Complex Virus Communication

The first signals from bacteria communicating to fellow comrades were discovered in the 1960s.  It took 60 years of research to realize the intelligence of bacteria. The first virus signal was discovered in 2017 and in the nine years since then a vast amount of information has been discovered about virus communication. Much of the research has been in phage viruses––viruses that interact with and inhabit bacteria.

It is clear now that phage viruses are not making decisions based on one or two signals. They are integrating information from at least a dozen distinct channels simultaneously, weaving together signals from their own secreted peptides, bacterial communication molecules, DNA damage pathways, metabolic indicators, biofilm messengers, immune alarm systems, and signals from multiple virus species into a coherent assessment of their situation.

Arbitrium: The Dedicated Phage Language

The story of phage communication began with the discovery of a signal molecule arbitrium. The arbitrium communication system has now been identified in more than a thousand phage species infecting both soil bacteria and human pathogens. This dedicated phage language, involves a system in which phages produce, secrete, accumulate, and sense their own communication molecules to coordinate decisions among many viruses. 

In the arbitrium system, each phage produces three signaling molecules that work in concert with each other. The signals inform the community of phage viruses about whether to choose to immediately kill the cell with the release of new viruses, called lysis or to be dormant and live peacefully inside the genome of a bacterium, called lysogeny.  This allows the phages to act as a sleeper agents, ensuring their survival when environmental conditions are harsh.

After the arbitrium discovery, it was initially thought that the lysis-lysogeny decision was simply based on whether the arbitrium signal was low or high and how many bacteria were available for phages to infect. As more was learned, it became apparent that phage viruses’ decisions are much more complex and that the arbitrium signal level is just one factor in a much more complex decision.

How to Make a Complex Decision 

The phage is not trying to estimate how many phages or bacteria exist––it is trying to estimate the probability that its offspring will find new hosts. The phage wants to know: Is this current bacterium healthy enough to support making new viruses? If the bacterium is killed, are there likely to be many uninfected bacteria available nearby for progeny? Is there another strategy more likely to maximize long-term descendants? 

Some examples of situations and decisions

1. Many bacteria with few infections and low amount of arbitrium signals. Phage determines bacteria are plentiful and therefore kills it.  

2. Many bacteria with many recent infections and therefore a high arbitrium level. Here the bacteria are plentiful now but may become scarce, so the decision to kill will depend on metabolism and other signals.

3. Few bacteria, but many infections with a high arbitrium count. Here the future number of bacteria are limited so the decision is to stay and incorporate into the genome. 

4. Bacterium is starving and damaged presenting a poor factory for virus production. Most phages would stay in genome at first and if more severe damage occurred, they would kill the cell. 

Recently, many new factors have been discovered.

Phages Eavesdropping on Bacterial Signals

Bacterial conversations use chemicals to coordinate biofilm formation, virulence factor production, mating, and countless other behaviors. Phages have learned to eavesdrop on these conversations and to utilize bacterial signals for their own purposes.

One species of phages can sense molecules produced by bacteria that indicate the density of E. coli bacteria. When these bacterial molecules accumulate to high levels, the phage detects them and kills the bacterium because high bacterial density means plenty of cells for the virus’s progeny. The phage has engineered a direct response to the bacterial signal that reflects how many bacteria are present.

Another example of eavesdropping exists with a difference species of phages that infects cholera bacteria. Cholera bacteria produce a molecule to describe density; this density is perceived by the bacteria colony via a receptor protein on the bacteria. The phage virus produces its own version of the receptor, which binds to the same signal molecules. This triggers a cascade of signals that kills the cell precisely at the moment when bacterial density is highest, which is when that cell is most likely to have neighbors that could serve as new infection targets. The phage has engineered a receptor that mimics the bacterial one, creating a bridge between the bacterial communication system and the phage decision-making apparatus.

When bacteria signal to each other about their population density, they are inadvertently instructing their parasitic phages about optimal timing for lysis. This is one-way control. The phage listens and responds, but the bacteria cannot hijack the phage system in return.

Interpreting The Cell’s Stress Signals

Beyond eavesdropping on bacterial density sensing, phages have also evolved to respond to bacterial stress responses. When a bacterial cell's DNA is damaged—by UV radiation or chemicals—it activates the SOS response, a cellular alarm system in which a protein becomes activated and triggers widespread changes in gene expression. Many phages have learned to interpret this SOS signal as a cue to kill the cell. If the host is dying, better to escape now than to persist in a failing cell.

One species of phage responds to both the molecule representing density and to the SOS response. Both signals lead to cell death, but they provide distinct information for the virus. The first tells the page whether other hosts are available. The SOS response tells the phage about whether this cell is viable. Together, these two signals allow the phage to make more sophisticated decisions than either could provide alone.

Phages also appear responsive to general physiological stress markers that bacterial cells produce in response to oxidative stress or other cellular injuries. Phages understand that these signals mean that the cell is compromised and that incorporation might be risky. The electrical and chemical gradients across the cell membrane provide another signal the phage can detect. A cell with declining membrane potential is a cell in trouble and less likely to provide a stable home.

Phages can also sense more subtle metabolic indicators of host cell health and incorporate that information into their decisions. If a host cell is starved for energy or amino acids, if ATP levels are low, or the cell is compromised, the phage decides that it is better to kill it, escape, and find a healthier host than to integrate into a dying cell. Conversely, if metabolic indicators suggest the host is robust and energetic, better to stay and live in the genome. 

Biofilm Signals

Bacteria use messages to coordinate the transition between free-floating and biofilm multicellular lifestyles. A bacterial stickiness signal favors biofilm formation. Some phages incorporate that into decision-making. When bacteria are embedded in biofilms, the phage may want to stay inside, betting that living in a biofilm will eventually prove advantageous. When bacteria are swimming alone outside a biofilm, the phage favors killing the cell and releasing progeny, since nearby bacteria are in a more mobile, accessible state.

Bacterial Immune Language

Phages can sense the activation of bacterial immune systems. Bacteria possess multiple layers of immune defense against phage infection producing characteristic molecular signals—alarm molecules that propagate through the bacterial cell and beyond. If a cell's defense is highly active, the cell is under phage attack. A phage that has just integrated in a cell’s genome might sense these signals and realize that competition for the cell is intense. Better to remain dormant, to hide from the immune system. This is phage eavesdropping on the conversation between bacteria and their defense systems.

Coinfection

Phages sense the number of other phages infecting a cell. If a single phage infects a cell, the phage is more likely to stay, but it is more likely to kill and leave if multiple phages infect simultaneously. With multiple phages competing for the same host there are less resources and less opportunity to incorporate. Any single phage's probability of successful incorporation is lower when competition is fierce.

The Phage Integrates Information and Uses It for Decision Making

Phage viruses are integrating information from at least a dozen distinct channels: their own arbitrium peptides, bacterial quorum sensing molecules, DNA damage signals, metabolic indicators, biofilm messengers, immune alarms, coinfection density, and general stress markers. Each channel carries different information and can provide a different perspective on the question of whether to kill or incorporate.

Different phage species can also interact with each other. Arbitrium systems from different phage species have variations in peptide sequences, yet their receptors can still recognize and bind peptides from other species. This recognition leads to ecological effects, such as one colony suppressing another. Phage ecological warfare involves producing signals that suppress the cycles of competitors, keeping rivals in dormant states while the signaler itself remains capable of rapid replication. A phage can also produce fake signals that disrupt the coordination of competitor populations.

It took many decades to see the intelligence of cells. Now, only nine years after the discovery of the first virus signal, it is hard not to think of communicating viruses as intelligent.