Genes Important for Catalase Activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly

Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation

Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected

Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems

BACKGROUND: Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. RESULTS: We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. CONCLUSION: Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of α(v)β(3 )integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection

Endocytosis of DNA-Hsp65 Alters the pH of the Late Endosome/Lysosome and Interferes with Antigen Presentation

BACKGROUND: Experimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies. METHODOLOGY AND PRINCIPAL FINDINGS: Using confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment. CONCLUSIONS AND SIGNIFICANCE: Taken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response

Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain