CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.     

Target site recognition by a diversity-generating retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.     

Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury

Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and glomerulosclerosis.     

MYCBP2 Is a Guanosine Exchange Factor for Ran Protein and Determines Its Localization in Neurons of Dorsal Root Ganglia

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway.     

Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl(2) at nominal concentrations of 0.01 and 1mg/L) for 4 days at either 15°C or 21°C. First, exposure to 1mg Cd/L induced a high mortality rate (67%) in fish held at 21°C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21°C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15°C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.     

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (～50%), but at a much lower frequency during vegetative growth (～0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ～90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.