Inhibition of Gene Expression by Peptide Nucleic Acids in Cultured Cells

Abstract

We have tested in cultured cells the capacity of antisense and antigene PNAs to inhibit, in a sequence specific manner, the expression of oncogenes in leukaemia and pancreatic carcinoma cells. The results observed appeared promising and suggest that PNA may play in the future an important role in targeting disease-related genes.     

Hybridization Studies with Chiral Peptide Nucleic Acids

A novel class of chiral peptide nucleic acids has been synthesized in which the sugar-phosphate backbone of DNA has been replaced with the glycyl-proline backbone of both the - and the -configurations, nucleobases being attached through the 4-position of proline withcis- andtrans-stereochemistry. TheT₁₀homopolymers withcis-stereochemistry in the - and -series bind strongly to poly(dA) withT_mvalues of 69 and 70°C, respectively. They bind more strongly to poly(rA) withT_mvalues of 73 and 72°C, respectively, and with apparent 1:1 stoichiometry. Using a mixed sequence decamer it was found that the thermal stability of the chiral peptide nucleic acid/oligonucleotide complex was comparable to that formed by Nielsen's polyamide nucleic acid.     

Intracellular Amounts of Nucleic Acids and Protein During Pollen Grain Growth in Tradescantia

The rapid growth, large organelles, and synchronous development of T. paludosa pollen grains make them ideal subjects for cytochemical analysis. A microphotometric study of the nucleoli, chromosomes, and cytoplasm fixed at daily intervals during pollen grain maturation indicated that: 1. DNA (Feulgen) synthesis in the generative nucleus occurred during the first third of interphase, while the DNA content of the vegetative nucleus remained unchanged. 2. Throughout development, changes in RNA (azure B) content, in general, paralleled changes in protein (NYS¹, Millon) content in each organelle of the vegetative cell. Initially, the RNA and protein of all organelles increased up to mid interphase, when chromosomal and nucleolar fractions began to decline despite a continued increase in cytoplasmic RNA and protein. At least 24 hours before anthesis, the vegetative nucleolus had disappeared and chromosomal protein and RNA of the vegetative nucleus were apparently in rapid decline. Such a system offered an opportunity to study the role of the nucleus, especially the nucleolus, in RNA and protein metabolism in the cytoplasm, by noting what cytoplasmic processes could and could not continue at a time when nuclear mechanisms were absent or minimal. It was found that at least 2 fundamental processes continued during this period: both RNA and protein accumulated in the cytoplasm at a rapid rate. It was concluded that the nucleus is not the sole source of cytoplasmic RNA, for the data suggest that there are at least 2 separate and independent, or remotely dependent synthesizing systems, one nuclear and the other cytoplasmic. It is evident that nuclear influence on cytoplasmic synthesis need be neither direct nor immediate.     

Phosphono Peptide Nucleic Acids with a Constrained Hydroxyproline-Based Backbone

Abstract

DNA mimics representing negatively charged analogues of peptide nucleic acids (PNAs), particularly hetero-oligomers constructed from alternating phosphono-PNA residues (pPNA) and monomers on the base of trans-4-hydroxy-L-proline (HypNA) as well as mimics composed of phosphono-HypNA monomers (pHypNA) were tested in a set of in vitro and in vivo assays, and they demonstrated a high potential for the use in nucleic acid based diagnostic, isolation of nucleic acids and antisense experiments.     

RNAi Reduces Expression and Intracellular Retention of Mutant Cartilage Oligomeric Matrix Protein

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.     

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

Background

Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular α-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs).

Methodology/Principal Findings

We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (∼1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions.

Conclusion/Significance

Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.     

Boost Protein Expression through Co-Expression of LEA-Like Peptide in Escherichia coli

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.     

Lactobacillus gasseri SBT2055 Reduces Infection by and Colonization of Campylobacter jejuni

Campylobacter is a normal inhabitant of the chicken gut. Pathogenic infection with this organism in humans is accompanied by severe inflammation of the intestinal mucosal surface. The aim of this study was to evaluate the ability of Lactobacillus gasseri SBT2055 (LG2055) to inhibit the adhesion and invasion of Campylobacter jejuni in vitro and to suppress C. jejuni colonization of chicks in vivo. Pretreatment with LG2055 significantly reduced adhesion to and invasion of a human epithelial cell line, Intestine 407, by C. jejuni 81–176. Methanol (MeOH)-fixed LG2055 also reduced infection by C. jejuni 81–176. However, proteinase K (ProK)-treated LG2055 eliminated the inhibitory effects. Moreover, LG2055 co-aggregated with C. jejuni 81–176. ProK treatment prevented this co-aggregation, indicating that the co-aggregation phenotype mediated by the proteinaceous cell-surface components of LG2055 is important for reducing C. jejuni 81–176 adhesion and invasion. In an in vivo assay, oral doses of LG2055 were administered to chicks daily for 14 days after oral inoculation with C. jejuni 81–176. At 14 days post-inoculation, chicks treated with LG2055 had significantly reduced cecum colonization by C. jejuni. Reduction in the number of C. jejuni 81–176 cells adhering to and internalized by human epithelial cells demonstrated that LG2055 is an organism that effectively and competitively excludes C. jejuni 81–176. In addition, the results of the chick colonization assay suggest that treatment with LG2055 could be useful in suppressing C. jejuni colonization of the chicks at early growth stages.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Discovery of Putative Small Non-Coding RNAs from the Obligate Intracellular Bacterium Wolbachia pipientis

Wolbachia pipientis is an endosymbiotic bacterium that induces a wide range of effects in its insect hosts, including manipulation of reproduction and protection against pathogens. Little is known of the molecular mechanisms underlying the insect-Wolbachia interaction, though it is likely to be mediated via the secretion of proteins or other factors. There is an increasing amount of evidence that bacteria regulate many cellular processes, including secretion of virulence factors, using small non-coding RNAs (sRNAs), but sRNAs have not previously been described from Wolbachia. We have used two independent approaches, one based on comparative genomics and the other using RNA-Seq data generated for gene expression studies, to identify candidate sRNAs in Wolbachia. We experimentally characterized the expression of one of these candidates in four Wolbachia strains, and showed that it is differentially regulated in different host tissues and sexes. Given the roles played by sRNAs in other host-associated bacteria, the conservation of the candidate sRNAs between different Wolbachia strains, and the sex- and tissue-specific differential regulation we have identified, we hypothesise that sRNAs may play a significant role in the biology of Wolbachia, and in particular in its interactions with its host.     

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.     

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.     

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved Quantification,Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

Background

Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.

Methodology

A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.

Key results

• Development of a standardised growth assay to quantify bacterial replication and viability in vitro.
• Quantitative comparison of different DNA extraction methods.
• Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.
• Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.
• Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.

Conclusions

Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.     

Rotavirus Infection Reduces Sucrase-Isomaltase Expression in Human Intestinal Epithelial Cells by Perturbing Protein Targeting and Organization of Microvillar Cytoskeleton

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268–8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.