Structure-function aspects of PstS in multi-drug-resistant Pseudomonas aeruginosa

The increasing prevalence of multi-drug-resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface-expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic DeltaPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%-100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant DeltaHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14-55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium.     

Wide distribution of Phycisphaera-like planctomycetes from WD2101 soil group in peatlands and genome analysis of the first cultivated representative

Phycisphaera-like WD2101 ‘soil group’ is one of the as-yet-uncultivated phylogenetic clades within the phylum Planctomycetes. Members of this clade are commonly detected in various terrestrial habitats. This study shows that WD2101 represented one of the major planctomycete groups in 10 boreal peatlands, comprising up to 76% and 36% of all Planctomycetes-affiliated 16S rRNA gene reads in raised bogs and eutrophic fens respectively. These types of peatlands displayed clearly distinct intra-group diversity of WD2101-affiliated planctomycetes. The first isolate of this enigmatic planctomycete group, strain M1803, was obtained from a humic lake surrounded by Sphagnum peat bogs. Strain M1803 displayed 89.2% 16S rRNA gene similarity to Tepidisphaera mucosa and was represented by motile cocci that divided by binary fission and grew under micro-oxic conditions. The complete 7.19 Mb genome of strain M1803 contained an array of genes encoding Planctomycetal type bacterial microcompartment organelle likely involved in l -rhamnose metabolism, suggesting participation of M1803-like planctomycetes in polysaccharide degradation in peatlands. The corresponding cellular microcompartments were revealed in ultrathin cell sections. Strain M1803 was classified as a novel genus and species, Humisphaera borealis gen. nov., sp. nov., affiliated with the formerly recognized WD2101 ‘soil group’.     

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H₂O₂, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H₂O₂ in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H₂O₂, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H₂O₂ is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H₂O₂-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H₂O₂ is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H₂O₂ was unable to induce this complex suggesting that H₂O₂ is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally     

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

The lethal genetic disease cystic fibrosis is caused predominantly by in‐frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide‐binding domain (NBD1) of CFTR, which functions as an ATP‐gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light‐scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg‐ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second‐site mutations that increase the solubility of isolated F508del‐NBD1 in vitro and suppress the trafficking defect of intact F508del‐CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del‐CFTR.     

Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor

Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine 6. In an effort to reduce the melting point in analogs of 6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of GNE-477 (8), a potent and efficacious dual PI3K/mTOR inhibitor.     

Vertical nontransovarial transmission of Bartonella in fleas

Pathogens use diverse pathways to infect host populations by vertical and/or horizontal routes. Horizontal transmission of bacteria belonging to the Bartonella genus via haematophagous vectors is well known. Vertical transmission of Bartonella species was also suggested to occur but its routes remain to be unveiled. In a previous study, we showed the absence of transovarial transmission of Bartonella species OE 1‐1 in Xenopsylla ramesis fleas, and that fleas feeding on Bartonella‐positive jirds produced Bartonella‐positive gut voids. This current study aimed to investigate whether vertical nontransovarial transmission of Bartonella occurs in fleas. For this aim, the X. ramesis–Bartonella sp. OE 1‐1 model was used. Four groups of fleas including Bartonella‐positive and Bartonella‐negative female fleas and larval offspring had access to either Bartonella‐negative or Bartonella‐positive gut voids and faeces. Sixteen per cent of flea offspring that had access to Bartonella‐positive faeces and gut voids became Bartonella positive. Our findings demonstrate that Bartonella‐positive flea faeces and gut voids are proper infection sources for flea larvae and indicate that vertical nontransovarial transmission of bartonellae occurs in fleas. This information broadens our understanding of Bartonella transmission routes in flea vectors and enlightens pathways of bartonellae transmission and maintenance in flea populations in nature.     

Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles

The Gram-negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1-L5), into the culture medium. These proteins are able to degrade Gram-positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram-positive and Gram-negative bacteria: Staphylococcus aureus 209-P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram-negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram-positive and Gram-negative bacteria.