Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms. After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles. In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells. Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells. Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells. We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization. We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L. Further, we report on differential processing of lysosomal hydrolases (such as cathepsin D and cathepsin L) associated with the isolated BCVs. Although there was some contamination of the S. typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y. pseudotuberculosis-containing vacuoles were predominately free of ER contamination. The Y. pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time. These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y. pseudotuberculosis traffics to lysosomes where they are degraded. The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y. pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.
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PMID:Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded. 980 87

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.
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PMID:The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence. 1532 89