Characterization of the pilin ortholog of the Helicobacter pylori type IV cag pathogenicity apparatus, a surface-associated protein expressed during infection

The Helicobacter pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) involved in host interaction and pathogenicity. Previously, seven cag PAI proteins were identified as homologs of Agrobacterium tumefaciens Vir proteins, which form a paradigm T4SS. The T pilus composed of the processed VirB2 pilin is an external structural part of the A. tumefaciens T4SS. In H. pylori, cag-dependent assembly of pili has not been observed so far, nor has a pilin (VirB2) ortholog been characterized. We have here identified, using a motif-based search, an H. pylori cag island protein (HP0546) that possesses sequence and predicted structural similarities to VirB2-like pilins of other T4SSs. The HP0546 protein displays interstrain variability in its terminal domains. HP0546 was expressed as a FLAG-tagged fusion protein in Escherichia coli, A. tumefaciens, and H. pylori and was detected as either two or three bands of different molecular masses in the insoluble fraction, indicating protein processing. As reported previously, isogenic H. pylori mutants in the putative cag pilin gene had reduced abilities to induce cag PAI-dependent interleukin-8 secretion in gastric epithelial cells. Fractionation analysis of H. pylori, using a specific antiserum raised against an N-terminal HP0546 peptide, showed that the protein is partially surface exposed and that its surface localization depended upon an intact cag system. By immunoelectron microscopy, HP0546 was localized in surface appendages, with surface exposure of an N-terminal epitope. Pronounced strain-to-strain variability of this predicted surface-exposed part of HP0546 indicates a strong selective pressure for variation in vivo.     

Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells

Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.     

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Background

Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease.

Design

Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.

Results

All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.

Conclusions

These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.     

Sequence-specific biosensors report drug-induced changes in epigenetic silencing in living cells

Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.     

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.     

Structure-activity studies of AtPep1, a plant peptide signal involved in the innate immune response

AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure-activity relationship of AtPep1 with its receptor, a 170 kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15 min with a half-maximal response of 0.25 nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9-23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9-23), resulted in a decrease in activity (12 max approximately 100 nM). AtPep1(9-23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A(15)]AtPep1(9-23) (12 max approximately 10nM), and a glycine, [A(17)]AtPep1(9-23) (12 max approximately 1000 nM). Neither [A(17)]AtPep1(9-23) nor the C-terminal truncated AtPep1, AtPep1(9-22), were able to compete with AtPep1(9-23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d-Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l- and d-proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.     

Biogeography of the Primates of Guyana: Effects of Habitat Use and Diet on Geographic Distribution

Species that exploit a wide range of resources or habitats (generalists) tend to be widely distributed, whereas species that exploit a narrow range of resources or habitats (specialists) often have a limited distribution. The distribution patterns are thought to result from specialists using relatively smaller habitats than those exploited by generalists. I used data from 1,725 km of primate surveys that I conducted in Guyana to test these hypotheses. Habitat breadth is the total number of different habitat types occupied by each species. I used the total number of different food categories exploited by each species to measure dietary breadth. Geographic range size is correlated with habitat breadth but not with dietary breadth or body size for the 8 primate species in Guyana. Habitat generalists—red howlers and wedge-capped capuchins—range into all habitats. Habitat specialists—spider monkeys, brown bearded sakis, and golden-handed tamarins—range only into large habitats. Habitat generalists tend to be dietary type specialists in Guyana. I suggest that only habitat generalists can subsist on the low-quality foods in small habitats in Guyana. Conversely, habitat specialists tend to be dietary type generalists in Guyana. They must feed on a variety of food types in large habitats. However, using the number of food categories exploited as a measure of dietary breadth may be only a weak aspect of multidimensional niche. Researchers testing biogeographic hypotheses associated with dietary breadth should consider including multivariate indicators of both the types of food categories eaten and the number of plant species exploited.     

Biogenesis of T Pili in Agrobacterium tumefaciens Requires Precise VirB2 Propilin Cleavage and Cyclization

VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens. The cleaved VirB2 protein is further cyclized to form the T pilin in A. tumefaciens but not in E. coli. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed.     

Composition and properties of the extracellular polysaccharide produced by arthrobacter stabilis NRRL B-3225

The extracellular polysaccharide produced by Arthrobacter stabilis NRRL B-3225 contains d-glucose, d-galactose, pyruvic acid, O-succinyl, and O-acetyl in the approximate molar ratio of 6:3:1:1:1.5. Succinyl is linked as its half-ester, making it a readily removable, acidic function that imparts versatility to the polysaccharide both for fundamental research and for commercial use. The viscosity of aqueous, salt-free solutions of both native and deacylated polymer is relatively low, but atypical of anionic polysaccharides, increases rapidly in the presence of salts, acids, or alkali.