NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a 70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.

We have investigated the specific contribution of protease-activated receptor-2 (PAR 2 ) to host defense during Porphyromonas gingivalis infection. Culture supernatants from P. gingivalis strains 33277 and W50 provoked Ca 2؉ mobilization in cells transfected with PAR 2 (PAR 2 -KNRK) and desensitized the subsequent responses to PAR 2 -selective agonist. In addition, culture supernatants of P. gingivalis E8 (RgpA/RgpB double knockout) did not cause calcium response in PAR 2 -KNRK cells, evidencing the involvement of the arginine-specific cysteine proteases RgpA and RgpB in PAR 2 activation by P. gingivalis. Injection of P. gingivalis into mouse subcutaneous chambers provoked an increased proteolytic activity, which was inhibited by serine protease inhibitors. Fluids collected from chambers of P. gingivalis-injected mice were able to activate PAR 2 and this activation was inhibited by serine protease inhibitors. P. gingivalis inoculation into subcutaneous chambers of wild-type mice induced an inflammatory response that was inhibited by a serine protease inhibitor and was significantly reduced in PAR 2 -deficient mice. Finally, mice orally challenged with P. gingivalis developed alveolar bone loss, which was significantly reduced in PAR 2 -deficient mice at 42 and 60 days after P. gingivalis infection. We conclude that PAR 2 is activated on P. gingivalis infection, in which it plays an important role in the host inflammatory response. (Am J Pathol

In this study, the plasminogen-binding activity of Streptococcus suis serotype 2 was investigated. Bound human plasminogen was activated by purified streptokinase, urokinase, or Streptococcus dysgalactiae subsp. equisimilis culture supernatant. Both human and porcine plasminogen were bound by S. suis. Binding was inhibited by -aminocaproic acid, and the plasminogen receptor was heat and sodium dodecyl sulfate resistant. One of the receptors was identified as glyceraldehyde-3-phosphate dehydrogenase. S. suis-associated plasmin activity was capable of activating free plasminogen, which in turn could contribute to degradation of fibronectin. This is the first report on the plasminogen-binding activity of S. suis. Further studies may reveal a contribution of this activity to the virulence of S. suis.Streptococcus suis, an important swine pathogen worldwide, is made up of 35 serotypes (1 to 34 and 1/2) (12). Although all serotypes can cause infections, serotype 2 is the most prevalent one isolated from diseased pigs (9, 11). Septicemia, arthritis, meningitis, and sudden death are the most important clinical signs associated with S. suis infections (11). Cases of meningitis and endocarditis caused by S. suis have also been reported in workers associated with the pig industry (11). Although several S. suis virulence factors have been identified and characterized, the exact mechanisms by which this bacterium invades the host and causes infections are still unclear. The only factor considered essential for the pathogenicity of S. suis is the polysaccharide capsule (3). An important feature, which could play an important role in the pathogenicity of S. suis, is its ability to bind host proteins, which may camouflage it and thus protect it from the host immune system. Fibronectin (7), albumin (21), and immunoglobulin G (1, 23) receptors have been identified on the cell surface of S. suis and have been proposed as virulence factors.Plasminogen is a 92-kDa protein that is an important component of the fibrinolytic system. Its activation into plasmin, a serine protease, is tightly regulated by the equilibrium between plasmin activators (urokinase plasmin activator [u-PA] as well as tissue plasminogen activator) and inhibitors (␣2-antiplasmin and ␣2-macroglobulin) (15). Plasminogen can also be activated by microbial products including streptokinase and staphylokinase produced by group A streptococci and Staphylococcus aureus, respectively (15). Many group A and C streptococci bind plasminogen on their cell surfaces (15). In a number of cases, the receptors have been characterized and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ␣-enolase (17,19,20,24). The plasmin activity generated on the bacterial cell surface is protected from host inhibitors and can activate latent u-PA and tissue plasminogen activator, which subsequently activate plasminogen (15). This mechanism of acquisition of a host proteinase activity increases the invasive potential of some pathogens, including Streptococcus pneumoniae (8). The ...