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Iron-Depletion prevents biofilm formation in Pseudomonas Aeruginosa through twitching mobility and quorum sensing

Influence of iron-depletion on twitching motility and quorum sensing (QS) system in P. aeruginosa was evaluated. The results demonstrated iron-depletion can retard biofilm formation and increase the twitching motility and expression of QS-related genes, suggesting a potential interaction between twitching motility and QS system in P. aeruginosa biofilm formation.

. In patients with these underlying diseases, it can cause chronic infections characterized by the formation of biofilms.
Biofilms are composed of a group of bacteria attached to the surfaces and are encased in a hydrated polymeric matrix (4).
Bacteria that form biofilms can withstand host immune responses and are much more resistant to antibiotics than their counterparts of nonattached, planktonic bacteria (20). Therefore, infections with biofilm-forming bacteria are persistent and difficult to treat with antibiotics. P. aeruginosa possesses polar filaments called type IV pili that are involved in attachment and surface translocation by twitching motility (15). Twitching motility, mediated by pilus extension and contraction, is required for the formation of biofilms in P. aeruginosa (10,21). Quorum-sensing (QS) system in bacteria detects cell density and regulates the expression of many genes at high cell densities. Many studies demonstrated that P. aeruginosa strains lacking functional QS system are less virulent than wild-type strains and form flat, undifferentiated biofilms that are less stable than the differentiated biofilms formed by wild-type P. aeruginosa. As a consequence, QS has been suggested as a potential target for new preventive and/or therapeutic strategies of P. aeruginosa infections (6).
Iron is essential for most pathogens because iron is an indispensable component of many proteins, especially some enzymes in bacteria. Therefore, iron acquisition from environment is important for the growth and metabolism of P. aeruginosa (5). Recently, many studies revealed that iron also play an important role in biofilm formation (1,23). In vitro experiments showed both iron-depletion (<1µM) and ironrepletion (>100µM) retarded biofilm formation (14).  Previous studies suggested that twitching motility is necessary for microcolony formation and thus essential for normal biofilm development (10,15,21). However, our study showed that IV pili mutant strain PAO-pilHIJK formed almost the same amount of biofilm compared to the wild-type strain, PAO1. The most distinct difference is that those studies focused on the initial stage of biofilm development (about 8~24 h), while we studied the mature biofilm formation (7 d). This may indicate that type IV pilus-mediated twitching motility mediates aggregation of cells in the initial stage of biofilm development, however given enough time (7 d in this study), twitching motility is not required for the formation of mature biofilm. Heydorn et al. (9) also reported that biofilm formed by the PAO-pilHIJK strain contained a large number of distinct microcolonies that were regularly spaced and almost uniform in size. As the biofilms growing, the microcolonies of the PAO-pilHIJK strain coalesced, leading to the formation of larger and larger microcolonies. They suggested that microcolonies formed by the PAO-pilHIJK strain was due to the clonal growth of an individual bacterium in the biofilm because these bacteria were unable to move by twitching motility. Another study also reported that mutant strains defective in twitching motility were not impaired in the early stages of biofilm development and eventually formed mature biofilms, although the morphology were distinguishable from those of wild-type strains (12). Therefore, we hypothesize that another pathway for the formation of microcolonies might exist, and further studies on how PAO-pilHIJK strain form a mature biofilm are necessary to test this hypothesis.
Biofilm formed by PAO1, PAO-pilHIJK and PAO-JP2 strains were significantly decreased under iron-depletion condition compared with that under normal condition. We also showed that twitching motility of PAO1 and PAO-JP2 increased under iron-depletion condition. Singh et al. (19) showed that lactoferrin acted as a chelator to bind iron, leading to the twitching and wandering of bacteria across the surface instead of forming cell clusters and biofilms. These findings suggested that iron plays an important role at a critical juncture biofilm development in which bacteria stop roaming as individuals and aggregate into durable communities.
Interestingly, as mentioned before, twitching motility is crucial in biofilm formation; however, too much twitching motility prevents biofilm development.
PAO-pilHIJK strain lacks IV pili and the results showed that twitching motility did not change significantly under irondepletion or iron-repletion conditions. Thus decrease of biofilm formation of PAO-pilHIJK strain is not due to the decrease in twitching motility. We then determined whether iron-depletion has an effect on QS system, another important system involved in biofilm formation. We showed that upon iron depletion, expression of QS-related genes, lasR and rhlR were significantly increased consistent with the results obtained in other studies (5,11,23). One of the contradictory results is that iron-depletion decreased biofilm formation ( Figure 1  Based on the results from this study, we concluded that twitching motility is not a prerequisite for the formation of mature biofilm and iron-depletion can decrease the formation of biofilm at least through both twitching motility and QS. The results also indicated there might be a correlation between QS and twitching motility. Further studies on the coordination and regulation of the two key pathways will enhance our understanding of P. aeruginosa biofilm formation and identify new targets to control biofilm formation.