Lindestam Arlehamn, Cecilia Sofie
Intracellular triggering of inflammation by the extracellular bacterium Pseudomonas aeruginosa.
PhD thesis, University of Glasgow.
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P.aeruginosa is an extracellular, Gram-negative opportunistic pathogen. One of the most important virulence factors during infection is the type III secretion system (T3SS). This system is found exclusively in Gram-negative bacteria and it forms a conduit between the bacteria and the host cell through which effector molecules can be translocated. These effectors alter the function of the host cell to promote survival of the bacterium. Infections are detected initially by the innate immune system via germ-line encoded receptors, pathogen recognition receptors (PRRs). These receptors recognise conserved microbial patterns, known as pathogen-associated molecular patterns and molecules which signal danger, danger-associated molecular patterns. PRRs are both membrane bound, such as Toll-like receptors (TLRs), and cytosolic, such as Nod-like receptors (NLRs). Some NLRs are involved in the formation of multimeric protein complexes, the Nod-signalosome and inflammasomes. These lead to the activation of NF-κB and the activation of caspase-1 and subsequent proteolytic processing of interleukin-1β (IL-1β) into its mature form. Both processes contribute to the inflammatory response following infection.
In this study we sought to elucidate whether P.aeruginosa is able to trigger cytosolic PRRs and the mechanism of this activation. Initially we studied inflammasome activation by P.aeruginosa. We demonstrated that P.aeruginosa is able to activate the NLRC4/ASC-inflammasome complex. This was found to be dependent on a functional T3SS, but independent of any effectors passing through the system. The activation was discovered by detection of processed, and thus active caspase-1 fragments, as well as by secretion of mature IL-1β.
The mechanism of the inflammasome activation was then investigated. We found that the NLRC4-dependent inflammasome activation is also dependent on extracellular potassium. An increase of extracellular potassium leads to a complete abrogation of inflammasome activation by P.aeruginosa and Salmonella. To further elucidate this finding, we investigated the leakiness of the pore formed by the T3SS in the host cell membrane. No flux of ions or small molecules could be detected in the host cell membrane following infection. However, host-membrane repair mechanisms were triggered, which could be detected by lysosomal-associated membrane protein (LAMP)-1-specific staining of the plasma membrane. We hypothesize a role for membrane potential in triggering of inflammasome activation by bacteria possessing a secretion system. Potassium-efflux has previously been identified as a activator of the NLRP3 inflammasome, but no changes in intracellular potassium could be found during this study.
The activation of the NLRC4 inflammasome by the Pseudomonal strain PA103 was shown, in this study, to be independent of flagellin. Instead, the bacterial molecule responsible for inflammasome activation was shown to be pilin. Pilin is important for attachment to the host cell and the function of the T3SS. We showed that a strain lacking pilin were still able to translocate effectors through its T3SS. However, it was unable to activate the inflammasome complex. Transfection of purified pilin into cells was shown to trigger inflammasome activation. This was found to be dependent on caspase-1 but independent of NLRC4 and ASC, which is not in agreement with the results found for live bacteria. We hypothesised that the reason for this is the delivery method used, since a T3SS and infection delivers proteins and molecules differently compared to a transfection reagent.
Finally, the role for Nod1 in infection by P.aeruginosa was explored. We could not identify Nod1-dependent NF-κB-activation using luciferase reporter gene assays. We therefore hypothesise that Nod1 does not have a role in the innate immune response to P.aeruginosa.
In conclusion, we have identified NLRC4- and ASC-dependent inflammasome activation by P.aeruginosa. This activation was shown to be dependent on a functional T3SS and the surface protein pilin, as well as extracellular potassium. This describes a novel NLRC4-activation mechanism dependent on potassium and identifies pilin as a PRR-trigger for the first time.
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