“It’s Alive!”- Resurrection from Dormancy in Pseudomonas aeruginosa

By: Carly Myers

Dr. Evil. Source

It is safe to say that we have all contemplated living longer. Or maybe, for those adventurous souls, hitting pause and waking up in some radically different future. As humans, it is only natural that we are, existentially rattled, yet fascinated by the immortal.

We have cultivated the likes of zombies, vampires, wizards- all sorts of human-like  entities that straddle the line between the living and the dead, or prevent death from ever occurring. In the 90’s cult classic, Austin Powers, he and Dr. Evil travelled through time by living out this transition from life, to near death, and back to life again by being cryogenically frozen. Is this capability strictly confined to the fictional domain? The lovely thing about life, is that it is always pushing past the limit of what is conceivably possible.

Certain strains of bacteria can manipulate this boundary between the living and dead. While some bacteria form internal spores, a highly resilient dormant structure that kills the mother cell. Others go into a stage of resilient dormancy, and then are capable of revival, without sacrificing themselves by creating a spore. The quest to understand this living and “dead” continuum within the bacterial realm has practical and potentially life saving applications for humans.

Pseudomonas aeruginosa. Source

Pseudomonas aeruginosa are common gram negative bacteria. Like humans, they are heterotrophic, meaning that they are bound to using organic carbon energy sources such as glucose. In moist environments, they are known to form communities known as biofilms. Human tissue and implants are hotspots for these P. aeruginosa communities, and people who suffer from Cystic Fibrosis easily succumb to infection in the lungs. Biofilms help make P. aeruginosa extremely virulent.

P. aeruginosa are rather clever when together in a group, such as they are when in a biofilm. They can police one another by poisoning certain members with cyanide. This helps to maintain stability. Another way they sustain their population is by maintaining a composition of clonal (from the same initial strain), yet differing cells. This is known as being heterogeneous. Within the biofilm community, some P. aeruginosa are more metabolically active and grow exponentially. Others in less favorable regions, often with reduced nutrients or oxygen, are in a stationary phase and grow much more slowly or are even dormant.

We are interested in stationary cells as they have multiple strategies for survival in poor conditions. One method these cells will utilize is the stringent response. Upon activation, this pathway targets the energy consuming ribosome, the protein complex that binds to mRNA and tRNA to make proteins and other amino acids. The first step in this process is the slowing of the ribosome, and subsequent binding of RelA and SpoT proteins that produce guanidine penta- and tetra- phosphate [(p)ppGpp]. The production of (p)ppGpp signals the expression of survival genes during the dormant phase.

A key mechanism stationary cells utilize to survive poor conditions is to decrease their protein synthesis. It is known that these cells are resistant to conditions that would kill a normal P. aeruginosa cell, such as the presence of antibiotics. When someone ceases their antibiotic treatment, dormant cells can reanimate and go on to repopulate the biofilm. P. aeruginosa infections are rarely cured in full by modern treatment protocols. Essentially we have a problem of trying to kill the undead.

So, if the undead do not respond to deadly environments, then what does P. aeruginosa need to come back to life when conditions are favorable to sustain it? In March of 2017, Akiyama et al. published their findings in the Proceedings of the National Academy of Sciences that illuminated an essential element of the resuscitation process in P. aeruginosa, a special protein known as the hibernation promoting factor PA4463. This protein aids in the preservation of the all important ribosome during dormancy. Remember, it is the ribosomes that have to survive, and be in sufficient numbers, to subsequently produce RNA and sustain the life of the cell when starvation ceases or oxygen supply returns.

Ribosomes of the prokaryotic cell are composed of the 30S subunit (pictured in yellow during exponential phase and green during stationary phase), and the larger 50S subunit (illustrated in blue), forming the 70S ribosome. While the cell must maintain the ribosome during dormancy, it must also keep the ribosome in an inhibited state to reduce the level of protein synthesis and conserve energy.

In the common lab bacteria, E. coli, Hpf acts as a hibernation promoting factor. In P. aeruginosa, PA4463 is the homolog of E. coli Hpf, and both are ribosome interacting proteins that inhibit translation by attaching to the 30S ribosomal subunit at the location where the tRNA and mRNA would bind. As seen in the image below, this is the spot where during exponential phase, an initiation factor IF-3 would bind, delaying the development of the 70S ribosome until further help is provided.

Bacterial ribosome transitioning from exponential phase, referred to as log phase, with functioning conformation, to stationary phase with Rmf and Hpf bound to the ribosomal subunits creating the 100S ribosome. Source

However, Hpf does not work alone. In E. coli, the ribosome modulation factor, Rmf, binds the 30S ribosome blocking the exit tunnel for mRNA. During this process, Rmf with the addition of the 30s ribosome, forms a 90s dimer. Hpf subsequently binds and provides stability by forming an inactive 100s ribosome consisting of the 30S, 50S, Hpf and Rmf together (as shown in the image above). Hpf as well as Rmf are common throughout the gamma proteobacteria class that comprises both E. coli and P. aeruginosa.

Akiyama et al. characterized the capacity for resuscitation after extensive nutrient deprivation in wild type (denoted in the figure as PAO1) P. aeruginosa as compared to deletion mutations of PA4463 (denoted in the figure as Hpf), Rmf, Hpf/Rmf, and RelA/SpoT (stringent response) genes. As expected, the wild type recovered fully from the starvation and stationary period. Unlike the results obtained from E. coli, Rmf mutants behaved similarly to the wild type strain. However, the Hpf mutant demonstrated a decrease in cell vitality, as well as varying cell morphology. This was found to be due to an increased delay of cell recovery versus the wild type strain.

The reduction of vitality, as well as the heterogenous morphology, was remedied by complementation of Hpf. However the colonies were smaller than noted in the wild type strain. Similarly, the Hpf/Rmf double mutant showed a reduction in growth after starvation, and was brought back to wild type levels with a plasmid containing Hpf, but not an Rmf plasmid.

The RelA/SpoT mutant performed similarly to the Hpf mutant, with a delayed growth rate compared to the wild type strain. These results reveal that Hpf as well as the stringent response are necessary for survival under nutrient deprived conditions. Rmf was shown to have no critical role in this process for P. aeruginosa.

23S rRNA is part of the 50S subunit of the ribosome, while the 16S rRNA is part of the smaller 30S subunit. The levels of these two subunits indicates the presence of functioning, non dimerized (combined) ribosomes. Akiyama et al. found that the wild type strain, as well as the Rmf mutant, did not show a reduction in either 23S or 16S rRNA. However, the Hpf mutant had a drastic decrease in both the 23S and 16S levels over a 4 day period that was reversed by Hpf complementation.

The ribosome is composed of multiple molecules bound together. This simplified image shows the 50S subunit containing the 23S and 5S components, as well as the 31 proteins. Also illustrated are the 16S component and 21 proteins that go into the 30S subunit. Source

Similarly, the Hpf/Rmf double mutant showed a decrease in these two ribosomal subunits that was again reversed by an Hpf plasmid. They further deduced that in the Hpf mutant, both 16S and 23S subunits were lowered in number. Thus, they were able to determine that Hpf is required for 23S rRNA preservation during nutrient starvation.

Akiyama et al. further found that the Hpf mutant led to an increase in non dividing cells, with 81% remaining single cells after a 4 day period of starvation. 98.7% of the wild type cells recovered with rather uniform, therefor normal, growth. While 84.4% of the Hpf mutants did not recover, and those that did had a heterogenous morphology due to the lag time of certain colonies growth. These findings indicate that without Hpf, cells are not able to grow normally.

While the RelA/SpoT mutant showed a reduction in growth, it did not have a reduction of the 23S rRNA as compared to the wild type strain. When an Hpf plasmid was introduced to the RelA/SpoT mutant, there was no observable effect on the initial phenotype. They concluded that Hpf is not solely regulated through (p)ppGpp, the product of the stringent response.

The authors illuminate an important feature for evolutionary studies and for medicinal application, that starvation response mechanisms could be nutrient specific. In previous studies, Rmf was shown to be transcribed into mRNA in high levels in P. aeruginosa biofilms, and in E. coli, Rmf is important for ribosome inactivation during the dormant stage. However, it does not appear to be a critical component of P. aeruginosa dormancy or resurrection. Furthermore, Hpf known to be regulated by at least one upstream promoter, and it is highly probable that it is under the influence of many other promoters immediately before it. Therefore, there are multiple locations for initiation of transcription under varying conditions.

There is no simple way to stay alive, and needless to say, there is no simple solution to going dormant and revitalizing oneself. Future studies concerning the resuscitation rates of P. aeruginosa from varying nutrient mediums should provide further insight into how organisms, including their macro and micro molecules, interact with the environment. A future study may seek to somehow mimic the conditions of the lungs during and after antibiotic treatment. This may provide a more encompassing view of human infection. Perhaps in the near future, with the further development of this research, as well as novel procedures such as presented by bacterial plasmid therapy, we will have the silver bullet needed to end the infection and put the undead P. aeruginosa to bed.

More Information: Akiyama et al. (2017) Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation. PNAS 114: 3204. https://doi.org/10.1073/pnas.1700695114