Pseudomonas aeruginosa is an opportunistic human pathogen. It is found in the cystic fibrosis (CF) lung, where it exerts chronic infection, but can also cause acute infection leading to bacteremia, necrosis, and gangrene. P. aeruginosa employs many different strategies to regulate the chronic/acute switch, signal to its neighbours, kill off competing bacteria, and evade antibiotic action.
Intrinsic drug resistance
P. aeruginosa assumes multidrug resistance through its many resistance-nodulation-division (RND) pumps. RND pumps are broad-specificity efflux pumps which extrude antibacterial agents out of the cell. Their expression is regulated by an unknown signal, which dissociates NalC from the armR locus. ArmR inhibits MexR and dissociates it from the mexAB-oprM locus. The RND pump is then expressed.
Latent pumps are RND pumps which are insensitive to any signalling. An example is the mexCD-oprJ locus, which is held repressed by NfxB. Natural selection potentiates the MexCD-OprJ pump by loss-of-function mutation in NfxB.
Resistance genes can be switched on in response to the drug itself. Normal cell wall turnover naturally produces 1,6-anhydro-MurNAc cell wall fragments, which are processed and recycled by AmpD in the normal case. In the normal case, the β-lactamase gene ampC is repressed by AmpR. In the presence of β-lactam antibiotics, cell wall degradation increases and AmpD is overwhelmed by 1,6-anhydro-MurNac fragments. It cannot process them all, so they are allowed to bind AmpR, switching it from a transcriptional repressor to a transcriptional activator of ampC. The β-lactamase AmpC is expressed and degrades the β-lactam.
P. aeruginosa chelate iron using siderophores. Siderophore biosynthesis clusters are regulated by the cellular concentration of iron. The ferric uptake regulator (Fur) acts as a sensor for this regulator. When cellular iron is high, Fur represses the transcription of the pyoverdine biosynthesis cluster σ factor PvdS. When cellular iron is low, Fur does not do this and the pvd cluster is expressed. Pyoverdine shuttles back and forth between the cell exterior and the periplasmic space, scavenging iron from its surroundings. Pyochelin moves between the cell exterior and the cytoplasm.
Type III secretion is used to inject toxins into host cells during acute infection. Type VI secretion is used to wipe out the competition from other bacteria during a chronic infection.
P. aeruginosa possess three quorum sensing systems, which are interlinked with each other and to regulation of biofilm formation. las and rhl are most similar to the canonical lux pathway in that the autoinducer synthase (LasI/RhlI) produces the quorum sensing molecule which is bound by LasR/RhlR. The third quorum sensing system in P. aeruginosa is the Pseudomonas quinalone signal (PQS), which is synthesised along a biosynthetic cluster. The final precursor to PQS is called HHQ; the final step for HHQ conversion into PQS is catalysed by PqsH. HHQ is more diffusible and so works well as a quorum sensing molecule; PQS induces a larger downstream signal as it is the more tightly bound by PqsR. However, there is evidence that PQS itself is transmitted between cells by means of extracellular vesicles.
Activated LasR feeds into both rhlR and pqsH transcription to coordinate switching into biofilm production for a chronic infection. Activated RhlR switches on rhamnolipid biosynthesis genes (see below), among other targets; while activated PqsR turns on pqsE for increased virulence.
Biofilms and cystic fibrosis
P. aeruginosa attack the CF lung by attaching onto the lung epithelium and producing a thick biofilm. Biofilms confer intrinsic resistance to antimicrobials as their thickness retards their movement into lower layers, and nutrient-poor, excretion product-rich regions can act to antagonise their action. Additionally, biofilms can promote the differentiation of cells to take on a resistant phenotype.
Biofilms are formed by reversible and then irreversible attachment of P. aeruginosa onto the substratum, followed by formation of mushroom structures. Mushrooms rupture, causing massive dispersal of cells to nucleate a new biofilm at another site. Flagella- and type IV pili mutants are unable to form a biofilm: flagella are needed to swim to explore a new site and establish the mushroom stalk; pili are needed to twitch to establish the mushroom cap.
The formation of biofilms is regulated with the acute/chronic switch via a two-component signalling system. Input signals cause phosphorylation of membrane-bound GacS, which transfers it phosphoryl group onto diffusible GacR. Phospho-GacR turns on the genes for the small RNAs rsmX, Y, and Z. These small RNAs bind and inhibit pro-plankonic (acute infection) RsmA, repressing type III secretion system expression and promoting biofilm formation, chronic infection, and type VI secretion system expression.
GacS is regulated positively and negatively by the homologous proteins LadS and RetS respectively. LadS transfers a phosphoryl group onto GacS, thus activating the pathway, while RetS binds GacS as a dominant-negative. (GacS functions normally as a homodimer.)
A second input comes from cyclic di-GMP (c-di-GMP), which is created by WspSR and destroyed by SadSR two-component systems, whose input signals are unknown.
The GacS system feeds positively into the LasR system. This upregulates rhamnolipid biosynthesis via RhlR. Rhamnolipids are used as a biological surfactant, aiding sliding of cells to find new nucleation sites for biofilm formation. Rhamnolipids are also used as a biological detergent, keeping channels clear of dirt.
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