Virus World

The Danish creeks, Odense Å and Lindved Å, have surprised researchers and students at SDU by containing previously unknown virus species. "We have found five new species that we believe are unknown to science," said associate professor Clare Kirkpatrick, who studies bacterial stress-response at the Department of Biochemistry and Molecular Biology at University of Southern Denmark. The somewhat surprising discovery was made during the coronavirus pandemic, when some of Kirkpatrick's students could not carry out their normal microbe studies in the laboratory and therefore went on field trips to local creeks to see if they had any interesting microbes to offer. The fact that viruses exist in nature is not surprising, as they are the world's most widespread organism. They are everywhere and part of all kinds of microbial cycles and ecosystems, but the fact that five potentially new species have appeared in local creeks, did surprise Clare Kirkpatrick. While four of the five have not yet had their genome mapped in a genome sequencing, one species has now been fully sequenced, scientifically described, named and published in Microbiology Resource Announcements. The name is Fyn8. Many viruses are so-called bacteriophages (or phages), meaning that they kill bacteria, and Fyn8 is no exception. It can attack and kill the bacteria Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a bacterium found naturally in soil and water. It is normally harmless towards healthy people, but like many other bacteria it has developed resistance to antibiotics and is found in hospitals. For example, patients with wounds (like burn patients) and ventilator patients are at risk of getting an infection that cannot be fought with antibiotics. The researchers have no doubt that Fyn8 can effectively kill Pseudomonas aeruginosa: "We could see it with the naked eye: Clear holes appeared in the layer of Pseudomonas aeruginosa bacteria in our petri dishes, where Fyn8 had infected the bacterial cells, killed them, multiplied and proceeded to attack the next." Considering that the world is facing a resistance crisis, where more people will die from an infection with resistant bacteria than from cancer, the new finding is of course interesting and raises an significant question: Can phages help us in the fight against resistant bacteria? Research in this field has been uncommon until recently, both in academic research institutions and in pharmaceutical companies. In the past and in other parts of the world however, there has been some research, and phages have also been used to treat infections in Eastern European countries in particular.

The phages were discovered at the beginning of the 20th century by researchers who had their bacterial cultures destroyed by virus infections. The benefits of that discovery were obvious, but antibiotics, not phages, became the most widespread cure against bacterial infections. One reason was perhaps that antibiotics were easy to produce and easy to use, while the phages were difficult to isolate and give to patients. Another reason was probably also that an antibiotic dose could kill many different bacteria, while a phage only matches with a single bacterial species. "But today it is relatively easy to make precision medicine for the individual patient. First you find out what exact bacteria a patient is infected with—and then you can treat the patient with exactly the phage that will kill the bacteria," explained Clare Kirkpatrick. She adds that this strategy works even on bacteria which are resistant to all known antibiotics. Time will tell whether there are more new virus species in the local creeks near University of Southern Denmark campus, but it is quite probable, Clare Kirkpatrick believes that "many, many more are waiting to be discovered."

Research published in Bacteriophages (Feb. 13, 2023):

Bacteria and the viruses that infect them are engaged in a molecular arms race as ancient as life itself. Evolution has equipped bacteria with an arsenal of immune enzymes, including CRISPR-Cas systems, that target and destroy viral DNA. But bacteria-killing viruses, also known as phages, have devised their own tools to help them outmaneuver even the most formidable of these bacterial defenses. Now, scientists at UC San Francisco and UC San Diego have discovered a remarkable new strategy that some phages employ to avoid becoming the next casualty of these DNA-dicing enzymes: after they infect bacteria, these phages construct an impenetrable "safe room" inside of their host, which protects vulnerable phage DNA from antiviral enzymes. This compartment, which resembles a cell nucleus, is the most effective CRISPR shield ever discovered in viruses. "In our experiments, these phages didn't succumb to any of the DNA-targeting CRISPR systems they were challenged with. This is the first time that anyone has found phages that exhibit this level of pan-CRISPR resistance," said Joseph Bondy-Denomy, PhD, assistant professor in the Department of Microbiology and Immunology at UCSF. Bondy-Denomy led the research team that made the discovery, which is detailed in a paper published Dec. 9, 2019 in the journal Nature.

The Hunt for DNA That CRISPR Can't Cut

To find CRISPR-resistant phages, the researchers selected viruses from five different phage families and used them to infect a common bacteria that had been genetically engineered to deploy four different Cas enzymes, the DNA-cutting component of CRISPR systems. These CRISPR-fortified bacteria emerged victorious against most of the phages they faced off against. But two jumbo phages -- so named because their genomes are five to 10 times larger than the genomes of the most well-studied phages -- were found to be impervious to all four CRISPR systems. The researchers decided to put these jumbo phages to the test and probe the limits of their CRISPR-resistance. They exposed them to bacteria outfitted with a completely different type of CRISPR, as well as bacteria equipped with restriction-modification systems -- a DNA-cleaving enzyme that's more common than CRISPR (restriction systems are found in about 90 percent of bacterial species, whereas as CRISPR is present in only about 40 percent) -- but which can only target a limited number of DNA sequences. The results were the same as before: petri dishes littered with the exploded remains of phage-infected bacteria. "It was really surprising because we engineered the bacteria to massively overproduce components of the immune system, but none of them could cut the phage DNA. These phages were resistant to all six bacterial immune systems tested. No other phage even comes close," said Bondy-Denomy.

Jumbo phages, it seemed, were virtually indestructible. But test tube experiments suggested otherwise -- jumbo phage DNA was, in fact, as vulnerable to CRISPR and restriction enzymes as any other DNA. The CRISPR resistance that was observed in phage-infected cells had to be the result of something the viruses were producing that interfered with CRISPR. But what?

Found: An Impenetrable CRISPR Shield

Microscope-based experiments finally revealed what was happening. When these jumbo phages infect bacteria, they build a spherical compartment in the middle of the host cell, which keeps antiviral enzymes at bay and provides a "safe room" for the viral genome to replicate. This compartment, it turns out, was identical to one first discovered in 2017 by UCSD Professor Joe Pogliano, PhD, and UCSF Professor David Agard, PhD, both of whom are co-authors of the new study. Though these researchers previously demonstrated that the phage genome replicated in this nucleus-like shell, nobody knew until now that the shell also serves as an impenetrable shield against CRISPR and other DNA-dicers....

Publisjed in Nature (09 December 2019):

https://doi.org/10.1038/s41586-019-1786-y

The U.S. Food and Drug Administration (FDA) cleared Armata Pharmaceuticals‘ investigational new drug (IND) application for a Phase 1b/2a clinical trial of AP-PA02 for the treatment of the Pseudomonas aeruginosa bacterial infections that are a hallmark of cystic fibrosis (CF).  “We are very pleased that the FDA has cleared our IND, and we plan to initiate clinical development of AP-PA02 by the end of this year,” Todd R. Patrick, Armata’s CEO, said in a press release. P. aeruginosa can colonize the lungs, causing difficult-to-treat infections, which can severely impact both the quality of life and survival rates among people with CF. Treatment for P. aeruginosa infections typically consists of antibiotics. AP-PAo2, conversely, utilizes a cocktail of viruses called bacteriophages to attack the infecting bacteria. Because phages are specific to bacteria — they cannot infect the cells of the patients’ organs — they cause fewer side effects than antibiotics and can target even antibiotic-resistant microbes. AP-PA02 delivers phages specific to P. aeruginosa directly to the lungs via inhalation.

The new trial — called SWARM-P.a. — is a multi-center, double-blind, randomized, placebo-controlled, single ascending dose (SAD) and multiple ascending dose trial that will assess the safety and tolerability of AP-PA02 in CF patients with chronic pulmonary P. aeruginosa infections. Armata expects to begin testing in the SAD group later this year. Of note, in a double-blind study, neither the participants nor the researchers know who is receiving the investigational therapy and who is given a placebo. “Results from this study, which we are calling SWARM-P.a. to reflect the manner in which phage attack dangerous pathogens, will be our company’s first clinical trial to evaluate a phage-based therapy as a potential treatment for Pseudomonas aeruginosa airway infections,” Patrick said. “This clinical trial will contribute to the evaluation of the potential of phage to combat multi-drug resistant infections, and potentially usher in a new era in the fight to develop alternatives to antibiotics,” he added. The Cystic Fibrosis Foundation is supporting Armata’s research through a $5 million grant, awarded earlier this year..