Detection and Characterization of the Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM Isolates from Humans and Cattle

The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an ~80-kb (six strains) or ~120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.     

Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2, compared with healthy human lymphocytes

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects' lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17(+ve) lymphocytes were exofacially peroxiredoxin 2(+ve) (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17(+ve) T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation.     

Does the disturbance hypothesis explain the biomass increase in basin‐wide Amazon forest plot data?

Positive aboveground biomass trends have been reported from old-growth forests across the Amazon basin and hypothesized to reflect a large-scale response to exterior forcing. The result could, however, be an artefact due to a sampling bias induced by the nature of forest growth dynamics. Here, we characterize statistically the disturbance process in Amazon old-growth forests as recorded in 135 forest plots of the RAINFOR network up to 2006, and other independent research programmes, and explore the consequences of sampling artefacts using a data-based stochastic simulator. Over the observed range of annual aboveground biomass losses, standard statistical tests show that the distribution of biomass losses through mortality follow an exponential or near-identical Weibull probability distribution and not a power law as assumed by others. The simulator was parameterized using both an exponential disturbance probability distribution as well as a mixed exponential–power law distribution to account for potential large-scale blowdown events. In both cases, sampling biases turn out to be too small to explain the gains detected by the extended RAINFOR plot network. This result lends further support to the notion that currently observed biomass gains for intact forests across the Amazon are actually occurring over large scales at the current time, presumably as a response to climate change.     

Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity

A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.     

Macroinvertebrate community structure across a wetland hydroperiod gradient in southern New Hampshire,USA

We conducted a field study to examine the influence of hydroperiod and concomitant changes in abiotic (wetland size, pH, conductivity, dissolved oxygen and water temperature) and biotic (predatory fish presence) characteristics on macroinvertebrate communities in isolated wetlands in southern New Hampshire. Invertebrates were sampled using dipnet sweeps in 42 wetlands with short (<4 months), intermediate (4–11 months) or long (permanent) hydroperiods in 1998 and 1999. We found that invertebrate genera richness, and to a lesser degree abundance, increased linearly along the hydrological gradient, and in response to temperature and dissolved oxygen. Relative abundance of genera also differed markedly with respect to hydroperiod. Most notably, invertebrate communities changed from Acilius-dominated communities to Notonecta-dominated communities. Invertebrate relative abundances in permanent wetlands also differed with respect to the occurrence of predatory fish. Some genera (e.g., Libellula, and Dytiscus) were more likely to occur in permanent wetlands without fish, whereas other genera (e.g., Buena, and Basiaeshna) were more likely to occur in wetlands with predatory fish. Because aquatic invertebrate communities differed markedly with respect to wetland hydroperiod, and in relation to the occurrence of predatory fish, it is essential to retain a diversity of wetlands in the landscape to ensure the long-term persistence of aquatic invertebrate biodiversity.     

Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157

The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.     

StcE,a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.