Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells.

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.

Internalization of Staphylococcus aureus by endothelial cells induces apoptosis.

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.

Chromatographic separation and partial characterization of microbial low-molecular-weight proteinase inhibitors.

A crude preparation, inhibiting trypsin (T), chymotrypsin (C) and papain (P), was isolated from the culture filtrate of Actinomyces sp. 9 by butanol extraction. Ion-exchange high-performance liquid chromatography (HPLC) of this preparation on a Mono S column resulted in the separation of two inhibitory fractions: one active against T (TI-9) and the other active against C and P (CPI-9). These two fractions, having the same inhibitory specificity, were also obtained from the crude extract by size-exclusion HPLC on a Superose 12 column. This method proved to be better in terms of the purity of the fractionated inhibitors than ion-exchange chromatography. Further purification of TI-9 and CPI-9 was achieved by using reversed-phase HPLC on a PEP RPC column. In this case, TI-9 was obtained as an apparently homogeneous peak. Studies on the physicochemical properties of the purified inhibitors showed them to be small molecules, similar in hydrophobicity, pH and thermal stability, but differing in solubility. Amino acid and spectral analyses provided the peptidic structure of TI-9 and gave a molecular weight of 1643, while parallel analyses of CPI-9 showed that it lacks the common amino acids.

Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum.

The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pI values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175,000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72,000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320,000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH.

Assay for enzyme inhibition: detection of natural inhibitors of trypsin and chymotrypsin.

A technique for quickly detecting nanogram quantities of low- and high-molecular-weight inhibitors of some serine proteases is described. The inhibitor solutions are spotted onto agar films which contain either L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or tosyl lysine chloromethyl ketone (TLCK)-chymotrypsin. Enzyme inhibition is visualized as colorless zones on a pink background after the films were stained with the chromogenic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester. The method is used for rapidly testing both high-performance liquid chromatography fractions and thin-layer chromatograms to identify the inhibitors of trypsin and chymotrypsin in complex microbial extracts. The assay is quantitative so that it is possible to compare the specificity of the inhibitory fractions for trypsin and chymotrypsin. Results with standard inhibitors demonstrate the high sensitivity of the method, e.g., inhibition is detected with 1 ng of soybean trypsin inhibitor and 0.3 ng of antipain or chymostatin.