Related Experiment Videos
Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism
J W Batanghari1, G S Deepe, E Di Cera
1Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA.
Abstract:
A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil. We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium-binding protein (CBP). Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium-binding sites. We also evaluated CBP1 expression by reverse transcription-polymerase chain reaction (RT-PCR) of yeasts grown in broth culture and within two host cell types, a macrophage-like cell line and respiratory epithelial cells. H. capsulatum yeasts expressed CBP1 in all of these settings. Splenocytes from mice immunized with H. capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts. In addition, after H. capsulatum yeasts were subjected to a calcium-free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP. These results suggest that CBP may function to provide yeasts with calcium when they are in a low-calcium environment, such as the phagolysosomal compartment within macrophages.
Insights
Histoplasma capsulatum yeast uses a calcium-binding protein (CBP) to survive in macrophages. This protein helps the fungus acquire calcium in low-calcium environments like the phagolysosome.
Area of Science:
- Mycology
- Cell Biology
- Infectious Diseases
Background:
- Histoplasma capsulatum exists as yeast in hosts and mycelia in soil.
- Yeast phase survives in macrophage phagolysosomes, differing in calcium needs.
- A calcium-binding protein (CBP) is released by the yeast phase.
Purpose of the Study:
- To investigate the role of CBP in Histoplasma capsulatum's survival within host cells.
- To characterize the CBP1 gene and its expression.
- To determine CBP's function in calcium acquisition by yeast.
Main Methods:
- Cloning and sequencing of the CBP1 gene.
- Reverse transcription-polymerase chain reaction (RT-PCR) to assess CBP1 expression.
- In vitro assays with purified CBP and yeast exposed to calcium-free conditions.
- Splenocyte proliferation assays from immunized mice.
Main Results:
- The CBP1 gene contains introns, a signal peptide, and calcium-binding sites.
- CBP1 was expressed by yeast in broth, macrophages, and epithelial cells.
- CBP was immunogenic in mice, and it aided yeast calcium uptake in low-calcium conditions.
Conclusions:
- CBP is produced by Histoplasma yeast during mammalian infection.
- CBP likely facilitates yeast survival by providing calcium in nutrient-poor phagolysosomes.