Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1

Abstract

Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.

Evaluation of the Synergistic Effect of Amikacin with Cefotaxime against Pseudomonas aeruginosa and Its Biofilm Genes Expression

“…Moreover, Suriyanarayanan et al [58] mentioned that the effects of fliC phosphorylation on biofilm attachment and dispersal led to two conclusions. Both initial attachment and detachment during the dispersal stage were delayed by the loss of fliC phosphorylation in static and dynamic flow biofilms.…”

Exoproteomics for Better Understanding Pseudomonas aeruginosa Virulence

“…Flagellins seem to be modified by a diversity of PTMs. It was detected, in the PAO1 strain, phosphorylated on Thr, Ser, and Tyr residues [93,94]. When no phosphorylation is present, the level of LasB, a T2SS-secreted protein, is increased.…”

Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

“…Beyond the necessity of filaments for bacterial attachment, they may affect biofilm formation through certain specific functions. In the P. aeruginosa PAO1 strain, the absence of phosphorylation at the conserved T27 and S28 residues of FliC does not affect swimming motility, but it does affect the secretion levels of extracellular proteases by the type 2 secretion system (T2SS) and biofilm formation [ 183 ]. Residues T27 and T28 are conserved in the N-terminal domain of flagellin across Pseudomonas sp., Escherichia sp.…”

“…The FliC-T2SS interaction is evident during biofilm growth of P. aeruginosa . Taken together, these data suggest that FliC phosphorylation could act to integrate environmental cues with the signals for attachment or dispersal of biofilms playing a key role in ecological adaptation of this opportunistic environmental pathogen [ 183 ].…”