Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.     

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1)

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.     

The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.     

Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.     

The Chlamydia pneumoniae Inclusion Membrane Protein Cpn1027 Interacts with Host Cell Wnt Signaling Pathway Regulator Cytoplasmic Activation/Proliferation-Associated Protein 2 (Caprin2)

We previously identified hypothetical protein Cpn1027 as a novel inclusion membrane protein that is unique to Chlamydia pneumoniae. In the current study, using a yeast-two hybrid screen assay, we identified host cell cytoplasmic activation/proliferation-associated protein 2 (Caprin2) as an interacting partner of Cpn1027. The interaction was confirmed and mapped to the C-termini of both Cpn1027 and Caprin2 using co-immunoprecipitation and GST pull-down assays. A RFP-Caprin2 fusion protein was recruited to the chlamydial inclusion and so was the endogenous GSK3β, a critical component of the β-catenin destruction complex in the Wnt signaling pathway. Cpn1027 also co-precipitated GSK3β. Caprin2 is a key regulator of the Wnt signaling pathway by promoting the recruitment of the β-catenin destruction complex to the cytoplasmic membrane in the presence of Wnt signaling while GSK3β is required for priming β-catenin for degradation in the absence of Wnt signaling. The Cpn1027 interactions with Caprin2 and GSK3β may allow C. pneumoniae to actively sequester the β-catenin destruction complex so that β-catenin is maintained even in the absence of extracellular Wnt activation signals. The maintained β-catenin can trans-activate Wnt target genes including Bcl-2, which may contribute to the chlamydial antiapoptotic activity. We found that the C. pneumoniae-infected cells were more resistant to apoptosis induction and the anti-apoptotic activity was dependent on β-catenin. Thus, the current study suggests that the chlamydial inclusion protein Cpn1027 may be able to manipulate host Wnt signaling pathway for enhancing the chlamydial anti-apoptotic activity.     

Century-scale Methylome Stability in a Recently Diverged Arabidopsis thaliana Lineage

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.