TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi

Abstract

Summary We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N-terminal regulatory SPX domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low affinity transporter with a Km for Pi in the millimolar range, and sodium-dependency. Epimastigotes overexpressing TcPho91-GFP have significantly higher levels of pyrophosphate (PPi) and short chain polyphosphate (polyP), suggesting accumulation of Pi in these cells. Moreover, when overexpressing parasites were maintained in a medium with low Pi, they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N-terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PPi and short-chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in Pi homeostasis in T. cruzi.

Polyphosphate and acidocalcisomes

“…After water release to the extracellular medium amino acids, cations and P i would return to the cytosol helping the regulatory volume decrease (RVD). Evidence in favor of this model is the presence of a phosphodiesterase C (PDEC), which would terminate cyclic AMP stimulation, in the spongiome of the contractile vacuole [51], the inhibition of RVD by PDEC inhibitors [52], microscopic evidence of fusion of these organelles [53], and the presence in the contractile vacuole of a sodium-phosphate symporter that could be involved in recycling of P i produced by the hydrolysis of polyP during RVD [54]. In addition, proteins involved in organellar fusion were detected in the contractile vacuole (SNAREs, TcRab32), and in acidocalcisomes (TcVAMP7) [31, 53].…”

The origin and evolution of the acidocalcisome and its interactions with other organelles

“…2). A sodium-phosphate transporter that could be involved in recycling of P i produced by the hydrolysis of polyP during RVD was found in the bladder of the CVC [82]. In addition, proteins involved in organellar fusion were detected in the CVC (SNAREs, TcRab32), and in acidocalcisomes (TcVAMP7) [66, 57].…”

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…These authors evaluated the efficiency of the method for knocking out the genes encoding a conserved hypothetical protein and calreticulin in T. cruzi epimastigotes, in the presence of a repair template containing a stop sequence. After evaluating several clones none of them were null mutants, although a few clones were single knockouts, a result that has been previously observed in attempts to generate knockouts in T. cruzi by conventional methods (Jimenez and Docampo 2015;Li et al 2011). Interestingly, this was useful for generating T. cruzi parasites exhibiting flagellar and cytokinesis defects in a substantial population of epimastigotes where RPN complexes were delivered to target proven essential genes for the flagellar structure, as observed by brightfield microscopy (Soares Medeiros et al 2017).…”

“…These authors evaluated the efficiency of the method for knocking out the genes encoding a conserved hypothetical protein and calreticulin in T. cruzi epimastigotes, in the presence of a repair template containing a stop sequence. After evaluating several clones none of them were null mutants, although a few clones were single knockouts, a result that has been previously observed in attempts to generate knockouts in T. cruzi by conventional methods (Jimenez and Docampo ; Li et al. ).…”