NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

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

Acquisition of Host Plasmin Activity by the Swine Pathogen Streptococcus suis Serotype 2

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 ɛ-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

The Chromosomal mazEF Locus of Streptococcus mutans Encodes a Functional Type II Toxin-Antitoxin Addiction System▿ †

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 σ70-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

Biochimica et biophysica acta

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in alpha-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp~10(5)) and that they are located just below the lipid headgroups (~10A) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability

Effectiveness of an asthma integrated care program on asthma control and adherence to inhaled corticosteroids

<div><p></p><p><i>Objective</i>: To measure the effectiveness of an integrated care program for individuals with asthma aged 12–45 years, on asthma control and adherence to inhaled corticosteroids (ICS). <i>Methods</i>: Researchers used a theoretical model to develop the program and assessed effectiveness at 12 months, using a pragmatic controlled clinical trial design. Forty-two community pharmacists in Quebec, Canada recruited participants with either uncontrolled or mild-to-severe asthma. One group was exposed to the program; another received usual care. Asthma control was measured with the Asthma Control Questionnaire; ICS adherence was assessed with the Morisky medication adherence scale and the medication possession ratio. Program effectiveness was assessed with an intention-to-treat approach using multivariate generalized estimating equation models. <i>Results</i>: Among 108 exposed and 241 non-exposed, 52.2% had controlled asthma at baseline. At 12-months, asthma control had improved in both groups but the interaction between study groups and time was not significant (<i>p</i> = 0.09). The proportion of participants with good ICS adherence was low at baseline. Exposed participants showed improvement in adherence and the interaction between study groups and time was significant (<i>p</i> = 0.02). <i>Conclusion</i>: An integrated intervention, with healthcare professionals collaborating to optimize asthma control, can improve ICS adherence.</p></div