A Sensitive Means to Detect and Identify Potential Phosphatase Substrates
Bacterial pathogens use a variety of strategies to interact with and manipulate host cell response to their advantage, allowing them to survive and propagate (1). One such response is phosphorylation of proteins containing hydroxyl group bearing amino acids (serine, threonine, tyrosine). Phosphorylation of proteins is a complex process carried out by specific protein kinases and reversed by their counter parts, i.e., specific phosphatases belonging to either the invading pathogen or infected host cell. Acinetobacter baumannii is a nosocomial pathogen that causes ventilator-associated as well as bloodstream infections in critically ill patients. The spread of multidrug-resistant Acinetobacter strains is cause for concern (5,6). In addition, an increase in carbapenem resistance, the last line of antibiotic therapy for treatment of multidrug-resistant Gram-negative bacteria is a major concern due to their often being resistant to all other commonly used antibiotics. Therefore, multi-drug resistant A. baumannii associated infections are difficult to effectively treat. Considering this emerging threat, we have pursued characterizing an Acinetobacter baumannii Acid Phosphatase (SurE), and our long-term goal is to assess its role in pathogenesis. Acid Phosphatases (Acp) have been implicated as intracellular virulence factors that enhance pathogen survival through suppression of the respiratory burst (4). Genomic sequence analysis of Acinetobacter baumannii reveals the presence of a putative Acid Phosphatase (AcpA). AcpA has been shown to remove phosphate in vitro from an array of artificial substrates as well as nucleotides, sugars, metabolites, vitamin derivatives, phosphorylated amino acids, and peptides (3). However, removal of phosphate from artificial substrates, e.g., para-Nitrophenylphosphate does not address the issue of phosphate removal from specific protein substrate in the cellular milieu. Moreover, protein BLAST and phylogeny analyses reveal that the AcpA from A.baumannii is closely related to the well-characterized E. coli SurE protein, but distant from other non-specific Acid Phosphatases. The E. coli SurE protein is a metal ion-dependent phosphatase that dephosphorylates various ribo- and deoxyribonucleoside 5'- and 3'-monophosphates, and plays an important role in bacterial stationary phase survival. Considering that A. baumannii expresses a phosphatase protein with 69.2% amino acid similarity (43.6% identity) to that of the E. coli SurE protein, and exhibits 47.3% amino acid similarity (21.9% identity) to the AcpA, this in concert with its emerging pathogenicity has prompted our continued characterization of the A.baumannii SurE-like protein. In order to assess the role of phosphorylated proteins of A.baumannii their identification is necessary. Interrogation of the effects of these enzymes on, i.e., phosphatases on the host phosphoproteome requires a simple and sensitive means for determining the presence of phosphate as well as its subsequent removal. Therefore, I investigated whether removal of phosphate from J774A.1 murine macrophage protein lysates could be detected using PAGE and a phosphotyrosine specific antibody. Cells were stimulated with Lipopolysaccharide (LPS) and Interferon gamma (IFN-γ), and the phospho-proteome was probed for changes in levels of phosphorylated-tyrosine protein. LPS and IFN-γ are known to produce an inflammatory response in macrophages . Cell lysates were also used to assess whether A.baumannii recombinant SurE removed phosphate supporting their being potential endogenous phosphatase substrates. From this study, we conclude that Two-Dimensional PAGE and Western Blot analysis is a sensitive means to determine the presence of cell lysate phosphorylated protein. Furthermore, data presented here albeit preliminary suggest the removal of phosphate by recombinant SurE phosphatase from lysate phospho-protein substrates, the identity and function of which remain to be determined, the focus of future work.