Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 30;13(3):e1006307.
doi: 10.1371/journal.ppat.1006307. eCollection 2017 Mar.

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Laura Jeacock  1 , Nicola Baker  1 , Natalie Wiedemar  2   3 , Pascal Mäser  2   3 , David Horn  1
Affiliations

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Laura Jeacock et al. PLoS Pathog. .

Abstract

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. T. b. brucei tolerates the loss of all three AQPs.
(A) The schematic maps indicate the AQP1 and AQP2-3 regions replaced by selectable markers as also indicated on the right. Δ indicates the regions deleted while the probes used for Southern blotting are shown above the maps. H, HpaI; S, SacII. (B) The Southern blots indicate deletion of the AQP1 alleles in aqp1 and three independent aqp1-2-3 strains. Wild-type (WT) is shown for comparison. Genomic DNA was digested with HpaI. (C) The Southern blots indicate deletion of the AQP2-3 alleles in aqp1-2-3 strains. WT is shown for comparison. Genomic DNA was digested with SacII.
Fig 2
Fig 2. The T. b. brucei AQPs have minimal impact on fitness or osmoregulation.
(A) Cumulative growth-curves for wild-type (WT), aqp1, aqp2-3 and aqp1-2-3 null-strains. (B) Dose-response curves for pentamidine. (C) Hypo-osmotic shock assay. Open symbols, Earle’s salt buffer; filled symbols, buffer diluted 50:50 with H2O. The recovery phase is shown. The phase-contrast images show two shocked and swollen cells (at left) and a recovered cell (at right). Scale-bar, 5 μm. DNA was counter-stained with DAPI (blue).
Fig 3
Fig 3. Glycerol uptake and utilisation is perturbed in aqp-null T. b. brucei.
(A) ATP levels were assessed in the strains indicated after incubation in 5 mM glucose or glycerol. Readings were taken in triplicate and normalised to substrate only. * indicates significantly different (P<0.001) to wild-type (WT) using an ANOVA test in GraphPad Prism. Error bars, SD. (B) Radiolabelled glycerol uptake was assessed in the strains indicated. Readings were taken in quadruplicate. * indicates significant difference (P<0.05) using a Student’s t-test. Error bars, SD.
Fig 4
Fig 4. aqp-null T. b. brucei display defective glycerol-efflux and respiratory inhibitor-sensitivity.
(A) Bloodstream T. brucei express a SHAM-sensitive mitochondrial trypanosome alternative oxidase (TAO). Under aerobic conditions, TAO activity allows ATP production without glycerol production as indicated by the black lines (left-hand blue ‘cell’). SHAM blocks TAO-activity, leading to the anaerobic production of glycerol, which is toxic if not removed, as indicated by the black lines (right-hand blue ‘cell’). SHAM dose-response curves for wild-type (WT) and aqp1-2-3 null-cells. EC50 values are indicated. (B) In the presence of SHAM and glycerol, the glycerol inhibits glycerol kinase (GK), also preventing ATP-production by the anaerobic route (blue ‘cell’). SHAM dose-response curves as in A but in the presence of 10 mM glycerol. (C) Propyl gallate and octyl gallate dose-response curves for wild-type (WT) and aqp1-2-3 null-cells. EC50 values are indicated. (D) SHAM EC50 values +/- 10 mM glycerol from A-B and also from aqp2, aqp2-3 and aqp1-2-3 cells re-expressing GFPAQP2. * indicates significantly different (P<0.01) to WT using an ANOVA test in GraphPad Prism. Pairwise comparisons +/- glycerol, except in the case of the aqp1-2-3 null, indicated significant (P <0.001) differences using a Student’s t-test. Error bars, SD. The images to the right show re-expression of GFPAQP2 in aqp1-2-3 null-cells.
Fig 5
Fig 5. Respiratory inhibitor-sensitivity in T. b. gambiense isolates and AQP-mediated glycerol transport.
(A) SHAM EC50 values for the T. b gambiense strains are indicated +/- glycerol. The inset shows pentamidine EC50 values. * indicates significantly different (P<0.05) to STIB930 using an ANOVA test in GraphPad Prism. All pairwise comparisons +/- 10 mM glycerol also indicated significant (P <0.001) differences using a Student’s t-test. Error bars, SD. (B) Propyl gallate and (C) Ascofuranone EC50 values. Other details as in A. (D) Model for glycerol transport by AQPs in T. b. gambiense. The weight of the arrows indicates relative impact on glycerol utilisation and efflux, with AQP2 being the major contributor; note that transport across both the plasma and glycosomal membranes contributes to glycerol utilisation and efflux, see the text for more details. The right-hand panel indicates the situation in melarsoprol-resistant (reduced melarsoprol uptake) and SHAM-sensitive (reduced glycerol efflux) clinical isolates where a chimeric AQP2/3 replaces AQP2 and AQP3.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Brun R, Blum J, Chappuis F, Burri C (2010) Human African trypanosomiasis. Lancet 375: 148–159. 10.1016/S0140-6736(09)60829-1 - DOI - PubMed
    1. Priotto G, Kasparian S, Mutombo W, Ngouama D, Ghorashian S, et al. (2009) Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet 374: 56–64. 10.1016/S0140-6736(09)61117-X - DOI - PubMed
    1. Iten M, Mett H, Evans A, Enyaru JC, Brun R, et al. (1997) Alterations in ornithine decarboxylase characteristics account for tolerance of Trypanosoma brucei rhodesiense to D,L-α-difluoromethylornithine. Antimicrob Agents Chemother 41: 1922–1925. - PMC - PubMed
    1. Kibona SN, Matemba L, Kaboya JS, Lubega GW (2006) Drug-resistance of Trypanosoma b. rhodesiense isolates from Tanzania. Trop Med Int Health 11: 144–155. 10.1111/j.1365-3156.2005.01545.x - DOI - PubMed
    1. Robays J, Nyamowala G, Sese C, Betu Ku Mesu Kande V, Lutumba P, et al. (2008) High failure rates of melarsoprol for sleeping sickness, Democratic Republic of Congo. Emerg Infect Dis 14: 966–967. 10.3201/eid1406.071266 - DOI - PMC - PubMed

Publication types

LinkOut - more resources