Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Abstract

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD 50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD 50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. Molecular & Cellular Proteomics

Global dynamics of Escherichia coli phosphoproteome in central carbon metabolism under changing culture conditions

“…Since then, investigations have flourished; reports have emerged of S/T/Y phosphorylation in Klebsiella pneumoniae [26], Pseudomonas spp. [27], Streptomyces coelicolor [28,29], Streptococcus pneumoniae [30], Mycoplasma pneumoniae [31], Listeria monocytogenes [32], Helicobacter pylori [33], Clostridium acetobutylicum [34],…”

Bacterial Protein-Tyrosine Kinases

“…In Bacteria, occupancy of protein-tyrosine phosphorylation sites is also very low, to the extent that this type of phosphorylation has not been identified at all by traditional proteomics approaches using 2D-gels and low-resolution mass spectrometry [42]. Only recently, the advent of gel-free analysis coupled to high-resolution mass spectrometry enabled us to systematically detect phospho-tyrosines in bacterial phosphoproteomes [27,[43][44][45]. In terms of the number of identified phosphorylation sites, phospho-tyrosines account for 3-10% of published bacterial phosphoproteomes and this is a considerable over-representation compared to the eukaryal systems.…”

“…Examples of BY-kinases phosphorylating enzymes involved in the synthesis of sugar polymers have rapidly emerged in other bacterial systems. S. thermophilus phosphoglycosyltransferase EpsE is phosphorylated and activated by the cognate BYkinase EpsD [25], Staphylococcus aureus BY-kinase Cap5B2 can phosphorylate and activate an endogenous UDP-acetyl-mannosamine dehydrogenase Cap5O [26], whereas tyrosine-phosphorylation of Klebsiella pneumoniae undecaprenolphosphate glycosyltransferase WcaJ has been reported as necessary for capsular polysaccharide synthesis [27]. The emergent phosphorylation networks based on BYkinases and their substrates hold promise of significant complexity.…”

Low Molecular Weight Protein Tyrosine Phosphatases Control Antibiotic Production in Streptomyces coelicolor A3(2)

“…However, over the last two decades evidence for thr/ser/tyr phosphorylation in bacteria has emerged [5]. Through recent studies that have focused on deducing the entire phosphoproteomes of bacterial species the existence of proteins phosphorylated on thr/ser/tyr has been well established [6][7][8]. Bacteria possess kinases and phosphatases that structurally resemble their eukaryotic counterparts [9] but have also developed idiosyncratic kinases and phosphatases without known homologues in eukaryotes [7].…”

“…Among the different classes, LMW-PTPs have been most often shown to control important physiological events. In Staphylococcus aureus and Klebsiella pneumonia these phosphatases are involved in production of a capsular polysaccharide, as a mechanism to overcome host defense mechanism during infection [6], while in B. subtilis LMW-PTPs are involved in the stress response [12] and production of teichuronic acid [13].…”