Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB inStaphylococcus aureus

Abstract

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn 2؉ as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.The phosphorylation of proteins is a key regulatory mechanism in the signal transduction pathways of both prokaryotes and eukaryotes. Typically, extracellular signals are translated into cellular responses. The phosphorylation of proteins is carried out by specific protein kinases and is coupled to dephosphorylation reactions catalyzed by protein phosphatases. In prokaryotes sensing of extracellular signals and transduction of information are usually mediated by two-component signal transduction systems consisting of histidine kinase sensors and their associated response regulators (42). In contrast, signal transduction in eukaryotes occurs via phosphorylation of serine, threonine, and tyrosine residues. Serine/threonine and tyrosine kinases and phosphatases control reversible phosphorylation of target proteins in eukaryotes and are essential for cell cycle control and differentiation (17,19).It has recently been shown in a number of studies that eukaryotic-type serine/threonine protein kinases (STPKs) and phosphatases are also expressed in many prokaryotes (2). Prokaryotic STPKs regulate various cellular functions, such as stress responses, biofilm formation, sporulation, and metabolic and developmental processes (20,23,30,34,37,39,46). STPKs also play a role in the virulence of many bacterial pathogens, such as streptococci, Mycobacterium tuberculosis, Yersinia pseudotuberculosis, and Pseudomonas aeruginosa (11,16,21,36,47). Although the functional roles of protein kinases have been described in previous studies, only a small number of target substrates have been identified so far. Moreover, the impact of phosphorylation and dephosphorylation of target protein functions has been investigated in only some cases (33,38).A single STPK has been found to be conserved in all sequenced strains of Staphylococcus aureus. Origi...

A Novel Mode of Regulation of the Staphylococcus aureus Catabolite Control Protein A (CcpA) Mediated by Stk1 Protein Phosphorylation

“…Stk1-mediated Phosphorylation of CcpA-The S. aureus genome encodes one Ser/Thr protein kinase named Stk1 or PknB (16,18). Although Stk1 appears to be involved in different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence (17)(18)(19)(31)(32)(33), only a little is known about the nature of the corresponding substrates.…”

“…Although Stk1 appears to be involved in different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence (17)(18)(19)(31)(32)(33), only a little is known about the nature of the corresponding substrates. Because of our interests in Ser/Thr kinase regulation in S. aureus and in central metabolic pathways of this pathogen, we wished to investigate whether the CcpA might be regulated by Ser/Thr phosphorylation in this organism.…”

“…Recent studies provided the first insights into the regulatory function of Stk1 and the molecular mechanisms of the Ser/Thr phosphorylation system in S. aureus (17). Interestingly, the Ser/Thr kinase Stk1 was identified to influence central metabolic processes in S. aureus (18) and the activity of important regulatory factors such as SarA (19), MgrA (20), and LuxS (21). Therefore, we investigated the possible regulation of CcpA by Stk1.…”

Implication of the NorB Efflux Pump in the Adaptation of Staphylococcus aureus to Growth at Acid pH and in Resistance to Moxifloxacin

“…Although acid stress responses do not include increases in major virulence factors or biofilm formation, there were noteworthy changes in several global regulators that control the expression of virulence genes and drug efflux transporters (5), such as the SaeRS and ArlRS, two-component regulators, the Ser/Thr kinase PknB, and its phosphatase Stp1. These global regulators have been shown to be important in modulating virulence factor and drug transporter expression (9,11,22,23,26). Other regulators exhibit increased expression at pH 4.5, including the transcriptional regulator of the iron regulator Fur family (SACOL1541), the histidine kinases PhoP and KdpD, and the VraS protein of the cell wall stimulon response (10,14,16).…”

Phosphorylation controls the functioning ofStaphylococcus aureusisocitrate dehydrogenase – favours biofilm formation

“…PknB is also implicated in the cell wall and polysaccharide intercellular adhesin (PIA) synthesis, functioning of various metabolic pathways, resistance to antibacterial agents and autolysis which are all required for biofilm formation. This pathogen survives in the human host mostly as biofilms contributing resistance to several antibiotics making S. aureus as a multidrug resistant strain 11,[15][16][17][18][19][20] . Further, in this organism, PknB is actively present in the external secretions, and along with phosphatases they interact with vast range of host tissue proteins aiding this pathogen to colonize in any anatomical locales in human host 21,22 .…”