Introduction
Even though you may not have heard of it, Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent Gram-negative bacteria, both in the environment and in its association with serious illness. These encapsulated, motile, rod-shaped bacteria have a predilection to moist environments, and so are primarily found in soil, water, and vegetation. However, they can also survive in hospital environments, leading to it being the fourth most commonly isolated nosocomial (meaning hospital-originated) pathogen.
The incredible versatility of P. aeruginosa is what allows it to adapt to these different surroundings. It is an aerobic organism, but can grow anaerobically if nitrate is present as an electron acceptor. Its incredible resilience means that in low nutrient availability, it can use ammonium and carbon dioxide as its nitrogen and carbon sources rather than its preferred source of amino acids. Meanwhile, its generation time is a speedy 30 minutes when placed in a rich media at 30 to 37°C, but it can still grow within a temperate range of 20 to 42°C. This high capacity to adapt may be due to P. aeruginosa’s large genome of 5.5 to 7 Mb, which may have many genes it can activate in specific situations. In fact, 8% of the genome are regulatory genes, which tightly control when the genes are expressed – this is important as expressing all the genes at once would be very energetically draining.
As well as being widespread in nature, P. aeruginosa is an opportunistic pathogen, meaning that it attacks those who are already medically compromised in some way. It is more common in those that are immunocompromised, are suffering from cancer, traumatic burns, or, as we will discuss in more detail, have a genetic condition known as cystic fibrosis. Along its evolutionary path, P. aeruginosa has adapted to become naturally resistant to a range of antibiotics. This, paired with its extraordinary survival skills, have cemented its place as a pathogen of great importance.
Acute vs. chronic infection
There are two forms of infection by P. aeruginosa, acute and chronic, and which one occurs depends on the environmental conditions the bacteria find themselves in. More specifically, regulatory gene networks respond to a global context of signals, not just one single stimulus, to which P. aeruginosa will respond accordingly.
Acute
In an acute infection, P. aeruginosa is being actively virulent (causing extremely severe disease), switching on genes encoding effectors that are capable of worming their way into host cells, wreaking havoc as they go. The aggressive behaviour of the bacteria results in the death of many host cells and the destruction of epithelium, a protective layer of cells covering surfaces.
Two key determinants for this violent lifestyle are the type II and type III secretion systems (abbreviated as T2SS and T3SS, respectively). Secretion systems are proteins that enable bacteria to secrete proteins that can carry out their functions outside of the cell and so are extremely pathogenically useful.
T2SS
The T2SS is the most common type of secretion system in Gram-negative bacteria, transporting proteins from the periplasm to the outside environment. There are components located in both leaflets of the bacterial membrane: an ATPase embedded in the inner membrane provides the energy for secretion through a channel in the outer membrane.
The T2SS produces and secretes multiple toxins, including exotoxin A, from the bacterium into the host cell. Exotoxin A has three domains: receptor-binding, translocation and catalytic. The translocation domain allows the toxin to be recognised by the T2SS and leave the bacterial cell. Once the exotoxin has been secreted from the bacterium, the receptor-binding domain binds the appropriate receptor on the target host cell. Once in the cytoplasm, the catalytic domain modifies a host protein called elongation factor 2. This modification results in abnormal protein translation in the host cell, which induces apoptosis.
Figure 1: The Type II Secretion System. Exotoxin A (ExoA) is secreted by the bacterial cell into the extracellular space, where it finds its receptor on a host cell plasma membrane and uses it to enter the host cell and modify EF2, ultimately leading to apoptosis.
T3SS
The T3SS is slightly more complex than the T2SS – it is a macromolecular system resembling a needle which extends out of the bacterial cell membrane and stretches all the way to the host cell’s plasma membrane. It injects four toxins straight into the target cell, where they get activated and then subvert cell signalling.
These four toxins are known as ExoS, ExoT, ExoU, and ExoY. ExoU is the only one with phospholipase activity, which allows it to disrupt the host cell membrane causing rapid cell death. This makes it the most potent and toxic effector. Meanwhile, ExoS and ExoT target the Ras family of small GTPases, causing widespread damage including but not limited to actin disruption, DNA synthesis inhibition, and reduction in cell to cell adherence. On the other hand, ExoY is, unfortunately, still poorly understood.
Figure 2: The Type III Secretion System. This diagram illustrates how the system’s apparatus is able to stretch over space from the bacterial cell to the host cell, where a set of proteins called the translocation apparatus injects the host cell and allows bacterial toxins to enter.
Chronic
Bacteria are often viewed as singular, planktonic, fully independent cells, happily floating around their environment individually. However, in reality, they much more commonly exist in a very different type of lifestyle that you might not recognise, which is called the biofilm.
Figure 3: A bacterial population in a biofilm.
A biofilm is a population of bacteria encased in a slimy, extracellular, auto-produced (meaning self-created) matrix, adhering with each other, and often also to a surface. Forming a biofilm is P. aeruginosa’s primary way of establishing a chronic infection, as they are incredibly difficult to eradicate. Their size and density give them mechanical resistance to flow, allowing them to persist even in areas where fluids are passing through. This allows them to reside in locations such as catheters and ventilators, potentially infecting already weak patients. Biofilms are also structurally organised – microcolonies within the biofilm are separated by aqueous channels, which allow the removal of waste and circulation of nutrients, allowing the biofilm to become sophisticated and established in a location.
The encasing matrix provides protection against various environmental stresses such as UV light, extreme pH, and free radicals. Furthermore, it also restricts antibodies and antibiotics from accessing the individual bacterial cells. As a matter of fact, the matrix is so effective at sealing the bacteria from the outside world that even adding 100 times the minimum inhibitory concentration of an antibiotic, which would usually kill planktonic bacteria, still won’t eradicate the biofilm. Even if an antibiotic was able to worm its way down into the slimy confines of the structure, it wouldn’t be able to diffuse homogenously through the bacterial population – so in some areas, the concentration of antibiotic will not be sufficient to destroy cells. Contrastingly, a sub-lethal concentration can lead to selection for resistance within the bacterial population.
The story doesn’t end there. Once a single bacterium has evolved resistance, these genes will be easily and favourably transferred to the rest of the population, making the biofilm even harder to remove. In addition, there is large metabolic heterogeneity within the biofilm. This means that the amount of chemical processes occurring is at different levels throughout the structure. For instance, the concentration of oxygen and nutrients is low at the bottom, meaning that many metabolic processes are not taking place. Given that antibiotics often target metabolic processes, this makes the biofilm incredibly resilient.
Figure 4: A diagram showing the five stages of biofilm formation, as explained below. The orange line at the bottom marks a surface, the blue bacteria represent P. aeruginosa, and the yellow area encasing the bacteria in the latter three stages is the extracellular matrix.
1. Initial attachment
Planktonic (meaning free-living) bacteria get transported to a surface by their flagella. The flagella also attach the bacterium to the surface, but reversibly – they are still able to leave at this stage. Different environmental cues will direct flagella to nearby surfaces.
2. Irreversible attachment
Adhesins on the bacteria, such as type IV pili and fimbriae mediate its irreversible attachment to the chosen surface. As multiple bacteria congregate, their pili will retract, pulling them together so they adhere and form a microcolony (a small colony/mass of bacterial cells). Multiple microcolonies may be forming on the surface – but at this stage, they will be far apart and have not yet united into a consistent biofilm population.
3. Early maturation
The microcolonies have grown in size and become one dense, homogenous population. At this stage, production of the matrix – which is primarily composed of exopolysaccharides – begins, to encase all of the bacteria and ‘glue’ them together.
Exopolysaccharides – as the name suggests – are polysaccharides that are secreted into the environment by their producer. The four main exopolysaccharides contributing to biofilm formation are:
Psl, which is mannose-rich
Pel, which is glucose-rich
Alginate, which is an acetylated polysaccharide composed of non-repetitive monomers
Extracellular DNA (eDNA), which is produced through the autolysis of selected cells and is very viscous
4. Maturation
Denser areas of bacteria start to protrude from the surface of the biofilm, forming mushroom-like structures. Bacteria can climb up to the top of the ‘mushroom’ by using their type IV pili. The aqueous channels which distribute nutrients and discard waste also form in between the mushrooms, finalising the formation of the biofilm.
5. Dispersal
The biofilm is not a static population – this means it can grow and shrink in size. Occasionally, some bacteria will be freed to explore the environment in search for more surfaces to colonise. To do this, the extracellular matrix is cleaved by alginate lyase and glycoside hydrolase, allowing the mushroom forms to disaggregate slightly and bacteria to leave. Alginate lyase degrades the alginate in the matrix by catalysing the breakage of the bonds between the monomers. Glycoside hydrolase works in a similar way, catalysing the hydrolysis of bonds in Psl and Pel. Once this has taken place, the formation cycle will then reinitiate to reform the part of the biofilm that has been disrupted.
Switching between acute and chronic lifestyle
There is no universal genetic programme that will switch bacteria from a planktonic lifestyle to a biofilm – the formation can be driven by many different determinants. However, there are three major regulatory systems which control the switch between the acute and chronic lifestyles of P. aeruginosa.
Quorum sensing
Quorum sensing is a type of signalling that bacteria carry out in order to evaluate their environment, and which responds to the bacterial population density.
Two particular proteins are involved in quorum sensing in P. aeruginosa: a regulator and a synthase enzyme. The enzyme, LasI, produces compounds known as N-acyl homoserine lactones (AHL), which are able to diffuse in and out of cells. LasI has a basal level of activity in the bacteria, resulting in a constant basal level of AHL. In high density populations, there is more bacteria and therefore more LasI, leading to a greater concentration of AHL.
When AHL reaches a threshold concentration, LasR (the regulator), can detect and bind to it. This binding activates LasR to bind to the promoter region of the LasI gene, hence causing even more LasI and therefore AHL production. This process is called autoinduction, as AHL is responsible for its own production. Therefore, AHL is known as an autoinducer. Through this signalling, the P. aeruginosa is aware of how many other bacterial cells are nearby and if there are enough to overwhelm the host’s immune system.
Crucially, LasR doesn’t just act on the LasI gene – it can also bind promoter regions of genes that encode virulence factors, which is how quorum sensing affects the lifestyle of P. aeruginosa. For example, exotoxin A is bound by LasR, as is RhlR, which is the regulator for the Rhl quorum sensing system. This system works in the same way as the LasR and LasI quorum sensing – but the regulator and enzyme are RhlR and RhlI instead. RhlR then binds genes that make up components of the T2SS, a key factor for the acute lifestyle, as well as further genes.
Figure 5: A diagram illustrating quorum sensing in P. aeruginosa. In situations with low cell density, there is a small amount of AHL in the environment due to the basal level of activity from the LasI enzyme. AHL is able to diffuse in and out of the cells, but the concentration of AHL is not high enough for the regulator, LasR, to be able to detect and bind to it. Therefore, LasR is also unable to bind to the promoter region of the LasI gene, meaning the level of LasI enzyme in the cell stays the same. Whereas in high cell density populations, the level of AHL in the environment is higher due to there being more bacterial cells in close proximity. The concentration of AHL has passed the threshold required for LasR to detect and bind it. When AHL is bound to LasR, LasR can bind to the promoter region of the LasI gene and increase production of LasI. With more LasI enzymes, the bacteria can then produce even more AHL. AHL-bound LasR will also be able to bind the promoter region of genes involved in the virulent lifestyle of P. aeruginosa.
Gac/Rsm cascade
The Gac/Rsm cascade is an example of a two-component regulatory system. This involves a stimulus interacting with a sensor, activating the autophosphorylation of the sensor (meaning that the sensor phosphorylates itself). The phosphate group then gets transferred to a response regulator, which binds promoters and activates a gene in answer to the original stimulus. In the case of P. aeruginosa, the exact signals that stimulate the sensor, GacS, are unknown and complex.
Figure 6: GacS, RetS and LadS are sensor kinases, with GacS being the main sensor. If LadS is activated, this also activates GacS. In contrast, the activation of RetS inhibits the activation of GacS. A central cascade started by the activation of GacS results in the phosphorylation and activation of GacA, the response regulator. GacA then activates two small regulatory RNAs known as RsmZ and RsmY. These small RNAs interact with and sequester RsmA, the translational regulator for the genes that control infection. A translational regulator controls how much of a protein is synthesised in a cell. If RsmA is sequestered, it will not bind to its target DNA; the ribosomal binding site (RBS) on the DNA will be free to be bound by the ribosome and translated. As a result, these genes promote chronic infection. Therefore, if the cascade wasn’t activated and RsmA was not sequestered, it would bind to the RBS and repress translation of the gene. The genes associated with the chronic lifestyle would then not be expressed, and the acute lifestyle would be activated.
c-di-GMP signalling
Cyclic di-GMP (c-di-GMP) is a secondary messenger that is used in multiple signalling cascades within many Gram-negative bacteria, and in P. aeruginosa, its concentration is key in determining whether a biofilm is formed or not.
A high c-di-GMP concentration is associated with mutants that have a knock-out of the RetS gene. RetS, as we discussed in the section on quorum sensing, represses biofilm formation. Exopolysaccharides, which form the biofilm matrix, rely on c-di-GMP as it activates the transport system for them. The effects of c-di-GMP are widespread and manifold, but the trend is that a high concentration in c-di-GMP results in the promotion of a chronic, persistent infection of P. aeruginosa.
Deep dive - P. aeruginosa in patients with cystic fibrosis
Cystic fibrosis (CF) is a recessive genetic condition in which the CFTR gene of individuals is defected. The CFTR gene encodes a chloride channel which sits in the plasma membrane of epithelial cells, and there are a number of possible mutations of the gene which result in CF – whereby the type of mutation determines the severity of the condition.
CF targets many organs, but primarily affects the lungs, causing them to be covered in a dehydrated, viscous layer of mucus. Efficient ion transport and beating of cilia for mucus movement requires a hydrated environment, so in a CF lung, the cilia aren’t moving. With that in mind, the immobilised cilia can result in a potentially fatal infection, as any pathogens that enter the lungs don’t get swept away.
P. aeruginosa is one such pathogen, and in a CF patient, it can prove deadly. In fact, by the time they reach 20 years old, 80% of CF patients will have a chronic P. aeruginosa infection. 90% of CF sufferers will die from respiratory failure due to established infections. However, the viscous mucus is not the only reason individuals with CF are more likely to be infected with P. aeruginosa. The cells lining the lungs of patients present higher levels of the receptor asialo-GM1 at their surface, which the type IV pili and flagella on P. aeruginosa initially use to attach to host cells and begin the process of colonisation. P. aeruginosa can also effectively fight off bacterial competition – it can induce a phospholipase and a redox-active metabolite that will kill Staphylococcus aureus, which may also be present.
The previously mentioned robustness and longevity of chronic P. aeruginosa infection means that the bacteria persist despite antibiotic treatment, and the excessive and ongoing inflammation triggered by their presence is what eventually destroys the lung. The pathogen can also survive in the upper respiratory tract, so even if a patient undergoes a lung transplant, they can become re-infected once again.
Treatment
Early detection of P. aeruginosa in CF patients allows its eradication using antibiotics. However, if the individual is re-infected it is usually systemic – meaning the infection will affect the whole body – and will persist despite antibiotic treatment. As previously mentioned, even lung transplants sometimes are not enough to cure the patient. However, if antibiotics are used, they are often used in combination to try and ensure that at least one of the drugs will be effective.
P. aeruginosa, however, has some innate (i.e. natural) resistance. Many of its pores are too small for drugs to enter through. 10 efflux pumps located in the cell membrane are able to rapidly recognise drugs and expel them out before they get a chance to do any damage, making treatment very difficult to carry out.
There are four groups of antibiotics which are still effective: aminoglycosides, which interfere with ribosomes; fluoroquinolones, which interfere with replication; polymyxins, which interfere with the cell surface; and beta-lactams, which interfere with the biosynthesis of the bacterial cell wall. CF patients will often receive multiple courses, which can be effective but if not will unfortunately result in the emergence of multi-resistant P. aeruginosa strains.
The extreme survival skills of P. aeruginosa make it an incredibly interesting organism to study, but a very dangerous pathogen to those that are susceptible. Its ability to evaluate its surroundings and vary its lifestyle accordingly are impressive, yet we still don’t understand all of its behaviour fully, and this will be crucial if we are to come up with a much-needed treatment for it. The capacity of biofilms to survive in even extreme environments poses a real threat, and there are hopes that new experimental therapeutics such as enzymes that can break down the biofilms may hold promise in the future.
Author: Sophie Ormiston, BSc Biochemistry
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