Cyclic-di-GMP effectors regulate type IV pili-mediated adherence and dissemination in Pseudomonas aeruginosa

To infect their human or animal hosts, bacterial pathogens need to be able to effectively colonize and breach epithelial barriers. During this process they attach to tissue surfaces, adapt their motility behavior and induce virulence factors. The opportunistic human pathogen Pseudomonas aeruginosa uses type IV pili (T4P) as primary adhesins and motility organelles to adhere to and disseminate on host tissue. How T4P are differentially regulated to coordinate distinct surface processes and behavior is not well understood. The second messenger cyclic-di-GMP (cdG) has emerged as an important coordinator in regulating pili activities during early surface sensing. Here, we analyze the regulation and activity of two cdG-dependent effectors, FimW and FimX, that differentially control T4P activity. While FimW stimulates T4P-dependent surface adherence and appears to act as a motility break, FimX was shown to promote T4P-dependent twitching motility. However, how FimW and FimX differs in their regulation on T4P activity is not known. In addition, how cdG individually controls distinct T4P functions has remained unclear. Here, we aim to identify protein interactors of FimW to investigate how FimW regulates T4P differently in comparison to FimX. In addition, we attempt to understand how FimW and FimX dynamics are differentially controlled by cdG and how this directs distinct pili-dependent behavior of P. aeruginosa on surfaces.
Our results suggest that FimW associates with T4P-assembly machinery at cell poles through interaction with the polar hub protein FimV after surface exposure. Furthermore, FimW can promote piliation and pili-dependent adherence through the FimV-homologue FimV2, while FimX activity is dependent exclusively on FimV. By correlating FimW and FimX dynamics with intracellular cdG in surface-adapted cells, we show that FimW localizes to cell poles when global cdG level is high, and this correlates with reduced surface motility, whereas FimX polar activity increases when global cdG level is low, and this correlates with increased surface motility. Thus, results suggest that effector dynamics respond differently to changes in global cdG and this drives their independent localization to the piliated pole where they can interact with different downstream components to regulate distinct pili function. It is possible that FimW and FimX are activated by different sources of cdG to balance attachment and motility. On the other hand, results suggest that FimX is also subject to additional regulation by the second messenger cyclic-AMP (cAMP) to promote pili-dependent motility when global cdG level is low.
In addition to the aforementioned results, this report includes supplementary data that investigate the function of PA5210 and PhaJ1, which were proteins identified in the FimW-interactor screen, and DgcP, a cdG-cyclase that was previously associated with FimW activation on surface. Results from this project suggest that DgcP is not responsible for cdG produced downstream of surface sensing that leads to FimW activation. On the other hand, results identify PA5210, an ATPase like protein, as a novel regulator that promotes pili function, and PhaJ1, previously characterized in the synthesis of carbon storage granules, as a novel regulator that promotes swarming motility. How FimW interacts with PA5210 and PhaJ1 remains unclear. However, these results highlight that FimW can associate with different interactors to regulate diverse surface behaviours, beyond just pili-mediated, in P. aeruginosa. Lastly, this report includes results from a CRISPR interference screen that aimed to identify genes involved in pili regulation in P. aeruginosa. Results show that by performing the screen in selective mutant backgrounds in addition to WT, genes that are active in selective pili-regulatory pathways can be isolated that would otherwise be masked by the global regulatory network.
Overall, results from this project contribute to an improved understanding of cdG-dependent regulation on T4P and cell behaviour mediated through two cdG-effectors during early surface sensing, as well as uncover new molecular components that are involved in the regulation of T4P in P. aeruginosa. Future work on this subject can aim to examine how the newly identified regulators contribute to pili regulation, and investigate how FimW and FimX-dependent regulation lead to different pili dynamics, including differences in pili length and extension and retraction rates, that promote surface attachment or motility.