FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

Metabolic flux analysis of<Emphasis Type="Italic"> pykF</Emphasis> gene knockout<Emphasis Type="Italic"> Escherichia coli</Emphasis> based on <Superscript>13</Superscript>C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

Metabolic flux analysis based on ¹³C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF^– mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF^– mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.     

Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy

Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these conditions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F. psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy. The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument. In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis. The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The erosion pattern suggests that the gliding motility of F. psychrophilum could be involved in this burrowing ability.     

Use of EQUORAL in de novo renal transplant recipients

This paper presents our experience to date with using a cyclosporine formulation Equoral (IVAX Pharmaceuticals) together with mycophenolate mofetil plus a steroid immunosuppressive regimen in the treatment of de novo renal transplant recipients. Ten cadaveric donor renal transplant recipients of mean age 51.6 years (range 37-66) were followed up over 6 months for the development of rejection attacks and side effects. All patients received prednisolone, mycophenolate mofetil (1 g/day during the first 5 days posttransplant and then 20 mg/kg/day) plus cyclosporine (3 mg/kg/day). Biopsy proven acute rejection episodes were observed in 2 out of 10 patients (20%). Six months patient as well as renal graft survival rate was 100%. The development of graft function was immediate after transplantation. The mean serum creatinine levels were gradually decreased. Over the 6-month posttransplant period, the function of the graft was satisfactory and stable. The majority of observed adverse events were those commonly reported with the use of cyclosporine and they resolved with a reduction in cyclosporine dose. Equoral treatment demonstrated an acceptable safety profile with maintenance of adequate renal function without incidence of malignancy/lymphoproliferative disease or serious infections. In conclusion, Equoral plus mycophenolate mofetil immunosuppression seems effective and safe on terms acute rejection rates, patient and renal graft survival rates and side profiles.     

Polyprotein processing protease of African swine fever virus: purification and biochemical characterization

The purified recombinant African swine fever virus polyprotein processing protease cleaves the two GG-X sites in polyprotein pp62 with the same efficiency. Cleavage at the site that is first recognized in vivo is not a requisite for cleavage at the second site, suggesting the existence of mechanisms that control the ordered processing of the polyprotein during infection.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.