Spacious phagosome formation within mouse macrophages correlates with Salmonella serotype pathogenicity and host susceptibility.

Light microscopic studies indicated a correlation between the virulence for mice of different Salmonella serotypes and the ability to form or maintain spacious phagosomes (SP) within mouse macrophages. Although Salmonella typhimurium induced membrane ruffling, macropinocytosis, and SP formation in macrophages from BALB/c mice, serotypes which are nonpathogenic for mice produced markedly fewer SP. SP formation correlated with both serotype survival within mouse macrophages and reported lethality for mice. Time-lapse video microscopy demonstrated that the human pathogen S. typhi induced generalized macropinocytosis and SP formation in human monocyte-derived macrophages, indicating a similar morphology for the initial phases of this host-pathogen interaction. In contrast to bone marrow-derived macrophages from BALB/c mice, macrophages from S. typhimurium-resistant outbred (CD-1) and inbred (CBA/HN) mice did not initiate generalized macropinocytosis after bacterial infection and formed markedly fewer SP. These deficiencies were not due to the Ihy resistance genotype of these mice, as macrophages from mice that were congenic except for the Ihy locus demonstrated equal SP formation in response to S. typhimurium. The observation that S. typhimurium-resistant CD-1 and CBA/HN mice are deficient in the ability to form and/or maintain SP indicates that a variable host component is important for SP formation and suggests that the ability to induce or form SP affects susceptibility to S. typhimurium. When serotypes nonpathogenic for mice were used to infect BALB/c macrophages, or when CD-1 or CBA/HN mouse macrophages were infected by S. typhimurium, some of the SP that formed shrank within seconds. This rapid shrinkage suggests that SP maintenance is also important for S. typhimurium survival within macrophages. These studies indicate that both host and bacterial factors contribute to SP formation and maintenance, which correlate with Salmonella intracellular survival and the ability to cause lethal enteric (typhoid) fever.

Molecular size-fractionation during endocytosis in macrophages.

The sorting of macromolecules within and between membranous organelles is often directed by information contained in protein primary or secondary structure. We show here that absent such structural information, macromolecules internalized by endocytosis in macrophages can be sorted by size. After endocytosis, small solute probes of fluid-phase pinocytosis were recycled to the extracellular medium more efficiently than large solutes. Using macropinosomes pulse labeled with fluorescent dextrans, we examined the ability of organelles to exchange solute contents. Dextran exchange was optimal between organelles of similar age, and small dextrans exchanged more efficiently than large dextrans. Efferent solute movement, from lysosomes or phagolysosomes toward the plasma membrane, occurred through the same endocytic vesicles as afferent movement, toward lysosomes and this movement was solute size dependent. Remarkably, uniform mixtures of different-sized dextrans delivered into lysosomes separated into distinct organelles containing only one dextran or the other. Thus, the dynamics of endosomes and lysosomes were sufficient to segregate macromolecules by size. This intracellular size fractionation could explain how, during antigen presentation, peptides generated by lysosomal proteases recycle selectively from lysosomes to endosomes for association with class II MHC molecules.