Putative cis-regulatory elements associated with heat shock genes activated during excystation of Cryptosporidium parvum

Abstract

Background: Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and C. parvum, leading to acute, persistent and chronic diarrhea worldwide. Although the complications of this disease can be serious, even fatal, in immunocompromised patients of any age, they have also been found to lead to long term effects, including growth inhibition and impaired cognitive development, in infected immunocompetent children. The Cryptosporidium life cycle alternates between a dormant stage, the oocyst, and a highly replicative phase that includes both asexual vegetative stages as well as sexual stages, implying fine genetic regulatory mechanisms. The parasite is extremely difficult to study because it cannot be cultured in vitro and animal models are equally challenging. The recent publication of the genome sequence of C. hominis and C. parvum has, however, significantly advanced our understanding of the biology and pathogenesis of this parasite.

Methodology/principal findings: Herein, our goal was to identify cis-regulatory elements associated with heat shock response in Cryptosporidium using a combination of in silico and real time RT-PCR strategies. Analysis with Gibbs-Sampling algorithms of upstream non-translated regions of twelve genes annotated as heat shock proteins in the Cryptosporidium genome identified a highly conserved over-represented sequence motif in eleven of them. RT-PCR analyses, described herein and also by others, show that these eleven genes bearing the putative element are induced concurrent with excystation of parasite oocysts via heat shock.

Conclusions/significance: Our analyses suggest that occurrences of a motif identified in the upstream regions of the Cryptosporidium heat shock genes represent parts of the transcriptional apparatus and function as stress response elements that activate expression of these genes during excystation, and possibly at other stages in the life cycle of the parasite. Since heat shock and excystation represent a critical step in the development of the infectious sporozoite form of Cryptosporidium, these results provide important insight into the pathogenicity of the parasite.

Figure 1. Distribution of C. parvum heat shock genes by chromosome.

The symbols represent the positions of each of the 12 single copy HSP genes on the eight C. hominis chromosomes. Genes oriented (5′ to 32) left to right are indicated by red triangles oriented to the right; genes oriented in the opposite orientation are indicated by black triangles oriented to the left.

Figure 2. Highest-Scoring motifs found by Gibbs-Sampling algorithms AlignACE and MEME.

A and B show the highest-scoring motifs found by AlignACE and MEME, respectively. Maximum a-priori Probability (MAP) and Log Likelihood Ratio (LLR) scores are displayed below their respective motifs and are well above cutoffs used in previous studies with the same algorithms. The first occurrence of the motif is located, on average, 125 bp upstream of the transcription start codon (see Table 1).

Figure 3. Excystation of Cryptosporidium parvum oocysts.

To induce exystation, C. parvum oocysts were incubated for one hour in excystation medium at: A) 25°C; or B) 37°C (see details in the Materials and Methods), and the resulting parasites were observed by phase microscopy at 40× magnification [inset at ∼100×]. Unexcysted oocysts appear as refractile spheres, sporozoites appear as dark crescent shaped cells, and oocyst ghosts appear as dark black spheres (see insets for close-up).