Research Article: Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

Date Published: November 12, 2015

Publisher: Public Library of Science

Author(s): Haili Zhang, Fengguang Guo, Guan Zhu, Laura J Knoll.

http://doi.org/10.1371/journal.ppat.1005250

Abstract

The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available.

Partial Text

Cryptosporidium parvum is a gastrointestinal parasite that can cause moderate to severe diarrhea in children and adults, and deadly opportunistic infection in AIDS patients [1, 2]. In addition, because Cryptosporidium oocysts are resistant to chemical stresses, such as chlorine treatment, it also frequently causes water-borne outbreaks around the world [3, 4]. Current treatment options for cryptosporidiosis are limited [1, 5]. In the United States, only nitazoxanide is approved by the Federal Drug Administration (FDA) to treat cryptosporidial infections in immunocompetent individuals, but this is not approved for immunocompromised patients [6–8].

In the present study, we found that the CpLDH protein is cytosolic during the motile, extracellular, stages of parasite growth, but associates with the PVM during the intracellular development. The PVM is a unique membrane structure, which hosts the intracellular development of apicomplexan parasites and facilitates interactions between these parasites and host cells. The Cryptosporidium PVM is unique from those of other apicomplexans, such as Plasmodium, Toxoplasma, and Eimeria, in that it localizes on the top of host cells, rather than in the cytosol. Therefore, Cryptosporidium is an intracellular, but extracytoplasmic, parasite. The protein composition of the cryptosporidial PVM is poorly understood. Currently, only a few proteins involved in fatty acid metabolism have been localized to the PVM, such as the long-type fatty acyl-CoA binding protein (CpACBP) [25], an oxysterol-binding protein-related proteins (CpORP1) [25], and the long chain fatty acid elongase (CpLCE) [26]. The discovery that CpLDH is a PVM-association protein suggests that this unique structure participates not only in fatty acid metabolism, but also in lactate fermentation. Since lactate cannot be reutilized by Cryptosporidium, we speculate that the PVM localization of CpLDH facilitates the quick release of lactate into host cells and/or the environment to eliminate the potential detrimental effect caused by the accumulation of this compound in the parasite.

 

Source:

http://doi.org/10.1371/journal.ppat.1005250

 

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