"Fragile Genes": Redefining the Key Genes in Mycobacterium Tuberculosis

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In the twenty-sixth issue of 2021 (the 63rd issue), a column article by Wang Jun, a researcher from the Institute of Microbiology of the Chinese Academy of Sciences, published a review of the papers in Cell
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Long press the picture and scan the QR code to read the paper.
The rapid growth of bacterial drug resistance is a major threat in the treatment of clinical infections.
It has been identified by the World Health Organization as one of the urgent human safety issues that need to be resolved in this century
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Mycobacterium tuberculosis, as a kind of pathogenic bacteria that has co-existed with humans for more than 8,000 years, still causes significant global loss of life every year under the current mature treatment methods, especially in low- and middle-income countries, causing a large number of clinical trials.
Infection and death
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In the context of increasing bacterial resistance, the creation of new drugs for tuberculosis and other bacteria requires new ideas
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Existing drug targets mainly focus on essential bacterial genes, but it is difficult for small molecules to achieve complete inhibition of these genes; another type of very potential target that has been overlooked is still incompletely inhibited.
Genes that can effectively reduce the viability of bacteria
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In response to the above problems, Professor Rock and others from Rockefeller University recently scanned the gene loci in Mycobacterium tuberculosis through CRISPRi technology and defined a new method for measuring the importance of bacterial genes: the vulnerability of genes, that is, if Achieving incomplete inhibition of this gene can still cause a significant decrease in the vitality (fitness, growth rate and competitiveness) of the bacteria
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The potential of this type of gene lies in the fact that in the future, even if the corresponding small molecule drugs cannot achieve complete gene expression inhibition, the viability and toxicity of the bacteria themselves are expected to be significantly reduced, thereby expanding the range of targets for drug screening, which is conducive to the development of new drugs
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The article was published in Cell Press’s flagship journal Cell on July 22
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Figure 1 is the main method of this research.
The authors used CRISPR technology-based gene interference (CRISPRi) library in Mycobacterium tuberculosis and other bacteria, and transferred it into cells by regulating the expression of sgRNA and the competitive growth test of bacteria.
, Use large-scale sequencing to detect the abundance of different sequences at different passage times
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Among them, the authors are most interested in a series of genes that have a relatively large impact on the viability of the bacteria when the gene expression is suppressed to the same degree
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The author established an estimation model based on Bayesian, which was able to estimate the vulnerability of genes, and based on this, judged the vulnerability of different gene loci in the genome of Mycobacterium tuberculosis, and further adopted experimental methods.
The accuracy of the method is verified
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▲Figure 1 The main technical route of this research
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By establishing a CRISPRi library containing more than 90,000 sgRNAs, large-scale and controllable gene knockout can be achieved
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Passage and competitive growth tests were carried out with and without induction of sgRNA expression, and the abundance of different mutants (sgRNA) was determined by large-scale sequencing
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Finally, calculate the growth inhibition of the bacteria itself (that is, the vulnerability of genes) when different genes are inhibited to a certain extent
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The authors further focused on the genes with obvious fragility, and the authors also conducted more in-depth further verification.
Independent experiments showed that these genes can indeed significantly reduce the growth of bacteria themselves when they are incompletely inhibited.
Rate
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The author further verified the conservation of other tuberculosis species and other bacterial genera, and found that these highly vulnerable genes are relatively conserved in species, and they also focus on bacterial transcription and translation in functional pathways.
And other key central laws related pathways
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Finally, the authors also found that in a highly virulent Mycobacterium tuberculosis, due to a series of gene mutations, the fragile genes found in the earlier stage of this article are inhibited, and the effect on the bacteria itself is relative to that of low virulence.
The smaller strain of the virus can partly explain the differences in the phenotype of this species, and it also has some enlightenment for future drug development and treatment
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▲Figure 2 Metabolic pathways (D) and physiological and biochemical processes (E) to which a series of highly vulnerable genes identified in this article belong
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In summary, the authors in this article use CRISPRi as a high-throughput screening method to define and determine the vulnerability of a large number of genes in an important human pathogen
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These highly fragile genes are evolutionarily conserved and can have a significant impact on the growth of bacteria themselves without being completely inhibited, so they are suitable as targets for the development of new small molecule antibacterial drugs in the future
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The highly vulnerable sites are mostly involved in the transcription and translation process of bacteria, and the differences in gene vulnerability between clinical strains also help explain and cope with differences in drug resistance and virulence
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Abstract: Antibacterial substances usually target essential genes of bacteria but rarely achieve complete inhibition; therefore, this "all-or-nothing" definition of gene necessity ignores another type of gene with great potential, namely, "fragile" or Gene targets that can significantly affect bacterial growth under incomplete inhibition
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The vulnerability of genes is a continuous and quantifiable indicator, which is significantly related to the viability of bacteria
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In this study, we established a high-throughput screening method on the CRISPR interference system and systematically studied the fragility of genes in Mycobacterium tuberculosis, and discovered multiple fragile gene targets that can be used for new drug development; The essential genes for non-fragility are explained to some extent the problems faced by drug development
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Further research also found that the reduced vulnerability of highly virulent strains is likely to be related to increased drug tolerance
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The results of this study quantify the key life processes of bacteria and provide valuable targets for drug development
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Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition.
Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs.
In contrast, gene ``vulnerability'' is a continuous, quantifiable trait that relates to the magnitude of gene inhibition to the effect on bacterial fitness.
We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes.
We identified highly vulnerable genes in various processes, including novel targets unexplored for drug dis- covery.
Equally important, we identified invulnerable essential genes,potentially explaining failed drug dis- covery efforts.
Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility.
Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Scroll down to read the abstract original Chinese content is for reference only, please refer to the original English text Head of Cooperative Research GroupOur results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Scroll down to read the abstract.
The original Chinese content is for reference only, please refer to the English original.
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cn Wang Jun, researcher at the Institute of Microbiology, Chinese Academy of Sciences, head of the cooperative research group of the Max Planck Society of GermanyOur results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
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The original Chinese content is for reference only, please refer to the English original.
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cn Wang Jun, researcher at the Institute of Microbiology, Chinese Academy of Sciences, head of the cooperative research group of the Max Planck Society of Germany
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Selected high-level overseas talent project (2017), Beijing "Technology Rising Star" (2020) and other project personnel, National Natural Science Foundation research project plans to cultivate new crown emergency research projects, research and development projects focused on topics such as the Ministry of Science
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Mainly engaged in bioinformatics and computational biology research with the microbiome as the core.
In recent years, he has published a series of articles on disease-related microbiome research and new technology applications.
The first and/or corresponding author is in Science, Nature Genetics (2 Articles), Cell Host Microbe, PNAS, Nature Communications, Protein Cell (2 articles), Microbiome (2 articles) and other important journals published a series of research papers
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Dr.
Jun Wang is a Principal investigator in CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, and also group leader of the Max-Planck Partner Group for Bioinformatics and Computational Biology.
He was awarded as “1000 Talent Junior Program" in 2017 and "Emerging Star of Science and Technology" of Beijing in 2020.
Dr.
Jun Wang's laboratory is mainly interested in studying the bioinformatic tools and computational methods for health and disease-related microbiome.
As the first or corresponding author , Dr.
Jun Wang has published a series of research papers in journals including Science, Nature Genetics (2), Cell Host Microbe, PNAS, Nature Communications, Protein Cell (2), Microbiome (2).
Swipe down to read English resume related The original information of the paper was published in the journal Cell under CellPress,