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Induction of novel protein synthesis by opsonized Histoplasma capsulatum ingested by murine peritoneal macrophages
K Kamei1, E Brummer, K V Clemons
1Department of Medicine, Santa Clara Valley Medical Center, San Jose, California, USA.
Abstract:
It is known that Histoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closely related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form of H. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingested H. capsulatum with [35S]-methionine labeling. Cycloheximide at 5 to 10 micrograms/ml was used to preferentially inhibit macrophage uptake of [35S]-methionine without affecting H. capsulatum uptake. Protein synthesis by H. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killed H. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingested H. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingested H. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.
Insights
Histoplasma capsulatum adapts to macrophage environments by altering protein synthesis. This study reveals specific metabolic changes in ingested yeast-form fungi, crucial for understanding their survival and multiplication.
Area of Science:
- Medical Mycology
- Cellular Microbiology
- Pathogen-Host Interactions
Background:
- Histoplasma capsulatum survives and replicates within macrophages, a key factor in its pathogenicity.
- The precise mechanisms enabling H. capsulatum to resist the intraphagolysosomal environment remain unclear.
Purpose of the Study:
- To investigate the metabolic state of yeast-form H. capsulatum (isolates G217B and CDC 105) after ingestion by macrophages.
- To elucidate the protein synthesis patterns of ingested H. capsulatum to understand its adaptation strategies.
Main Methods:
- Utilized [35S]-methionine labeling to track protein synthesis in ingested H. capsulatum.
- Employed cycloheximide to selectively inhibit host cell methionine uptake, ensuring focus on fungal protein synthesis.
- Analyzed protein synthesis changes using SDS-PAGE and fluorography, comparing results to controls (fungi in medium, heat-killed fungi).
Main Results:
- Ingested H. capsulatum (G217B and CDC 105) synthesized 4-5 novel proteins and increased synthesis of 9-17 existing proteins.
- Synthesis of 9-10 constitutive proteins was decreased in ingested fungi.
- Ten of the altered proteins were common to both H. capsulatum strains, indicating conserved adaptation mechanisms.
Conclusions:
- Metabolic reprogramming, specifically altered protein synthesis, is evident in H. capsulatum upon macrophage ingestion.
- These adaptive changes likely facilitate H. capsulatum's survival and multiplication within the hostile intraphagolysosomal environment.
- Understanding these fungal adaptations provides insights into Histoplasma pathogenesis.