Ruiguang Ge

Phosphoproteomic analysis reveals the multiple roles of phosphorylation in pathogenic bacterium Streptococcus pneumoniae.

Recent phosphoproteomic characterizations of Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pseudomonas putida, and Pseudomonas aeruginosa have suggested that protein phosphorylation on serine, threonine, and tyrosine residues is a major regulatory post-translational modification in bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the Gram-positive pathogenic bacterium Streptococcus pneumoniae. One hundred and two unique phosphopeptides and 163 phosphorylation sites with distributions of 47%/44%/9% for Ser/Thr/Tyr phosphorylations from 84 S. pneumoniae proteins were identified through the combined use of TiO(2) enrichment and LC-MS/MS determination. The identified phosphoproteins were found to be involved in various biological processes including carbon/protein/nucleotide metabolisms, cell cycle and division regulation. A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence.

Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post‐translational modification in the bacteria. To reveal the phosphorylation state in the Gram‐negative pathogenic bacterium Helicobacter pylori, we carried out a global and site‐specific phosphoproteomic analysis based on TiO2‐phosphopeptide enrichment and high‐accuracy LC‐MS/MS determination. Eighty‐two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over‐representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.

Crystal structure and metal binding properties of the lipoprotein MtsA, responsible for iron transport in Streptococcus pyogenes.

An ability to acquire iron is essential for the viability and growth of almost all organisms and in pathogenic bacteria is strongly correlated with virulence. The cell surface lipoprotein MtsA, a component of the MtsABC transporter of Streptococcus pyogenes, acts as the primary receptor for inorganic iron by this significant human pathogen. Iron is bound as Fe(2+), with the participation of bicarbonate. The crystal structure of MtsA has been determined and refined at 1.8 A resolution (R = 0.167, and R(free) = 0.194). MtsA has the classic bacterial metal binding receptor (MBR) fold, with the Fe(2+) ion bound to the side chains of His68, His140, Glu206, and Asp281, at a totally enclosed site between the two domains of the protein. The absence of bicarbonate from the binding site suggests that it is displaced during the final stages of metal binding. Both the fold and metal binding site are most similar to those of the manganese receptors PsaA and MntC, consistent with the similar coordination requirements of Fe(2+) and Mn(2+). Binding studies confirm a 10-fold preference for Fe(2+) over Mn(2+), although both may be carried in vivo. Mutational analysis of the binding site shows that His140 is critical for a fully functional binding site but that Glu206 is dispensable. The crystal structure explains the distinct roles of these ligands and also reveals potential secondary binding sites that may explain the binding behavior of MtsA for metal ions other than Fe(2+).

Putative copper- and zinc-binding motifs in Streptococcus pneumoniae identified by immobilized metal affinity chromatography and mass spectrometry.

The aim of metalloproteomics is to identify and characterize putative metal‐binding proteins and metal‐binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high‐accuracy linear ion trap‐Orbitrap MS to identify metal‐binding proteins and metal‐binding peptides. In total, 232 and 166 putative metal‐binding proteins were respectively isolated by Cu‐ and Zn‐immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu‐ and Zn‐binding peptides, putative Cu‐binding motifs were identified: H(X)mH (m=0–11), C(X)2C, C(X)nH (n=2–4, 6, 9), H(X)iM (i=0–10) and M(X)tM (t=8 or 12), while putative Zn‐binding motifs were identified as follows: H(X)mH (m=1–12), H(X)iM (i=0–12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma‐MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal‐binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.

Identification of Proteins Related to Nickel Homeostasis in Helicobater pylori by Immobilized Metal Affinity Chromatography and Two-Dimensional Gel Electrophoresis

Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed.

Lipoprotein MtsA of MtsABC in Streptococcus pyogenes primarily binds ferrous ion with bicarbonate as a synergistic anion

Lipoprotein MtsA is a critical component of MtsABC responsible for iron binding and transport in the Gram‐positive bacterium Streptococcus pyogenes. The present collective experimental data establish that Fe2+ is the primary binding ion for MtsA under optimal physiologically relevant conditions. The binding affinities of MtsA to metal ions are Fe2+ > Fe3+ >Cu2+ > Mn2+ > Zn2+. We report for the first time that bicarbonate is required as a synergistic anion for stable ferrous binding to MtsA, similar to the iron binding in human transferrin. This work provides valuable information, which helps to understand iron metabolism in bacteria, and creates a basis for developing strategies to suppress bacterial infection.

Iron trafficking system in Helicobacter pylori

Helicobacter pylori infections are closely associated with peptic ulcers, gastric malignancy and iron deficiency anemia. Iron is essential for almost all living organisms and the investigation of iron uptake and trafficking system is thus important to understand the pathological roles of H. pylori. Up to now, the iron trafficking system of H. pylori is not yet fully clear and merits further efforts in this regards. The available information about iron uptake and regulation has been discussed in this concise review, such as FeoB in ferrous transportation, FrpB2 in hemoglobin uptake, HugZ in heme processing, virulence factors (VacA and CagA) in transferrin utilization, Pfr and NapA in iron storage and Fur in iron regulation. The identified iron trafficking system will help us to understand the pathological roles of H. pylori in the various gastric diseases and iron deficiency anemia and stimulates further development of effective anti-bacterial drugs.

Iron depletion decreases proliferation and induces apoptosis in a human colonic adenocarcinoma cell line, Caco2.

Iron is essential for maintaining cellular metabolism of most organisms. Iron chelators such as desferrioxamine have been used clinically in the treatment of iron overload diseases. In the present study, we used human colon adenocarcinoma cells as a proliferating cell model to validate that desferrioxamine inhibits cell proliferation and induces apoptosis. Proteomic analysis revealed that proteins involved in cell proliferation, signal transduction, metabolism and protein synthesis were significantly regulated by the availability of iron, rendering a close correlation between cell apoptosis and the disturbance of mitochondrial, signaling and metabolic pathways. These results provide new insights into the mechanisms of cell proliferation inhibition attributed to iron depletion.

The low pKa value of iron-binding ligand Tyr188 and its implication in iron release and anion binding of human transferrin.

2D NMR–pH titrations were used to determine pK a values for four conserved tyrosine residues, Tyr45, Tyr85, Tyr96 and Tyr188, in human transferrin. The low pK a of Tyr188 is due to the fact that the iron‐binding ligand interacts with Lys206 in open‐form and with Lys296 in the closed‐form of the protein. Our current results also confirm the anion binding of sulfate and arsenate to transferrin and further suggest that Tyr188 is the actual binding site for the anions in solution. These data indicate that Tyr188 is a critical residue not only for iron binding but also for chelator binding and iron release in transferrin.

Inhibition of fumarase by bismuth(III): implications for the tricarboxylic acid cycle as a potential target of bismuth drugs in Helicobacter pylori

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations and gastric cancer. Triple therapy combining bismuth compounds with two antibiotics is the cornerstone of the treatment of H. pylori infections. Up to now, the molecular mechanisms by which bismuth inhibits the growth of H. pylori are far from clear. In the bacterial tricarboxylic acid (TCA) cycle, fumarase catalyses the reversible hydration of fumarate to malic acid. Our previous proteomic work indicated that fumarase was capable of bismuth-binding. The interactions as well as the inhibitory effects of bismuth to fumarase have been characterized in this study. The titration of bismuth showed that each fumarase monomer binds one mol equiv of Bi3+, with negligible secondary structural change. Bismuth-binding results in a near stoichiometric inactivation of the enzyme, leading to an apparent non-competitive mechanism as reflected by the Lineweaver–Burk plots. Our collective data indicate that the TCA cycle is a potential molecular target of bismuth drugs in H. pylori.