LasB and CbpD Virulence Factors of Pseudomonas aeruginosa Carry Multiple Post-Translational Modifications on Their Lysine Residues

Abstract

Pseudomonas aeruginosa is a multi-drug resistant human pathogen largely involved in nosocomial infections. Today, effective antibacterial agents are lacking. Exploring the bacterial physiology at the post-translational modifications (PTM) level may contribute to the renewal of fighting strategies. Indeed, some correlations between PTMs and the bacterial virulence, adaptation, and resistance have been shown. In a previous study performed in P. aeruginosa, we reported that many virulence factors like chitin-binding protein CbpD and elastase LasB were multiphosphorylated. Besides phosphorylation, other PTMs, like those occurring on lysine, seem to play key roles in bacteria. In the present study, we investigated for the first time the lysine succinylome and acetylome of the extracellular compartment of P. aeruginosa by using a two-dimensional immunoaffinity approach. Some virulence factors were identified as multimodified on lysine residues, among them, LasB and CbpD. Lysine can be modified by a wide range of chemical groups. In order to check the presence of other chemical groups on modified lysines identified on LasB and CbpD, we used 1-and 2-dimensional gel electrophoresis approaches to target lysine modified by 7 other modifications: butyrylation, crotonylation, dimethylation, malonylation, methylation, propionylation, and trimethylation. We showed that some lysines of these two virulence factors were modified by these 9 different PTMs. Interestingly, we found that the PTMs recovered on these two virulence factors were different than those previously reported in the intracellular compartment.

Exoproteomics for Better Understanding Pseudomonas aeruginosa Virulence

“…The presence of multiple PTMs on a same effector protein, for example LasB or CbpD, two major virulence factors in this bacterium, were also detected. Interestingly, there were above 20 phosphorylation sites identified on LasB or CbpD in the extracellular medium while no phosphorylation was detected in the intracellular medium [23,63]. Similarly, the acetylated and succinylated lysine residues pointed out between the intra-and the extracellular media were not always the same [23,88].…”

“…Interestingly, there were above 20 phosphorylation sites identified on LasB or CbpD in the extracellular medium while no phosphorylation was detected in the intracellular medium [23,63]. Similarly, the acetylated and succinylated lysine residues pointed out between the intra-and the extracellular media were not always the same [23,88]. Furthermore, for LasB, some lysine amino acids were detected modified with at least seven chemical groups (acetylation, succinylation, methylation, dimethylation, trimethylation, crotonylation, butyrylation, malonylation, and propionylation), meaning that a variety of proteoforms of LasB (and other effectors) exist.…”

“…PA0423 is annotated PasP, a serine protease that has been reported as a virulence factor in different studies on keratitis and human respiratory tract infections [76]. Recently, this protein was described acetylated or succinylated on lysine 42 in PA14 strain [23]. This PTM could play a role in the function of this protein.…”

“…Indeed, disruption of QS mostly inhibits the production of secreted proteins, resulting in the inability for the bacteria to metabolise extracellular nutrients [22]. Post-translational modifications (PTMs) on a protein may allow bacteria to answer in a short term to internal and external signals [23,24]. Their control would also prevent microorganisms from reacting to these signals and reduce their fitness.…”

“…Studies of PTMs on extracellular proteins are a research topic of interest in virulence but are less frequent than in intracellular media. Recently, Ser/Thr/Tyr phosphorylome [63], and both lysine acetylation and succinylation [23], have been investigated in the exoproteome of P. aeruginosa strain PA14. Taken together, these studies revealed that many virulence factors are modified (LasB, LasA, CbpD, azurin, or PrpL).…”