We demonstrated that fl-glucosidase and ,8-galactosidase can be trapped inside erythrocytes by rapid hemolysis of the cells in the presence of these enzymes. Enzyme enters only during liemolysis, and optimum uptake occurs within 60 sec. There is no loss in cell number after hemolysis-induced enzyme uptake, and the ghosts have only a slightly increased mean cell volume. Smaller proteins enter more readily than larger proteins, although enzymes with a molecular weight of at least 180,000 can be readily entrapped by erythrocytes. This finding may provide a useful approach to the problem of enzyme replacement in certain diseases, including Gaucher's disease.Enzyme therapy for certain diseases has attracted increasing attention recently. For example, modest success in treatment of certain asparaginase-sensitive leukemias (1) has been achieved by parenterally administered asparaginase. In addition, several human diseases are caused by the partial or complete absence of a particular enzyme activity. Presumably the consequences of these diseases could be alleviated if replacement enzymes could be introduced into such individuals to catalyze the missing reaction.However, the injection of free enzyme may be unsatisfactory in general because of the possibility of either an unfavorable immune response or because the enzyme will be rapidly cleared from the blood. An appreciation of these difficulties has led to the notion of encapsulating enzymes in some kind of semipermeable envelope that would sequester or retain enzyme while allowing substrates to enter (2). An additional problem in some cases may be the requirement that the enzyme reach an intracellular substrate. For example, Gaucher's disease is characterized by an accumulation of flglucocerebroside in cells of the reticuloendothelial system, including spleen, kidney, and bone marrow. The disease is due to an inherited deficiency in the enzyme ,3-glucocerebrosidase (3); membranous, glucocerebroside-rich storage deposits accumulate as a result of the incomplete catabolism of membrane glycolipids of erythrocytes and leukocytes (4, 5).We have found that f-glucosidase and fl-galactosidase can be trapped within sealed erythrocyte ghosts. When a hypotonic solution is added to erythrocytes, they hemolyse, and soluble intracellular contents including hemoglobin leave the cell. Depending upon conditions, the cell may be either reversibly or irreversibly ruptured. In the reversible case, pores of sufficient size to allow the escape of hemoglobin molecules open in the membrane and then close again. The resulting erythrocyte ghost is osmotically competent (6). These properties suggested to us that soluble enzyme might be able to enter the cell while the pores are open, providing a way of trapping the enzyme inside the cell. We suggest that cells loaded with #-glucocerebrosidase might be useful for treatment of Gaucher's disease because the erythrocytes will be phagocytized, thus introducing the enzyme into cells of the reticuloendothelial system. MATERIALS AND METHODSDeter...

Serratia marcescens, a gram-negative enteric bacterium, is capable of secreting a number of proteins extraceliularly. The types of activity found in the growth media include proteases, chitinases, a nuclease, and a lipase. Genetic studies have been undertaken to investigate the mechanisms used for the extracellular secretion of these exoproteins by S. marcescens. Many independent mutations affecting the extracellular enzymes were isolated after chemical and transposon mutagenesis. Using indicator media, we have identified loci involved in the production or excretion of extraceliular protease, nuclease, or chitinase by S. marcescens. None of the mutations represented general extracellular-excretion mutants; in no case was the production or excretion of multiple exoproteins affected. A variety of loci were identified, including regulatory mutations affecting nuclease and chitinase expression. A number of phenotypically different protease mutants arose. Some of them may represent different gene products required for the production and excretion of the major metalioprotease, a process more complex than that for the other S. marcescens exoproteins characterized to date.The movement of molecules from the site of synthesis to a new location is a fundamental property of biological systems. Protein export to the cell envelope has been the object of intensive study in Escherichia coli (4), and mutations affecting this process have been identified (18,30). Many of these mutations are conditional, demonstrating that protein export to the cell envelope is essential to the cell. The extracellular secretion (or excretion) of some proteins into the growth medium can also be achieved by some bacteria (29, 32), although enteric bacteria as a group are not renowned for their ability to excrete proteins. In fact, E. coli only excretes proteins when it carries extrachromosomal elements specifying exoproteins. For some proteins, such as a-hemolysin, bacteriocins, and toxins (14,19,22,26,32), the release of the exoprotein usually requires the presence of other gene products; this implies that E. coli is not normally endowed with a general extracellular secretory system. Since E. coli is such a limited system for studying extracellular proteins, a range of other organisms has been investigated (29,32). Usually the exoproteins these organisms excrete are either toxins or degradatory proteins like proteases and nucleases.Some steps, such as signal sequence (31) recognition and processing, may be common to both envelope and extracellular protein translocation. Genes required for the export of both membrane-bound and extracellular proteins are essential to the cell, but genes required only for excretion appear not to be essential. By isolating mutants that are defective only in the excretion of extracellular proteins, it may be possible to separate this mechanism from envelope secretion.Mutations in Pseudomonas aeruginosa strains defective in the excretion of certain extracellular proteins have been isolated (41) formation of many but not ...

Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations. The deforming force is probably provided by the polar flagella of these highly motile bacteria. Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria. Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm. Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded. Entry of the bacteria appears to be the result of a process of forced endocytosis.

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