Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, United States
- Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Germany
- Harvard Center for Mass Spectrometry, Harvard University, United States
Figures
Figure 1 with 5 supplements
A lack of supplemental glutamine in growth medium protects intracellular T. cruzi amastigotes from the cytocidal effects of ketoconazole.
(A) Dose response curves at 66 hpi of benznidazole and (B) ketoconazole treatment, in the indicated media compositions, normalized to the largest mean in each treatment group. Mean (symbols) and …
Figure 1—figure supplement 1
Experimental schematic for in vitro infection and readouts.
Trypomastigotes (Tula-βgal) are incubated with mammalian host cells for 2 hr to allow invasion. Remaining extracellular parasites are subsequently removed by thorough rinsing of monolayers. …
Figure 1—figure supplement 2
Sensitivity to additional azole drugs is modulated by glutamine.
(A) Dose response curves of itraconazole, (B) ravuconazole, and (C) posaconazole treatment measured at 66 hpi. Treatment including media compositions are indicated and growth is normalized to the …
Figure 1—figure supplement 3
Removal of supplemental glutamine but not glucose maintains the proportion of infected host cells in the presence of azoles.
The number of infected cells per field (n = 20) at (A) 66 hpi and (B) 90 hpi, mean and standard deviations shown. Comparisons of means (D,F) were performed using a one-way ANOVA and Bonferroni’s …
Figure 1—figure supplement 4
Proline or histidine supplementation do not sensitize amastigotes to ketoconazole in the absence of glutamine.
Dose response curves of (A) proline and (B) histidine in the absence of supplemental glutamine (n = 3).
Figure 1—figure supplement 5
Slowed amastigote growth, antioxidants, or hypoxia does not prevent the cidal effects of ketoconazole.
(A) Microscopic counts of amastigotes per host cell (n = 40) and (B) proportion of infected cells (n = 20) at 66 hpi following treatment at 18 hpi with ketoconazole (5 nM) and/or GNF7686 (150 nM) …
Figure 2 with 2 supplements
Glutamine-derived carbons are incorporated into amastigote sterols and influence the buildup of lanosterol.
(A) Chromatogram from GC-MS detection of samples. Host-cell-derived cholesterol is seen at retention time 12.21, eburicol at 13.30, lanosterol at 13.04 and the internal standard at 12.98. (B) Table …
Figure 2—figure supplement 1
Time course establishes 52 hpi as optimal time point to harvest intracellular amastigotes following ketoconazole treatment.
Time course following treatment with ketoconazole (5 nM) in complete media. (A) Amastigotes per infected host cell (n = 40) and (B) infected cells per 20 fields are shown. Fifty-two hpi identified …
Figure 2—figure supplement 2
Endogenous lanosterol and eburicol but not host derived cholesterol are reliable quantifiable from isolated intracellular amastigotes.
Isolated amastigotes (52 hpi) were prepared on three independent occasions (biological) and each isolation was extracted three separate times (sterol extraction). The coefficient of variation of …
Figure 3 with 1 supplement
Treatment with BPTES increases incorporation of carbons from glutamine into endogenously synthesized amastigote sterols.
(A) Table of detectable isolated amastigote sterol species from and the percentage of natural sterols (i.e. without detectable 13C). The proportion of species found with the indicated number of …
Figure 3—figure supplement 1
BPTES does not inhibit oxygen consumption of isolated amastigotes from glutamine and sensitizes amastigotes to ketoconazole.
(A) Measurement of the oxygen consumption rate (OCR) of isolated amastigotes when glutamine is the sole carbon source. BPTES was injected three times, increasing the BPTES concentration by 5 μM per …
Figure 4 with 1 supplement
Addition of metabolites re-sensitize intracellular T. cruzi amastigotes to ketoconazole in the absence of glutamine.
(A) Schematic of endogenous sterol synthesis. Dash lined arrows indicate omission of steps for simplicity. (B) Microscopic counts of amastigotes per infected cell (n = 40) at 90 hpi treated with …
Figure 4—figure supplement 1
Addition of metabolites re-sensitize infected cultures to ketoconazole in the absence of glutamine.
(A) Microscopic counts of infected cells per field (n = 20) at 90 hpi treated with BPTES (5 μM), (B) αKG (10 mM), (C) FPP or (D) farnesol. Comparisons of means were performed using a one-way ANOVA …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Trypanosoma cruzi) | Tula-βgal | ATCC | PRA-330 | Tulahuén LacZ clone C4: PMID:8913471 |
| Cell line (Macaca mulatta) | LLC-MK2 | ATCC | CCL-7 | PMID:14449902/14449901 |
| Cell line (Homo sapiens) | NHDF | Lonza | CC-2509 | Normal Human Neonatal Dermal Fibroblasts |
| Chemical compound, drug | ketoconazole | Enzo | Cat# EI107 | ≥99% (HPLC) |
| Chemical compound, drug | GNF7686 | Vitas-M Laboratory | Cat# STK393240 | PMID:26186534 |
| Other | glutamine | Gibco | Cat# A2916801 | |
| Other | 13C-glutamine | Cambridge Isotope Laboratories, Inc | Cat# CLM-1822 | Chemical Purity 98% |
