The evolutionary arms race
Bacteria are fierce competitors, constantly evolving and adapting to survive. Many bacteria are “naturally competent,” meaning that they can actively transport environmental DNA fragments across their cell membrane(s) and into their cytoplasm. Once in the cytoplasm, the DNA integrates into the bacterial genome in a process called recombination. Bacterial recombination is facilitated by horizontal gene transfer (HGT), the non-sexual movement of genetic information between organisms.1 For billions of years, bacteria have engaged in this evolutionary arms race, exchanging genetic material to evade predators, phages, and host immune systems. As in Lewis Carroll’s Through the Looking-Glass, when the Red Queen informs Alice that “here, you see, it takes all the running you can do to keep in the same place,” so too have bacteria constantly adapted through recombination to keep them one step ahead of their adversaries. This endless race has equipped bacteria with remarkable tools. But what if this evolutionary advantage, honed over eons, could be redirected to help humans in a different kind of battle: the fight against cancer?
Live bacterial therapeutics against cancer
Colorectal cancer is the second-leading cause of cancer-related death in the United States.2 Detecting it, however, remains a significant challenge. Colonoscopies are invasive and costly, and patients often do not exhibit symptoms, complicating diagnoses. Chemotherapy and radiation therapy are common treatments, but they often wound normal, healthy cells. Additionally, these therapies cannot penetrate solid tumors and patients can develop resistance to chemotherapy drugs. However, live bacterial therapeutics have emerged as a new kind of ally, and serve as a promising tool for detecting and potentially treating cancer.
In one study, researcher Dr. Omar Din in the Knight Lab at UC San Diego engineered the bacteria Salmonella enterica to act like a Trojan horse against tumors. Using a bacterial communication system called quorum sensing, in which bacteria release chemical signals to coordinate group behavior, the researchers engineered S. enterica to sense when their population reaches a certain threshold inside a tumor. At this threshold, they burst open, releasing a payload of chemotherapeutic cargo to the tumor.3 In another study, researcher Dr. Ting Fu, previously a postdoctoral scholar in the Evans Lab at the Salk Institute for Biological Sciences, engineered gut-native Escherichia coli to produce an enzyme that breaks down bile acids, or compounds made in the liver that can promote colorectal cancer. The researchers reintroduced the engineered E. coli into mice in an attempt to suppress the onset of colorectal cancer. This approach overcame the traditional challenge of lab-engineered bacteria that fail to survive in native physiological environments, though further research is needed to determine if these engineered E. coli can prevent cancer.4
At UC San Diego, Professor of Molecular Biology Dr. Jeff Hasty and scientist Dr. Robert Cooper are advancing this field of live bacterial therapeutics by engineering Acinetobacter baylyi bacteria and exploiting its natural competence to detect DNA shed from colorectal cancer cells. Their work culminated in a groundbreaking proof-of-concept study that demonstrates how bacterial biosensors can transform the battleground of cancer diagnostics, offering an accessible and affordable tool in humanity’s war against cancer.
CATCH: a two component system
Harnessing the power of natural competence, Dr. Cooper engineered A. baylyi to undergo HGT of cancer-related DNA sequences from colorectal tumors. These bacteria act as “biosensors” that detect and integrate tumor DNA directly into their genome using a cellular assay known as CATCH (CRISPR-discriminated horizontal gene transfer). The CATCH system has two components: DNA infiltration and mutation detection. The first component involves homologous recombination, where the incoming DNA integrates into the genome, much like bacteria spies intercepting messages. The second component involves CRISPR spacers, which act as the defensive line that only permits DNA with a specific mutation to pass, rejecting non-cancerous DNA. Together, these components enable CATCH to identify oncogenes.
The CATCH system targets the kras gene. This gene encodes for the K-Ras protein, which regulates cell growth, differentiation, and gene expression. Unfortunately, kras can be mutated to become an oncogene, driving the progression of colorectal adenomas (benign tumors) into advanced carcinomas (malignant tumors).7 The most frequently mutated codon in the K-Ras protein of carcinomas is the twelfth codon, where glycine is replaced by aspartic acid (KRASG12D).8 Dr. Cooper uses the A. baylyi biosensors to detect this single-codon mutation, creating a powerful tool for diagnosing colorectal cancer.
The first component of CATCH is to ensure that A. baylyi can intercept DNA from human cells. To do this, Dr. Cooper engineered the kras gene into A. baylyi and colorectal cancer cells. He designed a gene cassette, or small segment of DNA, containing a kanamycin resistance gene (kanR), a stop codon for kanR, and a green fluorescent protein (GFP). Then, he inserted this gene cassette in the middle of the kras gene. The segmented kras gene fragments serve as ‘homology arms,’ a pair of DNA sequences that are identical to corresponding regions in another gene cassette. Alignment of these pairs of homology arms allows for homologous recombination to occur. During homologous recombination, the sequences in between the pairs of homology arms are exchanged. The recipient genome incorporates the donor sequence, while the donor genome receives the recipient’s sequence.
To visualize the CATCH system in action, the cassette was first introduced into the A. baylyi genome. Then, the same cassette, this time without the kanR stop codon, was transduced into colorectal cancer cells. As DNA is constantly shed from cells due to the natural process of cell turnover, these DNA fragments act as genetic debris scattered across the battlefield. When the A. baylyi biosensors come into close proximity with DNA shed from engineered human colorectal cancer cells, the kras homology arms from both species line up, and homologous recombination occurs. The donor A. baylyi genome loses the kanR stop codon and can now express resistance to kanamycin, providing a clear readout for cancer detection.
The second component of CATCH wields A. baylyi’s native CRISPR-Cas system to detect the KRASG12D mutation. In bacteria, CRISPR functions as an adaptive immune system.9 It stores small DNA segments from viruses (spacers) into an array, enabling the bacteria to recognize and defend against future attacks. The array is transcribed into guide RNAs, which pair with Cas proteins to form a Cascade complex. This complex scans DNA for a matching sequence next to a short conserved sequence called PAM. If the PAM site is present and the sequence matches the spacer, the Cascade complex binds and degrades the viral DNA, effectively killing the virus.
Following homologous recombination between A. baylyi and colorectal cancer cells, the CRISPR-Cas system is deployed. If the incorporated kras sequence is wild-type, the PAM site remains intact. The Cascade complex then recognizes and cleaves the wild-type DNA, killing the A. baylyi cell. Conversely, if the incorporated sequence contains the KRASG12D mutation, then the PAM site is disrupted. The Cascade complex cannot cleave the mutant DNA, allowing these A. baylyi expressing kanR to survive and grow. Their survival serves as a battle flag raised in victory, signaling the presence of the KRASG12D mutation and providing a clear readout for cancer detection.
CATCHing cancer in vitro and in vivo
The A. baylyi biosensors successfully detected KRASG12D mutations in both engineered cancer cell lines on an agar plate and engineered tumorigenic organoid lines. Notably, the biosensors detected not only purified DNA, but also raw, unpurified DNA.7 In the chaos of a frenzied battleground, the A. baylyi biosensors wield a sword of precision. By recognizing a very specific mutation in an environment filled with contaminants, DNases, and other debris, the A. baylyi biosensors open the door to in vivo applications, where environments are similarly highly contaminated. To assess the A. baylyi biosensor in vivo, the engineered bacteria were rectally delivered to mice with and without colorectal tumors. The contents inside the gastrointestinal tract of the mice were analyzed for kanamycin-resistant A. baylyi by quantifying the number of colonies that grew on kanamycin selection agar plates. These results demonstrated that CATCH successfully distinguished between mice with or without colorectal cancer.
CATCHing non-engineered cancer DNA
The CATCH system represents a breakthrough in live bacterial therapeutics, coupling evolutionary ingenuity with modern biotechnology. Detecting engineered cancer DNA in vitro and in vivo is one achievement, but identifying non-engineered cancer DNA in patients presents an even greater challenge. In a new A. baylyi biosensor to detect non-engineered cancer DNA, a gene that encodes a tetracycline repressor protein (tetR) was inserted in the middle of the kras gene. Initially, the tetR gene is constitutively transcribed, continuously producing the repressor protein. Therefore, an output gene, in this case kanamycin resistance, is repressed.
Once again, when the biosensor encounters target DNA, homologous recombination initiates a tactical transfer. The tumor DNA delivers its kras gene to A. baylyi. If the kras sequence is wild-type with an intact PAM site, the Cascade complex recognizes and binds the sequence, degrading the kras DNA and output gene. This leaves the battlefield quiet, with no signal. However, if the kras sequence carries the KRASG12D mutation, the PAM site is altered and “camouflaged,” eluding detection. The Cascade complex is no longer able to degrade the DNA, allowing the output gene to produce a clear signal, a triumphant flare, marking the successful identification of colorectal cancer.7
Important considerations with the current biosensors
Bacterial biosensors represent a promising new weapon in the fight against cancer, but their development is still in the early stages. They are not yet poised to replace colonoscopies, which remain the gold standard for detecting polyps and early-stage colorectal cancers. Instead, these biosensors offer a less invasive, supplementary method for detection. However, this is not yet a battle-ready solution; current studies remain proof-of-concept and require further validation to ensure their effectiveness across diverse patient populations. To sharpen their battlefield precision, biosensors capable of detecting multiple genetic mutations—not just those in kras—would allow for a more comprehensive analysis of tumor DNA to identify multiple oncogenes or mutations within the same sample. Rigorous biocontainment measures, such as genetic “kill switches,” could prevent unintended consequences, ensuring biosensors retreat once their mission is complete.10 Optimizing delivery strategies is another key objective. While rectal delivery has been effective in animal studies, oral delivery would make these tools more practical for patients.
Positive outlooks for the biosensors
The vision for bacterial biosensors extends far beyond colorectal cancer detection. With further development, these systems could be adapted to detect a variety of cancers including breast, liver, and blood, or even other diseases characterized by specific genetic markers. Their adaptability and precision could open doors to applications in personalized medicine. Just as bacteria have engaged in the Red Queen’s relentless race for survival, constantly evolving to outpace their adversaries, we can now harness this evolutionary momentum for human health. By redirecting the tools bacteria have used over billions of years, we may turn their survival strategies into powerful allies in the fight against cancer. Though still in their infancy, bacterial biosensors exemplify the potential of evolution’s arsenal not to keep humanity running in place, but to bring us closer to victory in the ongoing battle against cancer.
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