2 h prior to incubation with primary antibodies diluted (1 : 20 000) in PBS with 2% BSA for 2 h. Membranes were washed in PBST FEMS Microbiol Lett && (2014) 1–8
Conditional silencing of M. tuberculosis topoisomerase I
(0.05% Tween-20) three times and then incubated with secondary antibodies for 2 h. HRP-conjugated secondary antibodies (GE Amersham) were added to detect the protein of interest using chemiluminescent substrate (Millipore). Growth analysis of MtbPptrtopoI
For monitoring the growth of Mtb cells, the cultures were spotted on 7H10-agar plates containing different concentrations of ATc (0 and 200 ng mL1) and incubated at 37 °C. For measuring the growth of Mtb and its conditional mutant in liquid medium, the exponential phase cultures (OD595 nm = 0.4–0.6) were taken and diluted in 7H9 media supplemented with ADC to OD595 nm = 0.05. The growth curve profiles were obtained by measuring the OD595 nm every 24 h and plotted using GRAPHPAD PRISM version 5.0. Fluorescence (DAPI) microscopy
Mtb cells were fixed by exposure to toluene (2%) and triton X-100 (1%) at 4 °C overnight. Prior to the staining of the DNA, the cells were washed, resuspended in PBS (PBS; 10 mM sodium phosphate, pH 7.4, 150 mM NaCl), and treated with lysozyme (2 mg mL1) for permeabilization. The cells were stained with DAPI (0.5 lg mL1) for 15 min at room temperature, and the samples were examined under a confocal microscope (Zeiss) equipped with a 1009 objective. Scanning electron microscopy (SEM)
Aliquots of concentrated cells (at densities of 5 9 108 CFU mL1) were placed on poly-L lysine-coated thermanox coverslips in 24-well tissue culture plates. Bacteria were allowed to settle for 30 min before gently decanting and adding 1 mL of a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 0.2 M sucrose. Samples were then treated with 2% OsO4 in 0.1 M cacodylate buffer for 2 h at room temperature. A series of sequential ethanol dehydrations were performed for 10 min each (30%, 50%, 70%, 95%, and 100%) before drying the samples under vacuum desiccators. Samples were gold sputter coated and imaged with a SEM Quanta 200 scanning electron microscope. Resazurin assay/isoniazid susceptibility assay
Conditional mutant was grown to OD595 nm = 0.6 in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.05% Tween-80, and 1% ADC. The cultures were FEMS Microbiol Lett && (2014) 1–9
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diluted to a final OD595 nm of 0.05 with fresh medium and aliquoted into a 96-well growth plate. 200 ng mL1 ATc was added to the MtbPptrtopoI cultures. The untreated culture was taken as a control. Serial dilutions of isoniazid (INH) were added to the culture, and the plates were incubated at 37 °C for 10 days. To monitor the effect of INH on cell growth, resazurin reduction microplate assay was carried out (Palomino et al., 2002). Resazurin dye was added to the cultures at a final concentration of 0.05%, and the cultures were incubated at 37 °C for 24 h.
Results TopoI is required for the M. tuberculosis growth
To confirm the essential function of the single topoI gene for cell survival, TetR/PipOFF system was used to generate a topoI conditional knockdown strain (Boldrin et al., 2010). To generate the mutant, the native promoters of topoI were replaced by Pptr – a Streptomyces pristinaespiralis repressible promoter (Fig. 1a), which is controlled by the transcriptional regulator Pip (Folcher et al., 2001). The fragment of topoI gene was cloned into the pFRA50 suicidal vector, and the construct was electroporated into the M. tuberculosis H37Ra cells. The single-site recombination event resulted in the replacement of the native topoI promoter with the ptr promoter, confirmed by PCR (Fig. 1b). Electroporation of promoter replaced strain with the plasmid containing the Tet/Pip regulatory circuit yielded the topoI conditional mutant. The depletion of TopoI in response to ATc was confirmed by the immunoblot analysis (Fig. 1c). As TopoI participates in essential cellular processes, one would expect it to be essential for cell growth and proliferation. To determine the essentiality of TopoI for Mtb survival, the WT and the conditional mutant were spotted on Middlebrook 7H10 agar plates with or without ATc. The presence of ATc did not affect the growth of WT cells, whereas the growth of the mutant was severely retarded in the plate containing ATc (Fig. 2a), indicating that the reduction in TopoI level by ATc could lead to the repression of growth. Although the spotting assay described above revealed the importance of TopoI for growth of Mtb on solid medium, to further test the effect of TopoI down-regulation on the bacterial growth in the liquid culture, experiments were carried out in a liquid broth with or without ATc as described in Materials and methods. The growth curve analysis showed that the growth of the mutant was drastically reduced in the presence of ATc (Fig. 2b). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
(b)
(c)
Fig. 1. Construction of TopoIMt conditional mutant. (a) Schematic representation of the single crossover recombination events employed to generate the MtbPptrtopoI conditional mutant. (b) Analysis of the genomic DNA isolated from WT (lane 1) and MtbPptrtopoI (lane 2–6) by PCR using the primers specific to promoter ptr (FP) and an internal coding region (RP) of the topoI gene. Specific amplification product obtained confirms the accurate integration of ptr promoter upstream of topoI gene. (c) Expression analysis by immunoblotting. The exponential phase cultures of the conditional mutant were exposed to ATc (200 ng mL1) for 24 h, and the TopoI level was monitored.
(a)
(b)
The final validation of TopoI essentiality for bacterial growth was carried out by determining the cell viable counts. The exponential phase cultures of conditional mutant were treated with ATc for 72 h, followed by the CFU determination. The cultures treated with ATc showed a significant decrease in the cell viable counts in comparison with the untreated culture (Supporting Information, Fig. S1a and b). From all these results, it is apparent that the TopoI expression is important for the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Fig. 2. TopoI is essential for the growth of Mycobacterium tuberculosis. (a) Effect of TopoI depletion on growth. Exponential phase cultures were 10-fold serially diluted and spotted on Middlebrook 7H10 media supplemented with or without ATc. The plates were incubated at 37 °C, and bacterial growth was monitored after 2 weeks of incubation. (b) MtbPptrtopoI cultures were grown in Middlebrook 7H9 medium with or without ATc (200 ng mL1) at 37 °C. The growth was monitored by measurement of OD595 nm every 24 h. Error bars represent the SD obtained in three independent experiments.
cell multiplication and its depletion by ATc led to the arrest of cell growth. Depletion of TopoI leads to the enhanced susceptibility to novobiocin and INH
DNA gyrase and topoisomerase I function in concert to maintain topological homeostasis, necessary for the accurate operation of DNA transaction processes. To reveal FEMS Microbiol Lett && (2014) 1–8
Conditional silencing of M. tuberculosis topoisomerase I
the influence of TopoI depletion on susceptibility of the mycobacterial cell to a gyrase inhibitor, the untreated and ATc treated, WT, and conditional mutant were grown in the presence of novobiocin for 48 h and CFU was determined. The conditional mutant culture treated with ATc was more susceptible to novobiocin compared with the WT (Fig. 3a). The enhanced susceptibility of the TopoIdepleted culture to novobiocin could be a result of the simultaneous reduction in the DNA gyrase level (Fig. 3b; see Discussion). From the aforementioned data, it is apparent that TopoI is essential for the bacterial growth and survival. With the reduction in TopoI expression, the growth of the cell was also affected. To explore the effect of TopoI deprivation on the viability of mycobacterial cell in the presence of a known anti-TB molecule, we evaluated the effect of INH on the TopoI-deprived cells. The ATc treated and untreated cultures were grown with various concentrations of INH and their viability was measured by the resazurin assay. In the presence of INH, the cultures without ATc treatment were able to metabolize the resazurin dye better than the TopoI-depleted cells (ATc treated), indicating the enhanced susceptibility of INH for the TopoI-deprived cultures (Fig. 3c). (a)
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Alteration in the TopoI level impacts the cell morphology
The appearance of the colony morphology of the conditional mutant was found to be altered (Fig. S2a). This alteration could be an attribute of the enhanced TopoI level in the mutant strain (Fig. S2b). Examination of the cells by SEM revealed that the suppression of TopoI expression resulted in the reduction in cell length and shape. TopoIdepleted cells appeared to be bulbous toward their poles (Fig. S2c), indicating that the perturbation of TopoI level confers the changes in the cell phenotype. Reduced levels of TopoI results in genome decompaction
The topology of the bacterial genome is regulated by the topoisomerases and nucleoid-associated proteins (Wang, 2002; Browning et al., 2010). To explore the effect of TopoI depletion on the genome organization, fluorescence microscopy was carried out. The conditional mutant retained a compact genome, and upon treatment with ATc for 24 h to reduce the TopoI level, the bacterial genome underwent decompaction (Fig. 4a). The diffused nucleoid (c)
(b)
Fig. 3. Depletion of TopoIMt leads to enhanced drug susceptibility. (a) Exponential phase cultures of WT and MtbPptrtopoI were diluted to OD595 nm = 0.05 in Middlebrook 7H9 medium with or without ATc in the presence of Novobiocin (2 lg mL1) at 37 °C. The growth was monitored by determining the CFU. The error bars represent the SD obtained in two experiments. MtbPptrtopoI (ATc-UT): conditional mutant culture grown to exponential phase in the absence of ATc (200 ng mL1) and MtbPptrtopoI (ATc-T): TopoI-deprived conditional mutant culture, that is, the conditional mutant culture grown to exponential phase in the presence of ATc (200 ng mL1); (b) The exponential phase cultures of the conditional mutant were treated with ATc (200 ng mL1) for 24 h and subjected to transcript analysis. The fold change in expression was normalized to the untreated culture; (c) MtbPptrtopoI cultures were grown in Middlebrook 7H9 medium with or without ATc (200 ng mL1) in the presence of INH at 37 °C. The growth was monitored by the resazurin reduction assay as described in Materials and Methods.
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(a)
(b)
Fig. 4. Reduction in TopoI level causes genome decompaction. (a) The genome of the conditional mutant was visualized by DAPI staining followed by fluorescence microscopy under 1009 objective. (b) Immunoblot analysis of TopoI and HU levels in the conditional mutant.
of the TopoI-depleted cells could be a consequence of the change in the level of the other genome architectural proteins. Immunoblot analysis of HU (Rv2986c) – a nucleoidassociated protein in Mtb – revealed the reduction in its level in the TopoI-deprived cells (Fig. 4b). From the data, we surmise that the optimal level of TopoI may be necessary to retain genome architecture.
Discussion Given the importance of TopoI for cellular processes, the presence of a single-type IA enzyme in Mtb indicates its essential role. Saturation transposon mutagenesis studies carried out earlier provided the first hint on topoI essentiality for Mtb (Sassetti et al., 2003). The gene encoding TopoIMt was among the c. 600 genes predicted to be essential for bacterial growth as insertional inactivation mutant in topoI gene was not obtained (Sassetti et al., 2003). Hence, we resorted to generate the conditional knockdown strain of topoI to study the function of TopoI for Mtb growth and survival. We demonstrate and validate the essential nature of TopoI for the Mtb growth and maintenance of cell and genome architecture. The decompaction of the nucleoid in the TopoIdeprived cells indicated the role of TopoI in the mainteª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
nance of genome architecture. The perturbation in the level of the enzyme involved in maintaining the DNA topology homeostasis could lead to changes in the expression of other topoisomerases and topology modulators such as nucleoid architectural proteins, resulting in a diffused nucleoid. Indeed, the expression of HU and DNA gyrase was found to be altered in the TopoI-depleted cells conferring the changes in the nucleoid. Being important components of the cell’s molecular machinery, topoisomerases not only solve the problems associated with the DNA topology, but also participate in the gene regulatory mechanism. Inhibition of DNA gyrase in E. coli and Streptococcus pneumonia affected the cellular transcriptome (Peter et al., 2004; Ferrandiz et al., 2010). Similarly, mutations in E. coli topoisomerases influenced the global proteome of the cell (Steck et al., 1993). Chromosomal supercoiling affects many cellular processes including replication, DNA segregation, recombination, etc. Thus, the perturbations in the genome supercoiling have numerous phenotypic implications (Dorman & Corcoran, 2009), which could be an attribute of the altered expression of the genes involved in metabolic pathways (Webber et al., 2013). The observed phenotype of the TopoIMt conditional mutant could be a consequence of the altered genome supercoiling and gene expression. Moreover, these changes may perturb the metabolic activity resulting into altered cell phenotype and enhanced susceptibility to anti-TB molecules. Thus, the increased susceptibility of TopoI-depleted cells could be due to a combination of reduction in topoisomerase levels and disturbance to metabolic pathways. More effective inhibition with novobiocin could be attributed to the former, whereas enhanced INH susceptibility correlates with the latter point. The TopoI-deprived cells appeared to be more susceptible to novobiocin indicating that the simultaneous targeting of TopoI and DNA gyrase can efficiently inhibit the Mtb growth. In addition, the enhanced sensitivity of the TopoI-deprived cells to INH signifies the synergistic targeting of TopoI and metabolic pathways to kill the mycobacteria. The dual targeting of an enzyme central to DNA transaction processes and the other one in a validated (like mycolic acid/lipid synthesis pathway) metabolic pathway may provide better ways of combating the pathogen. Altogether, the present study highlights the importance of TopoI for the Mtb growth and reveals the potential to exploit the enzyme as a potential drug target.
Acknowledgements The authors thank the members of VN laboratory for valuable suggestions and critical reading of the manuscript. Riccardo Manganelli is acknowledged for providing FEMS Microbiol Lett && (2014) 1–8
Conditional silencing of M. tuberculosis topoisomerase I
the Tet/Pip system constructs. W.A. is a recipient of Shyama Prasad Mukherjee fellowship of Council of Scientific and Industrial Research (CSIR), Govt. of India. V.N. is a recipient of J.C. Bose Fellowship of the Department of Science and Technology, Government of India and is partner 14 in EU consortium project MM4TB.
References Annamalai T, Dani N, Cheng B & Tse-Dinh YC (2009) Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol. BMC Biochem 10: 18. Azarova AM, Lyu YL, Lin CP, Tsai YC, Lau JY, Wang JC & Liu LF (2007) Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies. P Natl Acad Sci USA 104: 11014–11019. Boldrin F, Casonato S, Dainese E et al. (2010) Development of a repressible mycobacterial promoter system based on two transcriptional repressors. Nucleic Acids Res 38: e134. Browning DF, Grainger DC & Busby SJ (2010) Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression. Curr Opin Microbiol 13: f773–780. Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70: 369–413. Cole ST, Brosch R, Parkhill J et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537–544. Corbett KD & Berger JM (2004) Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct 33: 95–118. Corbett EL, Watt CJ, Walker N, Maher D, Williams BG, Raviglione MC & Dye C (2003) The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 163: 1009–1021. Dorman CJ & Corcoran CP (2009) Bacterial DNA topology and infectious disease. Nucleic Acids Res 37: 672–678. Dye C & Williams BG (2010) The population dynamics and control of tuberculosis. Science 328: 856–861. Dye C, Watt CJ, Bleed DM, Hosseini SM & Raviglione MC (2005) Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, and deaths globally. JAMA 293: 2767–2775. Ferrandiz MJ, Martin-Galiano AJ, Schvartzman JB & de la Campa AG (2010) The genome of Streptococcus pneumoniae is organized in topology-reacting gene clusters. Nucleic Acids Res 38: 3570–3581. Folcher M, Morris RP, Dale G, Salah-Bey-Hocini K, Viollier PH & Thompson CJ (2001) A transcriptional regulator of a pristinamycin resistance gene in Streptomyces coelicolor. J Biol Chem 276: 1479–1485. Gandhi NR, Moll A, Sturm AW et al. (2006) Extensively drugresistant tuberculosis as a cause of death in patients co-
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infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368: 1575–1580. Godbole AA, Leelaram MN, Bhat AG, Jain P & Nagaraja V (2012) Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity. Arch Biochem Biophys 528: 197–203. Gomez JE & McKinney JD (2004) M. tuberculosis persistence, latency, and drug tolerance. Tuberculosis (Edinb) 84: 29–44. Jacobs WR Jr, Kalpana GV, Cirillo JD et al. (1991) Genetic systems for mycobacteria. Methods Enzymol 204: 537–555. Laponogov I, Pan XS, Veselkov DA, McAuley KE, Fisher LM & Sanderson MR (2010) Structural basis of gate-DNA breakage and resealing by type II topoisomerases. PLoS ONE 5: e11338. LoBue P (2009) Extensively drug-resistant tuberculosis. Curr Opin Infect Dis 22: 167–173. Maxwell A (1997) DNA gyrase as a drug target. Trends Microbiol 5: 102–109. Mdluli K & Ma Z (2007) Mycobacterium tuberculosis DNA gyrase as a target for drug discovery. Infect Disord Drug Targets 7: 159–168. Narula G, Becker J, Cheng B, Dani N, Abrenica MV & TseDinh YC (2010) The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites. BMC Biochem 11: 41. Palomino JC, Martin A, Camacho M, Guerra H, Swings J & Portaels F (2002) Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46: 2720–2722. Peter BJ, Arsuaga J, Breier AM, Khodursky AB, Brown PO & Cozzarelli NR (2004) Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli. Genome Biol 5: R87. Sassetti CM, Boyd DH & Rubin EJ (2001) Comprehensive identification of conditionally essential genes in mycobacteria. P Natl Acad Sci USA 98: 12712–12717. Sassetti CM, Boyd DH & Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48: 77–84. Shenoi S, Heysell S, Moll A & Friedland G (2009) Multidrugresistant and extensively drug-resistant tuberculosis: consequences for the global HIV community. Curr Opin Infect Dis 22: 11–17. Steck TR, Franco RJ, Wang JY & Drlica K (1993) Topoisomerase mutations affect the relative abundance of many Escherichia coli proteins. Mol Microbiol 10: 473–481. Topcu Z (2001) DNA topoisomerases as targets for anticancer drugs. J Clin Pharm Ther 26: 405–416. Viard T & de la Tour CB (2007) Type IA topoisomerases: a simple puzzle? Biochimie 89: 456–467.
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Vos SM, Tretter EM, Schmidt BH & Berger JM (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12: 827–841. Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3: 430–440. Webber MA, Ricci V, Whitehead R, Patel M, Fookes M, Ivens A & Piddock LJ (2013) Clinically relevant mutant DNA gyrase alters supercoiling, changes the transcriptome, and confers multidrug resistance. mBio 4: e00273–13. doi: 10. 1128/mBio.00273-13.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. TopoI deprivation leads to reduction in CFU. Fig. S2. Altered cell and colony morphology of topoI conditional mutant.
FEMS Microbiol Lett && (2014) 1–8
Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival Wareed Ahmed1, Shruti Menon1, Adwait Anand Godbole1, Pullela V.D.N.B. Karthik1 & Valakunja Nagaraja1,2 1
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India; and 2Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
Correspondence: Valakunja Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Tel.: +91 080 2360 0668; fax: +91 080 2360 2697; e-mail: [email protected] Received 13 January 2014; revised 25 February 2014; accepted 27 February 2014. DOI: 10.1111/1574-6968.12412
MICROBIOLOGY LETTERS
Editor: Roger Buxton Keywords Mycobacterium tuberculosis; conditional silencing; essential genes; cell growth; topoisomerase I.
Abstract Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M. tuberculosis topoisomerase I (TopoIMt) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M. tuberculosis growth and open up new avenues for targeting the enzyme.
Introduction Tuberculosis (TB) is a life-threatening infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb; Dye et al., 2005). About one-third of the human population is estimated to be latently infected with this etiologic agent, and c. 8 million new cases of active TB and 2 million deaths are documented annually (Corbett et al., 2003; Dye & Williams, 2010). The success of the organism is due to its ability to evade the human immune response and then manipulate the host machinery to persist in the host (Gomez & McKinney, 2004). The elimination of the bacilli existing in the latent form has been largely unsuccessful by the currently used antimycobacterials. The emergence of multidrug resistance (MDR) and extremely drug-resistant TB (XDR) strains of the organism has compounded the problem further (LoBue, 2009; Shenoi et al., 2009). The success rate for the treatment of XDR-TB strain is meager, usually between 30% and 50%, and the mortality can be 100% in patients co-infected with HIV (Gandhi et al., 2006; LoBue, 2009). With this FEMS Microbiol Lett && (2014) 1–8
current scenario, new TB drugs have to be discovered urgently to combat the pathogen. The advent of new TB drugs would need better understanding of the in vivo function(s) of the target gene candidates, which are essential for the growth of the pathogen and can be battered by the chemical inhibitors. Topoisomerases are an important class of enzymes, which maintain topological homeostasis during different DNA transaction processes such as DNA replication, transcription, chromosome segregation, etc. (Wang, 2002; Vos et al., 2011). Based on their structure and mechanism of action, topoisomerases have been classified as type I and type II (Champoux, 2001; Corbett & Berger, 2004). Type I and type II enzymes follow a different mechanism of action but function synchronously to maintain the topological homeostasis inside the cell (Viard & de la Tour, 2007; Laponogov et al., 2010). Because of their essential role in cellular functions, topoisomerases from diverse organisms have been explored as drug targets. Indeed, both the type I and type II topoisomerases from human are being exploited extensively for anticancer therapy (Topcu, 2001; Azarova et al., 2007). Similarly, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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DNA gyrase – the bacterial type II enzyme – has been successfully targeted by various antibacterial agents (Maxwell, 1997). The Mtb genome encodes only one copy of type I and one copy of type II topoisomerase (Cole et al., 1998). The Mtb DNA gyrase has drawn a lot of attention for its chemotherapeutic potential, and newer fluoroquinolones which target the DNA gyrase, such as moxifloxacin and gatifloxacin, exhibit potent activity against Mtb and show promise to shorten the duration for TB treatment (Mdluli & Ma, 2007). In contrast, topoisomerase I which belongs to the type I class has not been explored to its potential as a candidate drug target. The high-throughput saturation mutagenesis studies have predicted the essentiality of TopoIMt (Rv3646c) for Mtb growth and survival (Sassetti et al., 2001, 2003). Validation of its role in Mtb growth and in vivo function is yet to be carried out, although TopoIMt is characterized biochemically (Annamalai et al., 2009; Narula et al., 2010; Godbole et al., 2012). To decipher the importance of TopoI for the cellular processes and Mtb growth, we have constructed an Mtb mutant, where the topoI expression can be conditionally regulated by the addition of anhydrotetracycline (ATc; Boldrin et al., 2010). Using this conditional knockdown mutant, we have examined the consequences of topoI down-regulation on the growth and survival of Mtb.
Materials and methods Bacterial strains, growth media, and transformation conditions
The following bacterial strains were used: Escherichia coli DH10B (Gibco BRL, Rockville, MD); Mycobacterium tuberculosis H37Ra (laboratory stock); Escherichia coli strains were grown at 37 °C in Luria–Bertani (LB) broth or on LB agar plates; Mycobacterium tuberculosis strains were grown at 37 °C in Middlebrook 7H9-broth (Difco) or 7H10-agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80. For growth of M. tuberculosis, 10% ADC (Albumin, Dextrose, Catalase, NaCl) and 10% OADC (Oleic acid, Albumin, Dextrose, Catalase, NaCl) were added in liquid media and 7H10-agar, respectively (Jacobs et al., 1991). Appropriate antibiotics were added to the media, at the following concentrations: streptomycin (Sm): 20 lg mL1; hygromycin (Hyg): 150 lg mL1 (E. coli) or 50 lg mL1 (M. tuberculosis). ATc (Sigma-Aldrich) was added at final concentrations of 200 ng mL1. Construction of conditional mutant of topoI in M. tuberculosis
From the 50 end, the first 750 bp of the TopoIMt gene was cloned downstream of the ATc repressible ptr ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
promoter in the suicide plasmid pFRA50 (Boldrin et al., 2010) to obtain pFRA50-TopoIMt. To replace the native promoter of topoI with the Pip controlled promoter (Pptr), M. tuberculosis H37Ra cells were electroporated with 5 lg of plasmid (pFRA50-TopoIMt). Recombinant colonies were selected on 7H10-agar plates, containing Hyg (50 lg mL1). Integration of the plasmid by singlesite integration was confirmed by PCR. The resulting recombinant strain was transformed with the integrative plasmid pFRA42B (containing the TetR/Pip system) to acquire M. tuberculosis topoI conditional mutant (MtbPptrtopoI). RNA extraction and quantitative PCR
The M. tuberculosis H37Ra (WT) and MtbPptrtopoI cells were grown to exponential phase (OD595 nm = 0.4–0.6). The cultures were treated with ATc (200 ng mL1) for the indicated period of time. Untreated cultures were used as a control. The cells were pelleted down, disrupted by bead beating using zirconia–silica beads, and processed by the SV RNA isolation kit (Promega) according to the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically. Purified total RNA (1 lg) was incubated with random hexamer primers at 70 °C for 5 min. After cooling on ice, 1 mM dNTPs, 40U RNase inhibitor (Ribolock; Fermentas), and 50 U MMLV reverse transcriptase (Fermentas) were added in a final volume of 20 lL. The reactions were incubated at 42 °C for 1 h and then terminated by incubation at 70 °C for 5 min. Further to carry out quantitative PCR, primers were designed using PRIMER3 software (http://frodo.wi.mit.edu/primer3/). The 20 lL PCR reactions consisted of 19 Master Mix (Applied Biosystems), 1 mM each primer, and 1 lL of cDNA. Reactions were carried out in duplicates in an Applied Biosystems Viia7 Real-Time PCR system with the following protocol: denaturation at 95 °C for 5 min and 95 °C for 15 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s, final extension at 72 °C for 5 min, 25 cycles of denaturation at 95 °C for 30 s, and annealing at 57 °C for 30 s with data collection. sigA was used as a reference gene. Immunoblot analysis
Proteins were separated on an 8% SDS PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated in PBS blocking buffer [10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 0.05% Tween-20] with 2% (w/v) BSA for 2 h prior to incubation with primary antibodies diluted (1 : 20 000) in PBS with 2% BSA for 2 h. Membranes were washed in PBST FEMS Microbiol Lett && (2014) 1–8
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(0.05% Tween-20) three times and then incubated with secondary antibodies for 2 h. HRP-conjugated secondary antibodies (GE Amersham) were added to detect the protein of interest using chemiluminescent substrate (Millipore). Growth analysis of MtbPptrtopoI
For monitoring the growth of Mtb cells, the cultures were spotted on 7H10-agar plates containing different concentrations of ATc (0 and 200 ng mL1) and incubated at 37 °C. For measuring the growth of Mtb and its conditional mutant in liquid medium, the exponential phase cultures (OD595 nm = 0.4–0.6) were taken and diluted in 7H9 media supplemented with ADC to OD595 nm = 0.05. The growth curve profiles were obtained by measuring the OD595 nm every 24 h and plotted using GRAPHPAD PRISM version 5.0. Fluorescence (DAPI) microscopy
Mtb cells were fixed by exposure to toluene (2%) and triton X-100 (1%) at 4 °C overnight. Prior to the staining of the DNA, the cells were washed, resuspended in PBS (PBS; 10 mM sodium phosphate, pH 7.4, 150 mM NaCl), and treated with lysozyme (2 mg mL1) for permeabilization. The cells were stained with DAPI (0.5 lg mL1) for 15 min at room temperature, and the samples were examined under a confocal microscope (Zeiss) equipped with a 1009 objective. Scanning electron microscopy (SEM)
Aliquots of concentrated cells (at densities of 5 9 108 CFU mL1) were placed on poly-L lysine-coated thermanox coverslips in 24-well tissue culture plates. Bacteria were allowed to settle for 30 min before gently decanting and adding 1 mL of a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 0.2 M sucrose. Samples were then treated with 2% OsO4 in 0.1 M cacodylate buffer for 2 h at room temperature. A series of sequential ethanol dehydrations were performed for 10 min each (30%, 50%, 70%, 95%, and 100%) before drying the samples under vacuum desiccators. Samples were gold sputter coated and imaged with a SEM Quanta 200 scanning electron microscope. Resazurin assay/isoniazid susceptibility assay
Conditional mutant was grown to OD595 nm = 0.6 in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.05% Tween-80, and 1% ADC. The cultures were FEMS Microbiol Lett && (2014) 1–9
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diluted to a final OD595 nm of 0.05 with fresh medium and aliquoted into a 96-well growth plate. 200 ng mL1 ATc was added to the MtbPptrtopoI cultures. The untreated culture was taken as a control. Serial dilutions of isoniazid (INH) were added to the culture, and the plates were incubated at 37 °C for 10 days. To monitor the effect of INH on cell growth, resazurin reduction microplate assay was carried out (Palomino et al., 2002). Resazurin dye was added to the cultures at a final concentration of 0.05%, and the cultures were incubated at 37 °C for 24 h.
Results TopoI is required for the M. tuberculosis growth
To confirm the essential function of the single topoI gene for cell survival, TetR/PipOFF system was used to generate a topoI conditional knockdown strain (Boldrin et al., 2010). To generate the mutant, the native promoters of topoI were replaced by Pptr – a Streptomyces pristinaespiralis repressible promoter (Fig. 1a), which is controlled by the transcriptional regulator Pip (Folcher et al., 2001). The fragment of topoI gene was cloned into the pFRA50 suicidal vector, and the construct was electroporated into the M. tuberculosis H37Ra cells. The single-site recombination event resulted in the replacement of the native topoI promoter with the ptr promoter, confirmed by PCR (Fig. 1b). Electroporation of promoter replaced strain with the plasmid containing the Tet/Pip regulatory circuit yielded the topoI conditional mutant. The depletion of TopoI in response to ATc was confirmed by the immunoblot analysis (Fig. 1c). As TopoI participates in essential cellular processes, one would expect it to be essential for cell growth and proliferation. To determine the essentiality of TopoI for Mtb survival, the WT and the conditional mutant were spotted on Middlebrook 7H10 agar plates with or without ATc. The presence of ATc did not affect the growth of WT cells, whereas the growth of the mutant was severely retarded in the plate containing ATc (Fig. 2a), indicating that the reduction in TopoI level by ATc could lead to the repression of growth. Although the spotting assay described above revealed the importance of TopoI for growth of Mtb on solid medium, to further test the effect of TopoI down-regulation on the bacterial growth in the liquid culture, experiments were carried out in a liquid broth with or without ATc as described in Materials and methods. The growth curve analysis showed that the growth of the mutant was drastically reduced in the presence of ATc (Fig. 2b). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
(b)
(c)
Fig. 1. Construction of TopoIMt conditional mutant. (a) Schematic representation of the single crossover recombination events employed to generate the MtbPptrtopoI conditional mutant. (b) Analysis of the genomic DNA isolated from WT (lane 1) and MtbPptrtopoI (lane 2–6) by PCR using the primers specific to promoter ptr (FP) and an internal coding region (RP) of the topoI gene. Specific amplification product obtained confirms the accurate integration of ptr promoter upstream of topoI gene. (c) Expression analysis by immunoblotting. The exponential phase cultures of the conditional mutant were exposed to ATc (200 ng mL1) for 24 h, and the TopoI level was monitored.
(a)
(b)
The final validation of TopoI essentiality for bacterial growth was carried out by determining the cell viable counts. The exponential phase cultures of conditional mutant were treated with ATc for 72 h, followed by the CFU determination. The cultures treated with ATc showed a significant decrease in the cell viable counts in comparison with the untreated culture (Supporting Information, Fig. S1a and b). From all these results, it is apparent that the TopoI expression is important for the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Fig. 2. TopoI is essential for the growth of Mycobacterium tuberculosis. (a) Effect of TopoI depletion on growth. Exponential phase cultures were 10-fold serially diluted and spotted on Middlebrook 7H10 media supplemented with or without ATc. The plates were incubated at 37 °C, and bacterial growth was monitored after 2 weeks of incubation. (b) MtbPptrtopoI cultures were grown in Middlebrook 7H9 medium with or without ATc (200 ng mL1) at 37 °C. The growth was monitored by measurement of OD595 nm every 24 h. Error bars represent the SD obtained in three independent experiments.
cell multiplication and its depletion by ATc led to the arrest of cell growth. Depletion of TopoI leads to the enhanced susceptibility to novobiocin and INH
DNA gyrase and topoisomerase I function in concert to maintain topological homeostasis, necessary for the accurate operation of DNA transaction processes. To reveal FEMS Microbiol Lett && (2014) 1–8
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the influence of TopoI depletion on susceptibility of the mycobacterial cell to a gyrase inhibitor, the untreated and ATc treated, WT, and conditional mutant were grown in the presence of novobiocin for 48 h and CFU was determined. The conditional mutant culture treated with ATc was more susceptible to novobiocin compared with the WT (Fig. 3a). The enhanced susceptibility of the TopoIdepleted culture to novobiocin could be a result of the simultaneous reduction in the DNA gyrase level (Fig. 3b; see Discussion). From the aforementioned data, it is apparent that TopoI is essential for the bacterial growth and survival. With the reduction in TopoI expression, the growth of the cell was also affected. To explore the effect of TopoI deprivation on the viability of mycobacterial cell in the presence of a known anti-TB molecule, we evaluated the effect of INH on the TopoI-deprived cells. The ATc treated and untreated cultures were grown with various concentrations of INH and their viability was measured by the resazurin assay. In the presence of INH, the cultures without ATc treatment were able to metabolize the resazurin dye better than the TopoI-depleted cells (ATc treated), indicating the enhanced susceptibility of INH for the TopoI-deprived cultures (Fig. 3c). (a)
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Alteration in the TopoI level impacts the cell morphology
The appearance of the colony morphology of the conditional mutant was found to be altered (Fig. S2a). This alteration could be an attribute of the enhanced TopoI level in the mutant strain (Fig. S2b). Examination of the cells by SEM revealed that the suppression of TopoI expression resulted in the reduction in cell length and shape. TopoIdepleted cells appeared to be bulbous toward their poles (Fig. S2c), indicating that the perturbation of TopoI level confers the changes in the cell phenotype. Reduced levels of TopoI results in genome decompaction
The topology of the bacterial genome is regulated by the topoisomerases and nucleoid-associated proteins (Wang, 2002; Browning et al., 2010). To explore the effect of TopoI depletion on the genome organization, fluorescence microscopy was carried out. The conditional mutant retained a compact genome, and upon treatment with ATc for 24 h to reduce the TopoI level, the bacterial genome underwent decompaction (Fig. 4a). The diffused nucleoid (c)
(b)
Fig. 3. Depletion of TopoIMt leads to enhanced drug susceptibility. (a) Exponential phase cultures of WT and MtbPptrtopoI were diluted to OD595 nm = 0.05 in Middlebrook 7H9 medium with or without ATc in the presence of Novobiocin (2 lg mL1) at 37 °C. The growth was monitored by determining the CFU. The error bars represent the SD obtained in two experiments. MtbPptrtopoI (ATc-UT): conditional mutant culture grown to exponential phase in the absence of ATc (200 ng mL1) and MtbPptrtopoI (ATc-T): TopoI-deprived conditional mutant culture, that is, the conditional mutant culture grown to exponential phase in the presence of ATc (200 ng mL1); (b) The exponential phase cultures of the conditional mutant were treated with ATc (200 ng mL1) for 24 h and subjected to transcript analysis. The fold change in expression was normalized to the untreated culture; (c) MtbPptrtopoI cultures were grown in Middlebrook 7H9 medium with or without ATc (200 ng mL1) in the presence of INH at 37 °C. The growth was monitored by the resazurin reduction assay as described in Materials and Methods.
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(a)
(b)
Fig. 4. Reduction in TopoI level causes genome decompaction. (a) The genome of the conditional mutant was visualized by DAPI staining followed by fluorescence microscopy under 1009 objective. (b) Immunoblot analysis of TopoI and HU levels in the conditional mutant.
of the TopoI-depleted cells could be a consequence of the change in the level of the other genome architectural proteins. Immunoblot analysis of HU (Rv2986c) – a nucleoidassociated protein in Mtb – revealed the reduction in its level in the TopoI-deprived cells (Fig. 4b). From the data, we surmise that the optimal level of TopoI may be necessary to retain genome architecture.
Discussion Given the importance of TopoI for cellular processes, the presence of a single-type IA enzyme in Mtb indicates its essential role. Saturation transposon mutagenesis studies carried out earlier provided the first hint on topoI essentiality for Mtb (Sassetti et al., 2003). The gene encoding TopoIMt was among the c. 600 genes predicted to be essential for bacterial growth as insertional inactivation mutant in topoI gene was not obtained (Sassetti et al., 2003). Hence, we resorted to generate the conditional knockdown strain of topoI to study the function of TopoI for Mtb growth and survival. We demonstrate and validate the essential nature of TopoI for the Mtb growth and maintenance of cell and genome architecture. The decompaction of the nucleoid in the TopoIdeprived cells indicated the role of TopoI in the mainteª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
nance of genome architecture. The perturbation in the level of the enzyme involved in maintaining the DNA topology homeostasis could lead to changes in the expression of other topoisomerases and topology modulators such as nucleoid architectural proteins, resulting in a diffused nucleoid. Indeed, the expression of HU and DNA gyrase was found to be altered in the TopoI-depleted cells conferring the changes in the nucleoid. Being important components of the cell’s molecular machinery, topoisomerases not only solve the problems associated with the DNA topology, but also participate in the gene regulatory mechanism. Inhibition of DNA gyrase in E. coli and Streptococcus pneumonia affected the cellular transcriptome (Peter et al., 2004; Ferrandiz et al., 2010). Similarly, mutations in E. coli topoisomerases influenced the global proteome of the cell (Steck et al., 1993). Chromosomal supercoiling affects many cellular processes including replication, DNA segregation, recombination, etc. Thus, the perturbations in the genome supercoiling have numerous phenotypic implications (Dorman & Corcoran, 2009), which could be an attribute of the altered expression of the genes involved in metabolic pathways (Webber et al., 2013). The observed phenotype of the TopoIMt conditional mutant could be a consequence of the altered genome supercoiling and gene expression. Moreover, these changes may perturb the metabolic activity resulting into altered cell phenotype and enhanced susceptibility to anti-TB molecules. Thus, the increased susceptibility of TopoI-depleted cells could be due to a combination of reduction in topoisomerase levels and disturbance to metabolic pathways. More effective inhibition with novobiocin could be attributed to the former, whereas enhanced INH susceptibility correlates with the latter point. The TopoI-deprived cells appeared to be more susceptible to novobiocin indicating that the simultaneous targeting of TopoI and DNA gyrase can efficiently inhibit the Mtb growth. In addition, the enhanced sensitivity of the TopoI-deprived cells to INH signifies the synergistic targeting of TopoI and metabolic pathways to kill the mycobacteria. The dual targeting of an enzyme central to DNA transaction processes and the other one in a validated (like mycolic acid/lipid synthesis pathway) metabolic pathway may provide better ways of combating the pathogen. Altogether, the present study highlights the importance of TopoI for the Mtb growth and reveals the potential to exploit the enzyme as a potential drug target.
Acknowledgements The authors thank the members of VN laboratory for valuable suggestions and critical reading of the manuscript. Riccardo Manganelli is acknowledged for providing FEMS Microbiol Lett && (2014) 1–8
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the Tet/Pip system constructs. W.A. is a recipient of Shyama Prasad Mukherjee fellowship of Council of Scientific and Industrial Research (CSIR), Govt. of India. V.N. is a recipient of J.C. Bose Fellowship of the Department of Science and Technology, Government of India and is partner 14 in EU consortium project MM4TB.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. TopoI deprivation leads to reduction in CFU. Fig. S2. Altered cell and colony morphology of topoI conditional mutant.
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