Chloramphenicol

Bioscientists learn to trigger 'silent' gene clusters in bacteria that could be rich sources of new antibiotic candidates. Silents are potentially golden in the search for antibiotics to slow the ongoing crisis of resistance in the treatment of disease. Rice University bioscientists have designed novel on and off switches to control the "silent" genes in a strain of bacteria. Their strategy could boost the perpetual search for new antibiotics. The researchers customized CRISPR tools to control the expression of genes in Streptomyces bacteria that, in nature, are only expressed when necessary. Until now, those genes have been challenging for synthetic biologists to access. "As labs started to sequence the genomes of these organisms that were known to produce one or a few antibiotics, we realized that the pathways responsible for the production of antibiotic and other molecules of interest are much more abundant than previously thought," said James Chappell, an assistant professor of biosciences whose lab studies bacteria and ways to engineer them. "Each Streptomyces strain is now predicted to be able to produce up to 40 different molecules of interest, including antibiotics, on average," he said. The work led by Chappell and graduate student Andrea Ameruoso may allow labs to quickly develop libraries of possible antibiotics to test on pathogens. Significantly, they said that while CRISPR-Cas9 has been used to create a platform to activate genes in organisms like Escherichia coli, this is the first time it's been applied to Streptomyces. Their study appears in Nucleic Acids Research. "Bacteria such as Streptomyces have evolved to only produce antibiotics when they need to, in natural environments like soil," Chappell explained. "When we grow them in the lab, it's an artificial environment and very different to how they naturally grow, so sets of genes are silenced. "They're a kind of genetic dark matter," he said. "We can't isolate the chemicals they express to perform a functional screen." The lab's new strategy eliminates the time-consuming task of exposing their proof-of-concept bacteria, S. venezuelae, the source of the common antibiotic chloramphenicol, to potential triggers for gene expression. "Andrea's technology adds synthetic regulators into the cell to artificially stimulate or repress the expression of these pathways," Chappell said. "Now we just need one protein and one small piece of RNA and we can go wherever we want to directly repress or activate a given target," Ameruoso added. The emergence of CRISPR technology, which adapts bacterial immune system mechanisms to locate specific genes along a strand of DNA, simplified access to the previously hidden gene clusters, he said. "Streptomyces is a genus of bacteria that encompasses up to 500 species, and each species can have between 20 and 40 of these clusters of genes able to produce antibiotics or other molecules of interest," Ameruoso said. "So once we figure out a way to scale up our technology, it can be incredibly powerful." Chappell said it's a simple matter to design CRISPR to bind to different DNA sequences. "We exploit that for gene expression control," he said. "If we want to do this in a bunch of different species on a bunch of different pathways, it should in theory be possible. So this paper lays the foundation for a new type of approach." Ameruoso said he's working on a fluorescent technique to observe the activation of clusters in real time. "The main challenge is that observing the depths of the activation of a cluster relies on the purification of the molecule from the extracts we generate," he said. "That's a low-throughput process that requires a lot of work. We want to develop a reporter to observe a fluorescent signal when a pathway is being activated." The researchers noted the process could be used to manufacture molecules for antifungal and anticancer agents or for agriculture. "We focus on antibiotics because at some point in history, we've observed that they kill microbes," Chappell said. "But that's not necessarily what they evolved for, because they're also frequently used as communication signals between cells. So there are many potential uses." He said the study demonstrates an important new approach to the activation of silent pathways. "The vision for the next generation of the work is to go big," he said. "We showed it works on a single silent pathway. Now let's do it on the 40 pathways in this one species, and then let's do it on thousands of microbes. "The power of CRISPR-Cas9 is that it's really scalable for that," Chappell said. Co-authors of the paper are Rice graduate student Maria Claudia Villegas Kcam and undergraduate alumna Katherine Cohen. The Welch Foundation (C-1982-20190330) and an Alfred P. Sloan Research Fellowship (FG-2018-10500) supported the research.

Mitochondria are the power plants of cells, and they contain their own genetic material and RNA molecules. Scientists have now discovered that certain modifications in mitochondrial RNA boost the invasive spread of cancer cells by supporting protein synthesis in mitochondria. They have established that a specific gene expression signature correlating with high levels of mitochondrial RNA modifications is associated with metastasis and poor prognosis in patients with head and neck cancer. When the researchers blocked the responsible RNA modifying enzyme in cancer cells, the number of metastases was reduced. Certain antibiotics that suppress protein synthesis in mitochondria were also able to prevent the invasive spread of cancer cells in laboratory experiments. Mitochondria are the power plants of cells, and they contain their own genetic material and RNA molecules. Scientists from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) have now discovered that certain modifications in mitochondrial RNA boost the invasive spread of cancer cells by supporting protein synthesis in mitochondria. They have established that a specific gene expression signature correlating with high levels of mitochondrial RNA modifications is associated with metastasis and poor prognosis in patients with head and neck cancer. When the researchers blocked the responsible RNA modifying enzyme in cancer cells, the number of metastases was reduced. Certain antibiotics that suppress protein synthesis in mitochondria were also able to prevent the invasive spread of cancer cells in laboratory experiments. The results have now been published in the journal Nature. Cancer cells in aggressive tumours invade the surrounding tissue in an attempt to form a new tumour in other organs. During this journey, cancer cells have to survive unfavourable conditions such as shortage of oxygen or shortage in nutrients. To overcome these stress factors, cancer cells adapt their energy production accordingly. The molecular mechanisms allowing this flexibility were poorly understood until now. "However, we suspected that this metabolic plasticity must be a key to the successful spread of the cancer cells," says Michaela Frye; cell biologist at the German Cancer Research Center. Mitochondria are tiny, membrane-enveloped structures known as the powerhouse of every cell in our body. For energy production, they use the so-called respiratory chain present in the mitochondrial membrane. Because mitochondria contain their own genetic material, they themselves produce key components of the respiratory chain. The production of components of the respiratory chain is tightly regulated by a specific machinery in the mitochondria -- with implications for the metastatic spread of cancer cells, as Michael Frye and her team have now discovered and published in the journal Nature. tRNA molecules are part of this machinery and are responsible for providing the individual amino acid building blocks during protein assembly. The research team identified the deposition of molecular modifications on mitochondrial tRNAs as the control mechanism to support production of proteins during metastasis. RNA modifications regulate mitochondrial function and drive metastasis Cancer cell invasion is a very energy consuming process. The team in Heidelberg discovered that a specific chemical modification found in mitochondrial tRNA, known as "m5C" (5-methylcytosine), is required for metastasis development. The m5C modification cranks up protein synthesis in the mitochondria. This enhances the production of components of the respiratory chain. As a result, the cell increases its pool of energy to fuel demanding cellular processes such as cancer cell dissemination from the tumour. Cancer cells lacking m5C, on the other hand, obtain their energy through a comparatively less efficient mechanism called glycolysis and have a limited ability to spread metastatically. The researchers demonstrated this using human tumors grown in mice. However, cell viability or growth in the primary tumour was not affected by the loss of m5C. RNA-modifying enzyme as a biomarker for metastatic tumours A specific enzyme, the methyltransferase NSUN3, is responsible for the m5C RNA modification. When the scientists switched NSUN3 off, the mitochondrial tRNA was less modified and the invasive spread of the cancer cells decreased. Could NSUN3 function as a biomarker for metastatic cancer? Gene expression signatures indicating high cellular NSUN3 levels and elevated m5C levels were indeed predictive for lymph node metastases and more severe disease progression in patients with head and neck cancer. Antibiotics repurposed to block mitochondrial protein synthesis slow down metastasis Certain antibiotics curb mitochondrial protein synthesis without affecting "general" protein synthesis in the cell's plasma. The researchers therefore assumed that these agents should affect cancer cells similarly to loss of NSUN3. Indeed, treatment with antibiotics such as chloramphenicol or doxycycline, reduced the invasive spread of cancer cells. Antibiotic administration also reduced the number of lymph node metastases in a mouse model. "The importance of mitochondrial RNA modifications was previously studied in certain metabolic diseases. But we now show for the first time that there is a direct link between mitochondrial tRNA modifications and invasive spread of cancer," says Michaela Frye. The researcher is pleased that her group's work identified new approaches to potentially combat the spread of advanced cancers; she adds: "Inhibition of NSUN3 is a promising way to slow down metastasis because the enzyme is solely responsible for metastasis-promoting m5C RNA label. However, the potential long-term side effects of blocking mitochondrial protein synthesis must first be further explored."