The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6 ⅐ CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.Mycobacterium tuberculosis is one of the most successful human pathogens, infecting nearly one-third of the world's population. Among the various factors that contribute, it is certainly the bacterium's ability to multiply and persist within professional phagocytic cells that is of primary importance (30).The extended RD1 region of M. tuberculosis encodes ESX-1, a novel protein secretion system involved in the immunogenicity and pathogenicity. The system, which is absent from the attenuated vaccines Mycobacterium bovis BCG and Mycobacterium microti (4, 6, 24), is responsible for the export of the 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10. ESX-1 is present in the saprophyte Mycobacterium smegmatis (9), where it acts in DNA uptake (11), which suggests that pathogenic mycobacteria might have adapted an ancestral conjugation system for protein secretion, which is required for survival and multiplication in the host cell.The importance of ESX-1 proteins for pathogenicity was shown by reintroduction of the extended RD1 region into BCG (34), deletion of RD1 from M. tuberculosis (23), signaturetagged and insertional mutagenesis (8,40,46), and targeted gene deletions (5,15,18). Several effects related to pathogenicity have been found to be associated with the expression of ESX-1 in M. tuberculosis and/or Mycobacterium marinum, a fish pathogen that harbors an ESX-1 system similar to that of M. tuberculosis (14,27). These ...
Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.
A cationic polymethacrylate with a guanidinium side group was designed in order to create a polymer with cell membrane-penetrating properties such as Tat or other arginine-rich peptides. The polymer, poly(3-guanidinopropyl methacrylate), abbreviated as pGuaMA, was synthesized by free radical polymerization. The DNA-condensing properties of pGuaMA (Mw 180 kDa) were investigated via dynamic light scattering and zeta potential measurements, and small, positively charged particles (110 nm, +37 mV) were found. It was shown that polyplexes based on pGuaMA were able to transfect COS-7 cells efficiently in the absence of serum, while under the same conditions poly(arginine) (pArg) polyplexes did not show detectable transfection levels. Addition of a membrane-disrupting peptide, INF 7, derived from the influenza virus, to preformed pGuaMA polyplexes did result in approximately 2 times increased transfection levels. DLS, zeta potential measurements, gel electrophoresis, and ethidium bromide displacement measurements indicated that serum induced aggregation of the polyplexes at high polymer/plasmid ratios, while at low polymer/plasmid ratios the polarity of the polyplexes reversed likely due to adsorption of negatively charged proteins on their surface. Likely, the unfavorable interactions of pGuaMA polyplexes with serum proteins is the reason for the absent transfection activity of these polyplexes in the presence of serum. Confocal laser scanning microscopy indicated cellular internalization via endocytosis of both polyplexes and free polymer. Thus, pGuaMA polyplexes enter cells, as reported for other polyplexes, by endocytosis and not, as hypothesized, via direct membrane passage.
Special delivery: Transporters constructed on a sorbitol scaffold with eight guanidine residues show significant translocation across the cell membrane and the blood–brain barrier and high affinities toward mitochondria in HeLa and KG1a leukemia cells. The picture shows colocalisation of one such transporter (T) with MitoTracker red in KG1a cells (scale bars: 10 μm).
For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.
Photochemical internalization (PCI) has been employed as a tool for site-specific intracellular delivery of a variety of molecules. In this study, for the first time, PCI has been employed to facilitate the endosomal escape of small interfering RNA (siRNA) molecules, which are the functional mediators of RNA interference (RNAi). In order to interact with the machinery that will induce post-transcriptional gene silencing, siRNA molecules need to enter the cytoplasm of the cells. This study shows that one of the important rate-limiting steps of siRNA silencing efficiency is the ability of siRNA molecules and/or complexes to escape from the endosomes into the cytosol of the cells. The target of this study, the epidermal growth factor receptor (EGFR), is known as an attractive target for cancer therapy. In this study, a 10-fold increased efficiency in knockdown of the EGFR protein was obtained when anti-EGFR siRNA treatment was combined with PCI as compared to siRNA treatment alone. The fact that this combined treatment resulted in a stronger silencing efficiency indicates that lower doses of siRNA can be used when PCI is employed to augment siRNA delivery. Lowering doses of siRNA would prevent saturation of the RNAi machinery and reduce off-target effects. In addition, local illumination of target tissue would only induce PCI in the desired cells, which can further increase the specificity of the treatment, supporting PCI as an attractive strategy to improve siRNA silencing efficiency.
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