Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of L. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

Resolution of the paradox of red cell shape changes in low and high pH.

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.

Cytoplasmic pH and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.

Shape response of human erythrocytes to altered cell pH.

Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH-titratable inner monolayer phospholipids were examined by assessing pH-dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.