Biomedical subjects

Daniel M Czajkowsky

Publications and source records attributed to Daniel M Czajkowsky.

6 recordsLinked to original sources

PubMed2006

Random mutagenesis of Helicobacter pylori vacA to identify amino acids essential for vacuolating cytotoxic activity.

VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.

Amino Acid Substitution↗

PubMed2006

Localization of linker histone in chromatosomes by cryo-atomic force microscopy.

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the "stem" structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is approximately 30 bp, slightly shorter than that estimated from nuclease digestion assays.

Binding Sites↗

PubMed2005

Mimicry of a host anion channel by a Helicobacter pylori pore-forming toxin.

Bacterial pore-forming toxins have traditionally been thought to function either by causing an essentially unrestricted flux of ions and molecules across a membrane or by effecting the transmembrane transport of an enzymatically active bacterial peptide. However, the Helicobacter pylori pore-forming toxin, VacA, does not appear to function by either of these mechanisms, even though at least some of its effects in cells are dependent on its pore-forming ability. Here we show that the VacA channel exhibits two of the most characteristic electrophysiological properties of a specific family of cellular channels, the ClC channels: an open probability dependent on the molar ratio of permeable ions and single channel events resolvable as two independent, voltage-dependent transitions. The sharing of such peculiar properties by VacA and host ClC channels, together with their similar magnitudes of conductance, ion selectivities, and localization within eukaryotic cells, suggests a novel mechanism of toxin action in which the VacA pore largely mimics the electrophysiological behavior of a host channel, differing only in the membrane potential at which it closes. As a result, VacA can perturb, but not necessarily abolish, the homeostatic ionic imbalance across a membrane and so change cellular physiology without necessarily jeopardizing vitality.

Anions↗

PubMed2004

Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane.

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113+/-5 A for the prepore but only 73+/-5 A for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the beta-barrel pore.

Bacterial Toxins↗

PubMed2002

Monomer-monomer interactions drive the prepore to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin.

Perfringolysin O (PFO), a cholesterol-dependent cytolysin, forms large oligomeric pore complexes comprised of up to 50 PFO molecules. In the present studies a mutant of PFO (PFO(Y181A)) has been identified that traps PFO in a multimeric prepore complex that cannot insert its transmembrane beta-hairpins and therefore cannot form a pore. Remarkably, PFO(Y181A) can be induced to insert its transmembrane beta-hairpins if functional PFO is incorporated into the PFO(Y181A) oligomeric prepore complex. Furthermore, the transition from prepore to pore appears to be an "all or none" process; partial insertion of the transmembrane beta-barrel does not occur. Therefore, cooperative interactions between the monomers of the prepore drive the prepore to pore conversion that results in the formation of the transmembrane beta-barrel.

Bacterial Toxins↗

PubMed2002

Supported lipid bilayers as effective substrates for atomic force microscopy.

Supported lipid bilayers offer a diverse set of substrates for AFM investigations of both water-soluble samples and integral membrane proteins. Although their amphipathic nature is necessary for the latter specimens, it is their robustness and easily changed surface characteristics that make these surfaces particularly attractive for the former ones. When starting any AFM investigation of water-soluble biological complexes, it is probably best to try mica first, owing to its remarkable effectiveness with a wide range of samples. Yet, if mica should prove inadequate, supported lipid bilayers are, as demonstrated here, a sensible second choice.

Aluminum Silicates↗