| Bacterial pathogenesis relies on the activity of effector protein that are either transferred into target cells or secreted into the extra-cellular environment. During those processes, bacterial pathogenesis should furnish nutrition, at the same time, should resist the clearance of host cell immune system. In Gram-negative pathogenic bacteria, the presence secretion systems evoking cell death are widespread in bacteria, serving a lot of different functions, such as phage defense systems, in virulence and biofilm formation, or as weapons in the interspecies competition and to ensure stable maintenance of genetic elements. Most of these systems are classical two-component systems in which one is an enzyme that interferes with cell vitality and can kill bacteria and the other component is a counteracting protein that can inhibit the toxic activity inducing cellular suicide (immunity protein or antitoxin).Up to now, seven types of secretion machines, termed T1SS-T7SS (type Ⅰ to type VII secretion systems) respectively, have been identified in the Gram-negative bacterial pathogens of animals or plants. One-step secretion systems, including T1SS, T3SS and T4SS, serve as directly conduits through the bacterial cell envelope. Two-step secretion systems, including T2SS and T5SS, require that a protein substrate first translocated across the inner membrane via either the Sec translocon or the twin-arginine translocation (Tat) pathway. Then, their N-terminal signal sequences are removed and delivered to the extracellular milieu. But the knowledge of T6SS, we cannot get enough and clearly. A minimal T6SS apparatus is formed by13conserved core components and other proteins assemble in the entire injection machinery. The conserved core components share similar structure with proteins of bacteriophage injection systems, indicating that T6SS is maybe derived from phages. Most importantly, the T6SS from the Gram-negative, pathogenic bacterium Pseudomonas aeruginosa was shown to inject at least three different effector molecules (Tse1, Tse2, and Tse3) into the periplasmic space of host cells. Tsel was shown to be an amidase that cleaves the c-D-glutamyl-L-meso-diaminopimelic acid amide bond of crosslinked of the peptide bridge of peptidoglycan, and Tse3is a muramidase that cleaves the peptidoglycan backbone between the N-acetylmuramic acid and the N-acet-ylglucosamine moieties. Tse2is a protein in P. aeruginosa which functions by a yet unknown toxic mechanism to induce host cell arresting of growth. Tse main physiological function is providing a competitive advantage for P. aeruginosa during fierce niche competition. Notably, Tsel and Tse3remain in the cytosol of the host cell and are separated from the PG in the cell membrane. As injection of effector proteins by the T6SS would also cause cell death of itself, P. aeruginosa co-synthesizes the cognate immunity proteins Tsi1and Tsi3(Type-6-Secretion-Immunity) and locates them into their periplasmic space to inactivate Tse1or Tse3.Our structural, and biochemical analyses of Tsel provide molecular insights into the specificity of this effector against Gram-negative bacteria and establish a general framework for defining the target range of the T6SS. Up to our study published, the structure of Tsi2has been solved. Crystal structure of Tsi2revealed that it functions as dimmer with stable helix model In the present paper, we report the crystal structures of Tse1and the Tse1-(6-148)-Tsi1-(20-end) complex at1.4A and1.6A (1A=0.1nm) resolutions respectively. The Tsel structure adopts a classical papain-like α+β fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tsel by structural comparison and mutagenesis studies. Through the structural and functional research about Tsel and Tsel-Tsil complex, we get the following results.1) The structure of Tsel reveals that it is a member of the NlpC/P60family, with an evolutionarily conserved catalytic centre and a primitive structural core. Tsel is considered to be a toxic component for the rival cells, and has no other regulative domains which may regulate their activity at different spatial and temporal terms, and, therefore, it is more promiscuous and destructive.2) Tse1has a unique characteristic which distinguishes it from other family members. It has four anti-parallel β strands compared with five in the NlpC/P60family. Instead of consisting of the core structure of the protein, the fifth β strand in Tse1becomes a long loop residing in petal2which interacts with Tsil in bacterial self-protection.3) Tsel adopts a deep and long Y-shaped active-site groove, but other NlpC/P60 member show a narrow and shallow V-shaped groove. The third residue orienting the catalytic histidine residue is a cysteine residue which has not been observed in this protein family before, although the function of cysteine seems to be dispensable. Thus the reaction centre in Tsel becomes a catalytic diad, compared with the classical catalytic triad in NlpC/P60family.4) There is an insertion which forms one helix following the conserved Gly67, which is not observed in the rest of the family members. These characteristics indicate that Tse1originated from the NlpC/P60family and acquired certain mutations and insertions during evolution.5) Tsi1shares little sequence similarity with the papain-like protease inhibitor of the cystatin superfamily.6) The structure of Tsi1is a whole β-strands fold, and three β-sheets constitute half of the β-propeller-like form. Unlike this three finger tips interaction manner, Tsi1uses a five finger tips contacting manner to bind Tsel. The middle finger tip (HI loop) directly inserts into the subsite of Tsel and interacts with the reaction centre residues His91and Cys30and, thus, it is similar to the first hairpin in the cystatin family. The JK loop (ring finger tip) and LMloop (little finger tip) are comparable with the N-terminal elephant trunk in cystatin which adopts a bstrate-like manner binding into the active site of papain-like cystein.7) The contrast between the Tse1-Tsi1complex and cystatin-peptidase (take stefin A-cathepsin H for example) complexes in binding specificity show that more hydrogen-bond network and more loop are involved. Tse1, is the NlpC/P60DL-endopeptidases are characterized as endogenous hydrolases involved in the peptidoglycan recycling process during bacterial.The other part of my thesis is expression, purification and preliminary crystallographic analysis of T6SS effector protein Tse3and Tsi3from Pseudomonas aerugmosa. |
PDF Full Text Request