Large-Scale,Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip

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Salmonella-induced enteritis: molecular pathogenesis and therapeutic implications

Salmonella-induced enteritis is a gastrointestinal disease that causes major economic and welfare problems throughout the world. Although the infection is generally self-limiting, subgroups of the population such as immunocompromised individuals, the young and the elderly are susceptible to developing more severe systemic infections. The emergence of widespread antibiotic resistance and the lack of a suitable vaccine against enteritis-causing Salmonella have led to a search for alternative therapeutic strategies. This review focuses on how Salmonella induces enteritis at the molecular level in terms of bacterial factors, such as the type III secretion systems used to inject a subset of bacterial proteins into host cells, and host factors, such as Toll-like receptors and cytokines. The type III secreted bacterial proteins elicit a variety of responses in host cells that contribute to enteritis. Cytokines form part of the host defence mechanism, but in combination with bacterial factors can contribute to Salmonella-induced enteritis. We also discuss animal and cell culture models currently used to study Salmonella-induced enteritis, and how understanding the mechanisms of the disease has impacted on the development of Salmonella therapeutics.     

In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.     

Actin-Based Motility of Burkholderia thailandensis Requires a Central Acidic Domain of BimA That Recruits and Activates the Cellular Arp2/3 Complex

Burkholderia species use BimA for intracellular actin-based motility. Uniquely, Burkholderia thailandensis BimA harbors a central and acidic (CA) domain. The CA domain was required for actin-based motility, binding to the cellular Arp2/3 complex, and Arp2/3-dependent polymerization of actin monomers. Our data reveal distinct strategies for actin-based motility among Burkholderia species.In common with selected species of Listeria, Shigella, Rickettsia, and Mycobacterium, some members of the genus Burkholderia are capable of intracellular actin-based motility (reviewed in reference 9). Such motility promotes cell-to-cell spread in the absence of immune surveillance and, in some cases, escape from autophagy. The melioidosis pathogen Burkholderia pseudomallei forms actin-rich bacterium-containing membrane protrusions (5), in a manner that requires BimA (Burkholderia intracellular motility A) (11). B. pseudomallei BimA exhibits C-terminal homology to the Yersinia autosecreted adhesin YadA and possesses motifs associated with actin binding, including WASP homology 2 (WH2) domains and proline-rich motifs (11). Actin-based motility is also a feature of infection by the glanders pathogen Burkholderia mallei and the avirulent B. pseudomallei-like species Burkholderia thailandensis (10). BimA homologs exist in these species that can compensate for the actin-based motility defect of a B. pseudomallei bimA mutant (10), and mutation of B. mallei bimA results in loss of function (7). BimA homologs from B. mallei and B. thailandensis (BimA_th) differ markedly in primary sequence from the B. pseudomallei protein (BimA_ps) and each other (10), raising the possibility that they may act in distinct ways. Intraspecies conservation of BimA is high in natural populations of B. pseudomallei, with the exception of a geographically restricted B. mallei-like BimA variant (8).Mechanisms of bacterial actin-based motility converge on activation of the Arp2/3 (actin-related protein 2/3) complex. Activation of Arp2/3 requires cellular nucleation-promoting factors (NPFs) such as Wiskott-Aldrich syndrome protein (WASP) family members, and pathogens capable of actin-based motility often mimic the activity of NPFs or recruit and activate them at the bacterial pole (reviewed in references 3 and 9). Arp2/3 is localized throughout B. pseudomallei-induced actin tails (2); however, the role that it plays in actin-based motility is unclear, as expression of an inhibitory domain of the cellular NPF Scar1 does not interfere with actin-based motility of B. pseudomallei (2). Moreover, BimA_ps can polymerize actin in vitro in an Arp2/3-independent manner (11).Recruitment and activation of the Arp2/3 complex by cellular and pathogen-associated NPFs require one or more WH2 domains and an amphipathic central and acidic (CA) domain (3, 6). Analysis of the primary sequence of BimA homologs revealed a CA domain in B. thailandensis BimA that was absent in the BimA proteins of other Burkholderia species and conserved relative to WASP family members, Listeria ActA, and Rickettsia RickA (10). Here we surveyed the conservation of the CA domain and probed its role in actin-based motility and the binding and activation of the Arp2/3 complex.Primers were designed to amplify an 87-bp region of the CA domain (corresponding to amino acid residues 102 to 130 inclusive) of the bimA gene of the sequenced B. thailandensis strain E264 (5′-AGGCGGGTAATCGACTCA-3′ and 5′-TTCGTCGTCCGACCATGA-3′). These primers, and universal bimA-specific primers (8), were used to screen 203 Burkholderia isolates for bimA and the CA domain by PCR using Platinum Taq DNA polymerase (Invitrogen, Paisley, United Kingdom) and boiled lysates of single colonies as template. The strain collection was described previously (8), supplemented by an additional 52 B. pseudomallei, 23 B. thailandensis, and 19 B. mallei isolates and 10 other isolates representing 5 other Burkholderia species. A bimA amplicon was detected for all isolates of species B. pseudomallei, B. thailandensis, B. mallei, and Burkholderia oklahomensis. Consistent with analysis of sequenced genomes (8), the CA domain was restricted to, and always found in, B. thailandensis isolates. Such a test may prove useful to rapidly discriminate between avirulent B. thailandensis and the closely related biothreat agents B. pseudomallei and B. mallei.To investigate the function of the CA domain, we deleted the region corresponding to residues 96 to 130 of the strain E264 BimA by PCR-ligation-PCR (1) using the cloned bimA gene of B. thailandensis strain E30 as a template (10). Pfu proofreading DNA polymerase was used to separately amplify the region 5′ of the CA domain with primers B_th-comp forward (5′-CATGAATTCCCATGCGTGCAACAGTTGCT-3′) and 5′-CGAGCCGCCCGCGCCTCGCGTGTT-3′ and the region 3′ of the CA domain with B_th-comp reverse (5′-CTTCTCGAGTCACCATTGCCAGCTCATGCCTACGC-3′) and 5′-TCCCCTCCGCCGACGCCGATCGCAA-3′ from pME6032- bimA_th (10). The PCR amplicons were ligated, and the desired recombinant was amplified by a further round of PCR with primers B_th-comp forward and B_th-comp reverse. The product was subcloned under the Ptac promoter in pME6032 (4) via EcoRI and XhoI sites incorporated in the primers (underlined), yielding pME6032-bimA_th-ΔCA. Faithful amplification and deletion of the CA domain were confirmed by nucleotide sequencing (data not shown).To evaluate the role of the CA domain in actin-based motility, pME6032-bimA_th and the ΔCA variant were introduced into a B. pseudomallei strain 10276 bimA::pDM4 mutant (11) by electroporation with selection for tetracycline resistance. Strains were amplified in Luria-Bertani (LB) medium, and J774.2 murine macrophage-like cells were infected at a multiplicity of 10 in RPMI medium containing 10% (vol/vol) fetal calf serum at 37°C in a 5% CO₂ atmosphere. After 30 min cells were washed and overlaid with medium containing 250 μg/ml kanamycin to kill extracellular bacteria. During bacterial culture and cell infection, expression of BimA proteins was induced with 0.25 mM isopropyl-β-d-thiogalactoside (IPTG). Eight hours postinfection cells were washed, fixed, permeabilized, and stained for bacteria, polymerized F-actin, and nuclei essentially as described previously (10). Images were captured using a Leica confocal laser scanning microscope with LAS AF v.2.0 software. B. pseudomallei 10276 and 10276 bimA::pDM4 were included as positive and negative controls, respectively (Fig. ​(Fig.11 A and B). As expected B. thailandensis E30 BimA restored the ability of the 10276 bimA::pDM4 mutant to form actin tails (10) (Fig. ​(Fig.1C).1C). Deletion of the CA domain of B. thailandensis BimA abolished this activity (Fig. ​(Fig.1D),1D), indicating that it is required for actin-based motility.Open in a separate windowFIG. 1.Representative confocal laser scanning micrographs of J774.2 cells infected with B. pseudomallei strain 10276 (A), an isogenic bimA::pDM4 mutant (B), or 10276 bimA::pDM4 trans complemented with pME6032-bimA_th (C) or pME6032-bimA_th-ΔCA (D) and induced to express the proteins under IPTG induction. Bacteria (red) were stained using mouse monoclonal anti-B. pseudomallei lipopolysaccharide antibody (Camlab, Cambridge, United Kingdom) detected with anti-mouse Ig-Alexa Fluor⁵⁶⁸ (Molecular Probes, Leiden, Netherlands). F-actin (green) was stained with Alexa Fluor⁴⁸⁸-conjugated phalloidin. DNA (blue) was stained with 4′,6-diamidino-2-phenylindole. Bars, 5 μm. Bacteria forming actin tails are marked with arrows.Monoclonal antibodies raised against BimA_ps (11) failed to recognize BimA_th on the bacterial pole (data not shown); therefore, we were unable to conclude that loss of actin-based motility upon deletion of the CA domain may be a consequence of failed secretion or polar localization. To investigate the role of the CA domain in actin binding and polymerization, the ΔCA variant of B. thailandensis BimA was PCR amplified from pME6032-bimA_th-ΔCA with primers 5′-GGGCCCGGATCCGCCGCTGACGAGACG-3′ and 5′-GGGCCCGAATTCTCACGCTCGCGCGTCG-3′. The product was first cloned by a topoisomerase-mediated process into pCR2.1-TOPO (Invitrogen, Paisley, United Kingdom) and then subcloned as a BamHI and EcoRI fragment into similarly digested pGEX-2T-1 (Amersham Biosciences, Buckinghamshire, United Kingdom), creating a fusion to the carboxyl terminus of glutathione S-transferase (GST). The subcloned region corresponds to amino acids 47 to 386 of BimA_th and was confirmed by sequencing to be identical to the pGEX-BimA_th plasmid described previously (10), except for the deleted CA region. The region encoding B. pseudomallei BimA residues 54 to 455 (lacking the signal peptide and membrane anchor) was amplified from pME6032-bimA_ps (10) with primers 5′-GCGCGCGGATCCATGAATCCCCCCGAACCGCCGGGC-3′ and 5′-GCGCGCGAATTCTTAGCGCGCGGTGTCGGTG-3′ and fused to GST in pGEX-4T-1 as described above. Plasmids encoding GST or GST fusions to BimA_ps, BimA_th, or BimA_th-ΔCA domain were separately introduced into Escherichia coli K-12 Rosetta2(DE3)pLysS cells expressing rare aminoacyl tRNAs (Merck Biosciences, Nottingham, United Kingdom). LB cultures were induced to express the proteins at late logarithmic phase for 3 h at ambient temperature, which proved optimal for recovery of intact proteins using glutathione Sepharose 4B beads (Fig. ​(Fig.2A2A).Open in a separate windowFIG. 2.SDS-PAGE analysis of fusions of BimA_ps (residues 54 to 455), BimA_th (residues 47 to 386), or BimA_th lacking the CA domain to the carboxyl terminus of GST (A). Sepharose beads coated with these proteins, or GST alone, were examined for their ability to sequester actin (B), p34-Arc/ARPC2 (C), or Arp3 (D) from murine splenic extracts by Western blotting of bead-associated proteins using specific antibodies. Coated beads were also examined for their ability to bind highly purified rhodamine-labeled actin in PBS (red) by confocal laser scanning microscopy (E). Mw, molecular weights in thousands.Beads coated with GST, GST-BimA_ps, GST-BimA_th, or GST-BimA_th-ΔCA were incubated with murine splenic cell lysate as described previously (10). After 15 min of incubation at ambient temperature, beads were washed with ice-cold Tris-buffered saline and analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and Western blotting with antiactin antibody (10). As reported previously, beads coated with GST-BimA_ps or GST-BimA_th, but not GST, sequestered actin from the splenic extract (Fig. ​(Fig.2B)2B) (10). Deletion of the CA domain did not impair actin binding, consistent with the fact that a predicted WH2 domain remains intact (10). It may be inferred that such binding is direct, since coated beads also bound highly purified rhodamine-labeled actin in phosphate-buffered saline (PBS) as assessed by confocal microscopy of beads treated as described previously (Fig. ​(Fig.2E)2E) (10). The ability of coated beads to sequester the Arp2/3 complex from murine splenic extracts was examined using rabbit anti-p34-Arc/ARPC2 (Millipore, Watford, United Kingdom) and goat anti-Arp3 (Autogen Bioclear, Wiltshire, United Kingdom) detected with species-specific Ig-horseradish peroxidase conjugates by a chemoluminescence method (Amersham Biosciences, Buckinghamshire, United Kingdom). GST-BimA_th, but not GST-BimA_ps, sequestered p34-Arc/ARPC2 (Fig. ​(Fig.2C)2C) and Arp3 (Fig. ​(Fig.2D).2D). Deletion of the CA domain abolished the ability to sequester Arp2/3 components (Fig. 2C and D), indicating that it plays a role in recruitment of the complex.The requirement for Arp2/3 and the CA domain in actin polymerization by B. thailandensis BimA was evaluated in vitro using pyrene-labeled actin. Polymerization of such monomers leads to an emission of fluorescence that can be sensitively recorded over time. Lyophilized pyrene-actin, Arp2/3, and the verprolin-like central and acidic (VCA) domain of WASP were obtained from Cytoskeleton (Universal Biologicals, Cambridge, United Kingdom) and prepared per the manufacturer''s instructions. Assay conditions were essentially as described previously (12). Briefly, 90-μl reaction mixtures were assembled in black opaque 96-well plates containing 100 nM GST or GST fusion protein, 2 μM pyrene-actin, and 30 nM Arp2/3 as required. Reactions were initiated by the addition of 10 μl of 10× polymerization buffer (100 mM Tris, pH 7.5, 500 mM KCl, 20 mM MgCl₂, 10 mM ATP), and the emission of fluorescence at 407 nm, after excitation at 365 nm, was followed every 30 s for 90 min using a Tecan Infinite M200 fluorescent plate reader with i-control software. The GST, GST-BimA_ps, GST-BimA_th, and GST-BimA_th-ΔCA proteins were prepared as described above but eluted from beads with 10 mM reduced glutathione. Triplicate determinations were performed with two independently purified sets of protein. Data from a representative assay are shown in Fig. ​Fig.3.3. Rates of polymerization were calculated as the rise in fluorescence units per second during the linear phase of polymerization, and the means of six values per protein ± standards of the means are recorded in Table ​Table1.1. Results were analyzed by pairwise Student t tests using R software (version 2.11), and P values of ≤0.05 were taken as significant.Open in a separate windowFIG. 3.Polymerization of pyrene-labeled actin monomers by GST (green lines), GST-BimA_ps (blue lines), GST-BimA_th (red lines), and GST-BimA_th-ΔCA (yellow lines) in the absence (solid lines) or presence (dotted lines) of the Arp2/3 complex. The Arp2/3 complex was confirmed to be active in the presence of the purified VCA domain of WASP (black line). The graph shows the increase in fluorescence units (at 407 nm) over time during a single representative experiment. Triplicate determinations were performed with two sets of independently purified proteins, and the mean rates of fluorescence are recorded in Table ​Table11.

TABLE 1.

Rates of polymerization of pyrene-labeled actin monomers by GST and GST-BimA fusion proteins in the absence or presence of the Arp2/3 complex

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Assignment of a gene (NEM1) for autosomal dominant nemaline myopathy to chromosome 1

Nemaline myopathy (NEM) is a neuromuscular disorder characterized by the presence, in skeletal muscle, of nemaline rods composed at least in part of alpha-actinin. A candidate gene and linkage approach was used to localize the gene (NEM1) for an autosomal dominant form (MIM 161800) in one large kindred with 10 living affected family members. Markers on chromosome 19 that were linked to the central core disease gene, a marker at the complement 3 locus, and a marker on chromosome 1 at the alpha-actinin locus exclude these three candidate genes. The family was fully informative for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage to APOA2, with a lod score of 3.8 at a recombination fraction of 0. Recombinants with NGFB (1p13) and AT3 (1q23-25.1) indicate that NEM1 lies between 1p13 and 1q25.1. In total, 47 loci were investigated on chromosomes 1, 2, 4, 5, 7-11, 14, 16, 17, and 19, with no indications of significant linkage other than to markers on chromosome 1.     

Effects of nest paper hydrocarbons on nest and nestmate recognition in colonies ofPolistes metricus say

The role of nest paper hydrocarbons in nest and nestmate recognition for the social waspPolistes metricus was examined. Newly emergedP. metricus workers maintained in the laboratory spent four days alone on a fragment of nest paper that was subjected to one of the following tretments: untreated, extracted with hexane to remove surface hydrocarbons, or extracted with extract reapplied. Test wasps were returned to their natal nest with nestmates and observed for 1 h. Time spent on nest by test wasp and its behaviors were recorded. Wasps exposed to untreated and reapplied nest fragments spent an average of 34.13 and 31.75 min on their nests, respectively, while wasps from extracted fragments averaged 17.19 min. Behavior of wasps exposed to extracted paper differed significantly from wasps exposed to paper with hydrocarbons. These results suggest that exposure to nest paper hydrocarbons is important for both nest and nestmate recognition.     

Transmission of aerosolized seasonal H1N1 influenza A to ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID₅₀) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID₅₀ for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.