Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

Bhowmick, Tuhin; Ghosh, Soumitra; Dixit, Karuna; Ganesan, Varsha; Ramagopal, U.A.; Dey, Debayan; Sarma, Siddhartha P.; Ramakumar, Suryanarayanarao; Nagaraja, Valakunja

doi:10.1038/ncomms5124

Cited by 96 publications

(156 citation statements)

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“…Mutations in either of them can cause perturbation in topology and nucleoid structure (24,28,40–42). The two proteins play different roles in the nucleoid organization process.…”

Section: Discussionmentioning

confidence: 99%

Direct regulation of topoisomerase activity by a nucleoid-associated protein

Ghosh

Mallick

Nagaraja

2014

Nucleic Acids Research

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The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.

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“…Mutations in either of them can cause perturbation in topology and nucleoid structure (24,28,40–42). The two proteins play different roles in the nucleoid organization process.…”

Section: Discussionmentioning

confidence: 99%

Direct regulation of topoisomerase activity by a nucleoid-associated protein

Ghosh

Mallick

Nagaraja

2014

Nucleic Acids Research

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“…Noteworthy is an increase in the transcript level of the histone-like DNA binding protein Hlp, associated with compaction of DNA [13, 14], mirroring the nucleoid condensation observed in LARCs and SMRCs shown in Fig. 1 [8].…”

Section: Resultsmentioning

confidence: 99%

Developmental transcriptome of resting cell formation in Mycobacterium smegmatis

Gengenbacher

Chung

et al. 2016

BMC Genomics

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BackgroundMycobacteria, along with exospore forming Streptomyces, belong to the phylum actinobacteria. Mycobacteria are generally believed to be non-differentiating. Recently however, we showed that the mycobacterial model organism M. smegmatis is capable of forming different types of morphologically distinct resting cells. When subjected to starvation conditions, cells of M. smegmatis exit from the canonical cell division cycle, segregate and compact their chromosomes, and become septated and multi-nucleoided. Under zero nutrient conditions the differentiation process terminates at this stage with the formation of Large Resting Cells (LARCs). In the presence of traces of carbon sources this multi-nucleoided cell stage completes cell division and separates into Small Resting Cells (SMRCs). Here, we carried out RNA-seq profiling of SMRC and LARC development to characterize the transcriptional program underlying these starvation-induced differentiation processes.ResultsChanges among the top modulated genes demonstrated that SMRCs and LARCs undergo similar transcriptional changes. The formation of multi-nucleoided cells (i.e. LARCs and the LARC-like intermediates observed during SMRC formation) was accompanied by upregulation of septum formation functions FtsZ, FtsW, and PbpB, as well as the DNA translocase FtsK. The observed compaction of chromosomes was accompanied by an increase of the transcript level of the DNA binding protein Hlp, an orthologue of the Streptomyces spore-specific chromosome condensation protein HupS. Both SMRC and LARC development were accompanied by similar temporal expression patterns of candidate regulators, including the transcription factors WhiB2, WhiB3, and WhiB4, which are orthologues of the Streptomyces sporulation regulators WhiB, WhiD and WblA, respectively.ConclusionsTranscriptional analyses of the development of mycobacterial resting cell types suggest that these bacteria harbor a novel differentiation program and identify a series of potential regulators. This provides the basis for the genetic dissection of this actinobacterial differentiation process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3190-4) contains supplementary material, which is available to authorized users.

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“…Since then, the TetR/Pip‐OFF system has been successfully used by several groups to study essential genes and validate drug targets in vitro and in vivo in different mycobacterial species (Serafini et al ., 2009, 2013; Cortes et al ., 2011; Di Luca et al ., 2012; Mondino et al ., 2013; Ventura et al ., 2013; Ahmed et al ., 2014, 2016; Bazet Lyonnet et al ., 2014; Bhowmick et al ., 2014; Boldrin et al ., 2014; Kolly et al ., 2014a,b; Pandey and Rodriguez, 2014; Verma and Chatterji, 2014; Gola et al ., 2015; Gupta et al ., 2015; Mori et al ., 2015; Hu et al ., 2016; Degiacomi et al ., 2017). This broad experience taught us that the main drawback of this otherwise very succesful system was the strength of the P ptr promoter, which causes overexpression of the gene of interest, when this is physiologically expressed at low level, with resulting accumulation of its product.…”

Section: Introductionmentioning

confidence: 99%

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Boldrin

Degiacomi

Serafini

et al. 2017

Microbial Biotechnology

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SummaryA range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip‐OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr, a strong Pip‐repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild‐type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip‐OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole‐cell screening assay to identify inhibitors of this enzyme.

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Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

Cited by 96 publications

References 54 publications

Direct regulation of topoisomerase activity by a nucleoid-associated protein

Direct regulation of topoisomerase activity by a nucleoid-associated protein

Developmental transcriptome of resting cell formation in Mycobacterium smegmatis

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

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