CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

Nature Communications ◽

10.1038/s41467-021-21748-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Poushali Chakraborty ◽

Sapna Bajeli ◽

Deepak Kaushal ◽

Bishan Dass Radotra ◽

Ashwani Kumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune System ◽

Biofilm Formation ◽

Antimycobacterial Activity ◽

Drug Tolerance ◽

Host Immune System ◽

Tissue Sections ◽

Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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Drug Tolerance to EGFR Tyrosine Kinase Inhibitors in Lung Cancers with EGFR Mutations

Cells ◽

10.3390/cells10071590 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1590

Author(s):

Kenichi Suda ◽

Tetsuya Mitsudomi

Keyword(s):

Lung Cancer ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Drug Tolerance ◽

Lung Cancers ◽

Egfr Tyrosine Kinase ◽

Egfr Tyrosine Kinase Inhibitors ◽

Egfr Mutated

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) serve as the standard of care for the first-line treatment of patients with lung cancers with EGFR-activating mutations. However, the acquisition of resistance to EGFR TKIs is almost inevitable, with extremely rare exceptions, and drug-tolerant cells (DTCs) that demonstrate reversible drug insensitivity and that survive the early phase of TKI exposure are hypothesized to be an important source of cancer cells that eventually acquire irreversible resistance. Numerous studies on the molecular mechanisms of drug tolerance of EGFR-mutated lung cancers employ lung cancer cell lines as models. Here, we reviewed these studies to generally describe the features, potential origins, and candidate molecular mechanisms of DTCs. The rapid development of an optimal treatment for EGFR-mutated lung cancer will require a better understanding of the underlying molecular mechanisms of the drug insensitivity of DTCs.

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Extracellular DNA of slow growers of mycobacteria and its contribution to biofilm formation and drug tolerance

Scientific Reports ◽

10.1038/s41598-021-90156-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aleksandr Ilinov ◽

Akihito Nishiyama ◽

Hiroki Namba ◽

Yukari Fukushima ◽

Hayato Takihara ◽

...

Keyword(s):

Biofilm Formation ◽

Drug Tolerance ◽

Extracellular Dna ◽

Exponential Growth Phase ◽

Human Pathogens ◽

Pellicle Formation ◽

Phage Dna ◽

Physiological Impact ◽

Slow Growing ◽

Growth And Reproduction

AbstractDNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria’s eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

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