Shigella Effector IpaB-Induced Cholesterol Relocation Disrupts the Golgi Complex and Recycling Network to Inhibit Host Cell Secretion

Shigella infection causes destruction of the human colonic epithelial barrier. The Golgi network and recycling endosomes are essential for maintaining epithelial barrier function. Here we show that Shigella epithelial invasion induces fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport in the infected host cell. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting cell surface receptor recycling. The molecular process underlying the observed damage to the Golgi complex and receptor recycling is a massive redistribution of plasma membrane cholesterol to the sites of Shigella entry. IpaB, a virulence factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. Shigella infection-induced Golgi disorganization was also observed in vivo, suggesting that this mechanism affecting the sorting of cell surface molecules likely contributes to host epithelial barrier disruption associated with Shigella pathogenesis

Meropenem/colistin synergy testing for multidrug-resistant Acinetobacter baumannii strains by a two-dimensional gradient technique applicable in routine microbiology

Objectives Precise assessment of potential therapeutic synergy, antagonism or indifference between antimicrobial agents currently depends on time-consuming and hard-to-standardize in vitro chequerboard titration methods. We here present a method based on a novel two-dimensional antibiotic gradient technique named Xact™. Methods We used a test comprising a combination of perpendicular gradients of meropenem and colistin in a single quadrant. We compared test outcomes with those obtained with classical chequerboard microbroth dilution testing in a study involving 27 unique strains of multidrug-resistant Acinetobacter baumannii from diverse origins. Results We were able to demonstrate 92% concordance between the new technology and classical chequerboard titration using the A. baumannii collection. Two strains could not be analysed by Xact™ due to their out-of-range MIC of meropenem (>128 mg/L). Conclusions The new test was shown to be diagnostically useful, easy to implement and less labour intensive than the classical metho

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

International audienceVarious pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins

Oxidative protein folding in bacteria

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli , DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75150/1/j.1365-2958.2002.02851.x.pd

RNA profiling in host-pathogen interactions

The development of novel anti-bacterial treatment strategies will be aided by an increased understanding of the interactions that take place between bacteria and host cells during infection. Global expression profiling using microarray technologies can help to describe and define the mechanisms required by bacterial pathogens to cause disease and the host responses required to defeat bacterial infection

Cortactin overexpression results in sustained epidermal growth factor receptor signaling by preventing ligand-induced receptor degradation in human carcinoma cells

The chromosome 11q13 region is frequently amplified in human carcinomas and results in an increased expression of various genes including cortactin, and is also associated with an increased invasive potential. Cortactin acts as an important regulator of the actin cytoskeleton. It is therefore very tempting to speculate that cortactin is the crucial gene within the 11q13 amplicon that mediates the invasive potential of these carcinomas. Cortactin also participates in receptor-mediated endocytosis, and recent findings have shown that, during receptor internalization, cortactin overexpression inhibits the ubiquitylation-mediated degradation of the epidermal growth factor receptor, resulting in a sustained ligand-induced epidermal growth factor receptor activity

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes

Non-canonical CRP sites control competence regulons in Escherichia coli and many other γ-proteobacteria

Escherichia coli's cAMP receptor protein (CRP), the archetypal bacterial transcription factor, regulates over a hundred promoters by binding 22 bp symmetrical sites with the consensus core half-site TGTGA. However, Haemophilus influenzae has two types of CRP sites, one like E.coli's and one with the core sequence TGCGA that regulates genes required for DNA uptake (natural competence). Only the latter ‘CRP-S’ sites require both CRP and the coregulator Sxy for activation. To our knowledge, the TGTGA and TGCGA motifs are the first example of one transcription factor having two distinct binding-site motifs. Here we show that CRP-S promoters are widespread in the γ-proteobacteria and demonstrate their Sxy-dependence in E.coli. Orthologs of most H.influenzae CRP-S-regulated genes are ubiquitous in the five best-studied γ-proteobacteria families, Enterobacteriaceae, Pasteurellaceae, Pseudomonadaceae, Vibrionaceae and Xanthomonadaceae. Phylogenetic footprinting identified CRP-S sites in the promoter regions of the Enterobacteriaceae, Pasteurellaceae and Vibrionaceae orthologs, and canonical CRP sites in orthologs of genes known to be Sxy-independent in H.influenzae. Bandshift experiments confirmed that E.coli CRP-S sequences are low affinity binding sites for CRP, and mRNA analysis showed that they require CRP, cAMP (CRP's allosteric effector) and Sxy for gene induction. This work suggests not only that the γ-proteobacteria share a common DNA uptake mechanism, but also that, in the three best studied families, their competence regulons share both CRP-S specificity and Sxy dependence

Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates

Genotype-Phenotype Correlations in Charcot-Marie-Tooth Disease Due to MTMR2 Mutations and Implications in Membrane Trafficking

Charcot-Marie-Tooth type 4 (CMT4) is an autosomal recessive severe form of neuropathy with genetic heterogeneity. CMT4B1 is caused by mutations in the myotubularin-related 2 (MTMR2) gene and as a member of the myotubularin family, the MTMR2 protein is crucial for the modulation of membrane trafficking. To enable future clinical trials, we performed a detailed review of the published cases with MTMR2 mutations and describe four novel cases identified through whole-exome sequencing (WES). The four unrelated families harbor novel homozygous mutations in MTMR2 (NM_016156, Family 1: c.1490dupC; p.Phe498IlefsTer2; Family 2: c.1479+1G>A; Family 3: c.1090C>T; p.Arg364Ter; Family 4: c.883C>T; p.Arg295Ter) and present with CMT4B1-related severe early-onset motor and sensory neuropathy, generalized muscle atrophy, facial and bulbar weakness, and pes cavus deformity. The clinical description of the new mutations reported here overlap with previously reported CMT4B1 phenotypes caused by mutations in the phosphatase domain of MTMR2, suggesting that nonsense MTMR2 mutations, which are predicted to result in loss or disruption of the phosphatase domain, are associated with a severe phenotype and loss of independent ambulation by the early twenties. Whereas the few reported missense mutations and also those truncating mutations occurring at the C-terminus after the phosphatase domain cause a rather mild phenotype and patients were still ambulatory above the age 30 years. Charcot-Marie-Tooth neuropathy and Centronuclear Myopathy causing mutations have been shown to occur in proteins involved in membrane remodeling and trafficking pathway mediated by phosphoinositides. Earlier studies have showing the rescue of MTM1 myopathy by MTMR2 overexpression, emphasize the importance of maintaining the phosphoinositides equilibrium and highlight a potential compensatory mechanism amongst members of this pathway. This proved that the regulation of expression of these proteins involved in the membrane remodeling pathway may compensate each other's loss- or gain-of-function mutations by restoring the phosphoinositides equilibrium. This provides a potential therapeutic strategy for neuromuscular diseases resulting from mutations in the membrane remodeling pathway