Phosphorylation of Extracellular Proteins in Acinetobacter baumannii in Sessile Mode of Growth

Abstract

Acinetobacter baumannii is a problematic nosocomial pathogen owing to its increasing resistance to antibiotics and its great ability to survive in the hospital environment, which is linked to its capacity to form biofilms. Structural and functional investigations of post-translational modifications, such as phosphorylations, may lead to identification of candidates for therapeutic targets against this pathogen. Here, we present the first S/T/Y phosphosecretome of two A. baumannii strains, the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, cultured in two modes of growth (planktonic and biofilm) using TiO2 chromatography followed by high resolution mass spectrometry. In ATCC 17978, we detected a total of 137 (97 phosphoproteins) and 52 (33 phosphoproteins) phosphosites in biofilm and planktonic modes of growth, respectively. Similarly, in AB0057, 155 (119 phosphoproteins) and 102 (74 phosphoproteins) phosphosites in biofilm and planktonic modes of growth were identified, respectively. Both strains in the biofilm mode of growth showed a higher number of phosphosites and phosphoproteins compared to planktonic growth. Several phosphorylated sites are localized in key regions of proteins involved in either drug resistance (β-lactamases), adhesion to host tissues (pilins), or protein secretion (Hcp). Site-directed mutagenesis of the Hcp protein, essential for type VI secretion system-mediated interbacterial competition, showed that four of the modified residues are essential for type VI secretion system activity.

"The Biofilm Resistance: Many Responses but Still Many Questions"

“…Thus, Massier, et al [33] reported significant differences in the phosphorylation rates of extracellular proteins in planktonic and sessile Acinetobacter baumannii cells. These authors also demonstrated that some phosphosites were located in key regions of proteins involved in antibiotic resistance, such as in betalactamases [33].…”

“…Low penetration of the antimicrobial Decrease of the local concentration of the antimicrobial [15,16] Low growth rate Decrease of the efficacy of some antimicrobials (dormancy) [19][20][21] Metabolism alterations Accumulation of proteins involved in adaptation, target modifications [25,26] Oxygen gradient Decrease of the efficacy of some antimicrobials (intracellular penetration, dormancy) [33][34][35] Presence of persisters Decrease of the efficacy of some antimicrobials (dormancy) [37][38] Quorum sensing Activation of efflux pumps [43] Stress responses Cross-resistance against antimicrobials [50] A Diffusional Resistance to Inhibitors…”

“…These differences can of course be explained by the weak correlation between the quantity of mRNA and protein, but could also indicate the existence of key proteins which would not yet be identified because modified qualitatively and not quantitatively, via post-translational modifications such as phosphorylations and/or acetylations, for example [32]. Thus, Massier, et al [33] reported significant differences in the phosphorylation rates of extracellular proteins in planktonic and sessile Acinetobacter baumannii cells. These authors also demonstrated that some phosphosites were located in key regions of proteins involved in antibiotic resistance, such as in betalactamases [33].…”

Lysine Trimethylation in Planktonic and Pellicle Modes of Growth in Acinetobacter baumannii

“…baumannii resistance. , Recently, the phospho-secretomes of two A. baumannii strains, the reference strain ATCC 17978 and the virulent multidrug resistant strain AB0057, highlighted a higher number of phosphoproteins in the biofilm mode of growth compared to the planktonic growth . Moreover, five phosphosites were detected on the T6SS tube protein Hcp .…”

“…baumannii strains, the reference strain ATCC 17978 and the virulent multidrug resistant strain AB0057, highlighted a higher number of phosphoproteins in the biofilm mode of growth compared to the planktonic growth . Moreover, five phosphosites were detected on the T6SS tube protein Hcp . After mutagenesis, specific phosphorylated residues were clearly shown to be required for Hcp secretion and therefore T6SS activity.…”