Cruz-Bustos T et al. (2018), An Intracellular Ammonium Transporter Is Necess...

XB-ART-54575

mSphere 2018 Jan 17;31:. doi: 10.1128/mSphere.00377-17.

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An Intracellular Ammonium Transporter Is Necessary for Replication, Differentiation, and Resistance to Starvation and Osmotic Stress in Trypanosoma cruzi.

Cruz-Bustos T , Potapenko E , Storey M , Docampo R .

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Trypanosoma cruzi, the etiologic agent of Chagas disease, undergoes drastic metabolic changes when it transits between a vector and mammalian hosts. Amino acid catabolism leads to the production of ammonium (NH4+), which needs to be detoxified. However, T. cruzi does not possess a urea cycle, and it is unknown how intracellular levels of ammonium are controlled. In this work, we identified an intracellular ammonium transporter of T. cruzi (TcAMT) that localizes to acidic compartments (reservosomes, lysosomes). TcAMT has 11 transmembrane domains and possesses all conserved and functionally important amino acid residues that form the pore in other ammonium transporters. Functional expression in Xenopus oocytes followed by a two-electrode voltage clamp showed an inward current that is NH4+ dependent at a resting membrane potential (V h ) lower than -120 mV and is not pH dependent, suggesting that TcAMT is not an NH4+/H+ cotransporter but an NH4+ or NH3/H+ transporter. Ablation of TcAMT by clustered regularly interspaced short palindromic repeat analysis with Cas9 (CRISPR-Cas9) resulted in significant defects in epimastigote and amastigote replication, differentiation, and resistance to starvation and osmotic stress. IMPORTANCETrypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH3), which needs to be detoxified. We report here the identification of an ammonium (NH4+) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress.

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Genes referenced: dnai1 mapt pam

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	FIG 1 . Localization of TcAMT in epimastigotes. (A) Overexpressed TcAMT (AMT-OE) detected with anti-HA antibodies (red) colocalizes with anticruzipain antibodies (green). (B) Overexpressed TcAMT (green) colocalizes with LysoTracker (LT; red). (C) Endogenously tagged TcAMT (AMT-3×HA; red) does not colocalize with anticruzipain (green). (D) Endogenously tagged TcAMT (green) colocalizes with LysoTracker (red). DIC, differential interference contrast microscopy; CZP, cruzipain. Yellow indicates merged results. DAPI staining appears in blue. (A to D) Bars = 5 µm.
	FIG 2 . Localization of TcAMT in infective stages. (A) TcAMT-OE detected with anti-HA antibodies (red) partially colocalizes with anticruzipain antibodies (green) in trypomastigotes. (B) Endogenously tagged TcAMT (AMT-3×HA; green) colocalizes with LysoTracker (LT; red) in trypomastigotes. (C) TcAMT-OE (red) colocalizes with anticruzipain (green) in amastigotes. (D) Endogenously tagged TcAMT (green) colocalizes with LysoTracker (red) in amastigotes. DIC, differential interference contrast microscopy. DAPI staining appears in blue. (A to D) Bars = 5 µm.
	FIG 3 . Protein and mRNA expression after hyposmotic stress. (A) Western blot analysis of TcAMT-OE epimastigotes maintained under isosmotic (Iso) or hyposmotic (Hypo) conditions for up to 6 h using antibodies against HA. Reaction mixtures with antitubulin antibodies (Tub.) were used as a loading control (lower panel). Molecular weight markers are shown on the left. Relative densities (RD) compared to those of the corresponding isosmotic control are shown below the blot. Lane C is time 0. (B) qRT-PCR of epimastigotes maintained under isosmotic or hyposmotic conditions for 30 min. (C) Western blot analysis of TcAMT-OE epimastigotes maintained in PBS or LIT medium for 20 h using antibodies against HA. Antitubulin antibodies were used as a loading control (lower panel). Molecular mass markers (lane M) are shown on the left. WT, wild type. (D) qRT-PCR of epimastigotes maintained in PBS or LIT medium for 20 h. Values are means ± standard deviations (SD) (n = 3). *, P < 0.001; **, P < 0.0001 by two-way analysis of variance (ANOVA).
	FIG 4 . Ammonium-induced currents in TcAMT-expressing oocytes. (A) Average step protocol data recorded in oocytes injected with TcAMT cRNA (red and pink) and DEPC (blue and light blue) in the presence of 10 mM NH4+ (red and blue) and in regular ND96 solution (pink and light blue) (I, intensity); (B) same data (most hyperpolarized part of curve) normalized as a percentage of the current amplitude increase in the presence of ammonium in control oocytes (blue) and those expressing TcAMT (red).
	FIG 5 . Voltage and ammonium concentration dependence of TcAMT. (A) Inward and outward current transients induced by brief (10-s) application of NH4+ at different concentrations (1, 2, 5, 10, and 20 mM) and Vhs (−60, −90, −130, and −150 mV); (B, C) averaged amplitudes of transients induced by application of NH4+ in different concentrations recorded at Vhs equal to −130 mV (B) and −60 mV (C).
	FIG 6 . pH dependence of TcAMT. (A, B) Inward and outward current transients induced by brief (10-s) application of 10 mM NH4+ at different pHs (5.5, 6.0, 7.0, and 8.0) and Vhs (−60, −130, and −150 mV) in DEPC (A)- and TcAMT cRNA (B)-injected oocytes; (C) averaged amplitudes of transients induced by application of NH4 at different pHs recorded at Vhs equal to −130 mV and −60 mV; (D) dose dependence of currents recorded at a Vh of −120 mV in the presence of low concentrations of ammonium (1, 0.5, and 0.1 mM) in DEPC (blue)- and TcAMT cRNA (red)-injected oocytes.
	FIG 7 . TcAMT knockout cells. (A) Schematic representation of the strategy used to generate a TcAMT-KO mutant by CRISPR-Cas9-induced homologous recombination. A double-stranded genomic DNA (gDNA) break was produced by Cas9 at nucleotide (nt) 5 of the TcAMT ORF. DNA was repaired with a blasticidin S-deaminase (BSD) cassette containing ~500-bp homologous regions from the TcAMT locus. Arrows show primers that were used to verify gene deletion by PCR. 5′ and 3′ UTR, 5′ and 3′ untranslated regions, respectively; Fwd, forward; Rv, reverse; PAM, photospacer adjacent motif. (B) PCR analysis using gDNA isolated from wild-type (WT) and TcAMT-KO (KO) cell lines. The intact locus generates a PCR product of 1,908 bp, while the replaced locus generates a fragment of 762 bp. Lane C, PCR negative control; lane L, molecular ladder. The control was from the same gel and is separated because duplicate samples of the TcAMT-KO cell lines that were in the original gel were deleted.
	FIG 8 . Phenotypic changes in TcAMT-KO mutant epimastigotes. (A) Growth of wild-type (WT) and TcAMT-KO epimastigotes in LIT medium. Values are means ± SD (n = 3), and differences are significant from 3 to 15 days. *, P < 0.001 by Student’s paired t test. (B) Percentages of metacyclic trypomastigotes in epimastigote cultures after incubation in TAU 3AAG medium. (C) Effect of TcAMT knockout on trypomastigote infection of Vero cells. There were no significant differences in percentages of infected Vero cells. (D) Effect of TcAMT knockout on amastigote replication after 48 h. Differences were significant. (B to D) Values are means ± SD (n = 3, 5, and 4, respectively). n.s., no significant difference. *, P < 0.0001; , P < 0.05 by one-way and two-way ANOVA with multiple comparisons.
	FIG 9 . Autophagy in TcAMP-KO mutant epimastigotes. (A) Representative fluorescence microscopy images of wild-type (WT) and TcAMT-KO (AMT-KO) epimastigotes labeled with anti-TcATG8.1 antibody after incubation in LIT medium or PBS for 20 h. DAPI staining and DIC images are also shown. Bars = 5 µm. (B) Numbers of autophagosomes per cell under the different conditions shown in panel A. (C) Percentages of cells with autophagosomes under the conditions shown in panel A. (D) Regulatory volume decreases in epimastigotes. Cells suspended in isosmotic buffer were diluted with water to a final osmolarity of 150 mosM after 2 min, and relative changes in cell volume were monitored by determining absorbance at 550 nm as described in Materials and Methods. All experiments were done with the same amount of cells. Values are means ± SD from three independent experiments and are expressed as arbitrary absorbance units. Values are means ± SD (n = 3). *, P < 0.001; **, P < 0.0001 by one-way and two-way ANOVA with multiple comparisons.

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