Newswise — Viruses, much like antagonists in movies, follow one of two approaches: either a stealthy infiltration or an aggressive assault.
In the stealthy mode, viruses remain low-key, quietly penetrating the body's defense mechanisms. Alternatively, they can switch to kill mode, bursting out of their hiding places and launching attacks in all directions. However, such viral attacks are often akin to suicide missions, as they cause the destruction of the host cell on which the virus relies. To succeed, the virus must find enough healthy cells nearby to infect. If the viral particles fail to find suitable targets, the virus becomes unable to sustain itself. Although viruses aren't considered living entities, they essentially stop functioning.
Hence, the critical challenge for a virus is determining the opportune moment to transition from chill mode to kill mode.
In a groundbreaking discovery made by Princeton biologist Bonnie Bassler and her former graduate student Justin Silpe four years ago, a particular virus was found to possess a significant advantage—it can eavesdrop on bacterial communication. The virus specifically tunes into the chemical signal released by bacterial cells when they achieve a critical population size, known as "We have a quorum!" This communication process among bacteria is called quorum sensing and was initially uncovered by Bassler and her team, leading to numerous prestigious awards for their contribution to the field.
Building upon their previous work, Bassler, Silpe, and their fellow researchers have now uncovered a fascinating revelation. They have identified dozens of viruses that respond to either quorum sensing or other chemical signals emitted by bacteria. The results of their extensive research have been published in the current issue of the scientific journal Nature. This new finding opens up exciting possibilities for understanding the intricate interactions between viruses and bacteria, shedding light on the complexities of microbial ecosystems.
According to Bassler, the Squibb Professor in Molecular Biology and the chair of the Department of Molecular Biology at Princeton, the world is teeming with viruses capable of monitoring and gathering information from suitable host organisms. While the complete range of stimuli triggering this behavior remains unknown, their latest research illustrates that this is a widespread mechanism among viruses.
Moreover, their study not only revealed the prevalence of this strategy but also uncovered the tools that govern it. These tools can send signals instructing the viruses to switch from their passive state (chill mode) into an aggressive mode (kill mode). This discovery highlights the sophisticated and dynamic ways in which viruses interact with their host organisms, providing valuable insights into viral behavior and its control mechanisms.
Bacteriophages, commonly known as phages, are viruses that target bacterial cells. They attach to the surface of a bacterial cell and transfer their genetic material into it. Interestingly, multiple types of phages can infect a single bacterium simultaneously, given that they are all in a dormant state, referred to as lysogeny, by biologists. When multiple phages coexist peacefully within a single bacterium, this phenomenon is known as polylysogeny.
In a polylysogenic state, the phages peacefully cohabitate with the bacterial cell, allowing it to undergo continuous replication just like healthy cells do. The viral DNA or RNA remains tucked away inside the bacterium's own genetic material, replicating in synchrony with the host cells. This intricate relationship demonstrates how phages can quietly integrate their genetic material with the bacterial cell, leading to a harmonious coexistence with their host.
However, the phages that infiltrate the bacterial cells are not precisely peaceful; it's more akin to a state of mutually assured destruction. The delicate balance between the phages and the bacterium remains intact until a certain trigger prompts one or more of the phages to transition into kill mode.
Researchers investigating phage warfare have been aware for some time that significant disruptions to the system, such as intense UV radiation, carcinogenic chemicals, or specific chemotherapy drugs, can cause all the resident phages to activate their kill mode simultaneously. This event results in a widespread attack on the bacterial host, leading to its demise and the release of newly replicated phages, ready to engage in the next phase of their life cycle.
Up until this point, scientists believed that when triggered, the phages would compete, racing to seize the bacterial resources. The prevailing assumption was that the fastest phage would emerge victorious, releasing its viral particles.
However, contrary to these expectations, Bassler's research team made a surprising discovery.
To observe the behavior of infected bacterial cells under the influence of a universal kill signal, Grace Johnson, a postdoctoral research associate in Bassler's research group, employed high-resolution imaging. She carefully monitored individual bacterial cells that were simultaneously infected with two different phages. As she introduced the kill signal, both phages swiftly sprang into action, attacking and breaking down the host cell.
In order to discern the outcome, Johnson creatively used special fluorescent tags to "paint" each phage's genes. These tags emitted distinct colors, allowing her to identify and distinguish which phage was actively replicating during the process. The use of these fluorescent markers helped shed light on the intricate dynamics between the two phages within the host cell and provided valuable insights into their replication strategies.
Upon observation, Grace Johnson was taken aback by the unexpected results. Contrary to the assumption of a clear victor or even a tie between the two phages, she witnessed a surprising outcome. The bacterial cells did not display a straightforward outcome; instead, they revealed a complex pattern. Some bacteria emitted a distinct color, others glowed with the second color, and remarkably, some displayed a blended appearance, simultaneously producing both phages at once.
The revelation of three distinct subpopulations within the bacterial cells left the researchers astounded. As Bassler remarked, no one had ever anticipated such a multifaceted and diverse response from the infected cells. This groundbreaking discovery challenged conventional notions and opened up new avenues of inquiry into the intricate mechanisms governing phage interactions within their host cells.
Johnson expressed her enthusiasm, recalling that particular day as truly exhilarating. Through high-resolution imaging, she witnessed the diverse array of responses from the bacterial cells, engaging in various phage production combinations. Some cells induced one phage, while others activated the other, and intriguingly, certain cells even induced both simultaneously. Equally remarkable were the cells that did not trigger either of the phages.
Another significant hurdle for the research team was to devise a method to selectively activate only one of the two phages at any given time. This challenge added an extra layer of complexity to their investigation, as they sought to decipher the intricate mechanisms that govern the phage behavior within the bacterial cells.
After completing his postdoctoral studies at Harvard, Silpe returned to Bassler's lab as a postdoctoral research associate and took charge of identifying the triggers for the phages. Although the natural signals that prompt these phages to react remain unknown, Silpe ingeniously devised specific artificial chemical triggers for each phage. Contributing significantly to the investigation, Grace Beggs, another postdoctoral fellow in the Bassler group, played a vital role in the molecular analyses of these artificial systems.
In their experiments, Silpe introduced the artificial cue to the polylysogenic cells. Astonishingly, only the phage responsive to his specific trigger initiated replication, and it occurred in all the cells. On the other hand, the other phage remained entirely dormant in chill mode, unaffected by the artificial signal. This breakthrough in selective phage activation showcased the team's progress in understanding the intricacies of phage behavior, setting the stage for further exploration into the fascinating world of viral-host interactions.
"I honestly didn't have much confidence that it would work," Silpe admitted. "Given that my approach deviated from the natural processes, I expected both phages to replicate. The fact that we observed only one phage replicating was a genuine surprise. As far as I know, no one had attempted anything like this before."
Bassler also expressed her amazement at the results, emphasizing that the concept of exploring phage-phage warfare within a single cell was never considered because it seemed impractical. Grace J. and Justin's experiment, however, opened up new possibilities. The microscopic scale of bacteria posed a significant challenge in itself, as observing individual bacteria was already difficult. Adding to the complexity, visualizing phage genes inside bacteria proved to be an even more formidable task due to their minuscule size – smaller than small, as Bassler described it.
Nonetheless, the groundbreaking experiment conducted by Grace J. and Justin showcased the power of their approach, enabling researchers to delve into uncharted territory and unravel the mysteries of phage interactions within a single cell.
Johnson had been adapting the fluorescence in situ hybridization (FISH) imaging platform for a different research project related to biofilms and quorum sensing. However, when she attended Silpe's presentation during a group meeting and learned about his research on eavesdropping phages, she had an epiphany. Johnson realized that FISH could be the key to unraveling the enigmatic secrets surrounding Silpe's phages, which had been difficult to study until then.
While many bacterial cells worldwide host multiple phages simultaneously, manipulating and imaging them remained an elusive feat until Johnson and Silpe's breakthrough. Their ingenious approach allowed them to induce one phage, the other, or both phages as needed, a feat that impressed Bassler and the research team. Furthermore, the ability to visualize this phage warfare at the single-cell level was a remarkable achievement, credited to Johnson's contributions.
Bassler pointed out that a large portion of viral genome genes remains shrouded in mystery, and their functions remain unknown. The complexity of viral genes presents an ongoing challenge for researchers, leaving much to explore and discover in the realm of virology.
"Indeed, in this research, we were able to unveil the functions of several phage genes, revealing their roles in orchestrating this remarkable and unexpected chill-kill switch. This switch plays a crucial role in determining the victor during phage-phage warfare," she explained. "Our discovery strongly suggests that there are even more fascinating processes awaiting discovery in this field."
She further emphasized the significance of phages in the field of molecular biology. Having initiated the molecular biology era 70 years ago, phages are now experiencing a resurgence in interest, both as potential therapies and as an incredible repository of molecular mechanisms that have evolved over time. Their untapped potential presents a treasure trove of possibilities, yet to be fully explored by the scientific community. With so much yet to be uncovered, the realm of phages holds immense promise for future breakthroughs and advancements in various fields.
“Small protein modules dictate prophage fates during polylysogeny,” by Justin E. Silpe, Olivia P. Duddy, Grace E. Johnson, Grace A. Beggs, Fatima A. Hussain, Kevin J. Forsberg and Bonnie L. Bassler, was published in Nature on July 26 (DOI:10.1038/s41586-023-06376-y). The research was supported by Princeton University's endowment, the Howard Hughes Medical Institute (HHMI), the National Institutes of Health (NIH), the National Science Foundation (NSF), the Jane Coffin Childs Memorial Fund for Medical Research (JCC Fund), the NIH Office of Extramural Research (OER) and the Damon Runyon Cancer Research Foundation.
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