- Volume 157, Issue 10, 2011

Enterohaemorrhagic Escherichia coli (EHEC), serotype O157 : H7 is a non-invasive, pathogenic bacterium that employs a type III secretion system (T3SS) to inject effector proteins into infected cells. In this study, we demonstrate that EHEC blocks tumour necrosis factor-alpha (TNFα)-induced NF-κB signalling in infected epithelial cells. HEK293T and INT407 epithelial cells were challenged with EHEC prior to stimulation with TNFα. Using complementary techniques, stimulation with TNFα caused activation of NF-κB, as determined by luciferase reporter assay (increase in gene expression), Western blotting (phosphorylation of IκBα), immunofluorescence (p65 nuclear translocation) and immunoassay (CXCL-8 secretion), and each was blocked by EHEC O157 : H7 infection. In contrast, subversion of host cell signalling was not observed following exposure to either enteropathogenic E. coli, strain E2348/69 (O127 : H6) or the laboratory E. coli strain HB101. Heat-killed EHEC had no effect on NF-κB activation by TNFα. Inhibition was mediated, at least in part, by Shiga toxins and by the O157 plasmid, but not by the T3SS or flagellin, as demonstrated by using isogenic mutant strains. These findings indicate the potential for developing novel therapeutic targets to interrupt the infectious process.

Cupriavidus necator H16 (DSM 428), whose genome has been sequenced, was found to degrade N-acetyltaurine as a sole source of carbon and energy for growth. Utilization of the compound was quantitative. The degradative pathway involved an inducible N-acetyltaurine amidohydrolase (NaaS), which catalysed the cleavage of N-acetyltaurine to acetate and taurine. The degradation of the latter compound is via an inducible, degradative pathway that involves taurine dehydrogenase [EC 1.4.2.–], sulfoacetaldehyde acetyltransferase [EC 2.3.3.15], phosphotransacetylase [EC 2.4.1.8], a sulfite exporter [TC 9.A.29.2.1] and sulfite dehydrogenase [EC 1.8.2.1]. Induction of the expression of representative gene products, encoded by at least four gene clusters, was confirmed biochemically. The acetate released by NaaS was activated to acetyl-CoA by an inducible acetate–CoA ligase [EC 6.2.1.1]. NaaS was purified to homogeneity; it had a K m value of 9.4 mM for N-acetyltaurine, and it contained tightly bound Zn and Fe atoms. The denatured enzyme has a molecular mass of about 61 kDa (determined by SDS-PAGE) and the native enzyme was apparently monomeric. Peptide-mass fingerprinting identified the locus tag as H16_B0868 in a five-gene cluster, naaROPST (H16_B0865–H16_B0869). The cluster presumably encodes a LysR-type transcriptional regulator (NaaR), a membrane protein (NaaO), a solute : sodium symporter-family permease [TC 2.A.21] (NaaP), the metal-dependent amidohydrolase (NaaS) and a putative metallochaperone (COG0523) (NaaT). Reverse-transcription PCR indicated that naaOPST were inducibly transcribed.

Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H2S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H2S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H2S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H2S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K m of 0.32±0.02 mM and a k cat of 0.69±0.01 s−1. Based on current and published data, the enzymic activity for H2S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H2S produced by Fn1419 was estimated to be 1.9 % of the total H2S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H2S production (87.6 %).