Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum

Abstract

Background: The apicomplexan parasite Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium.

Results: We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum.

Conclusions: Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite.

Figure 1

Phylogenomic analysis pipeline. The procedures used to analyze, assess and manipulate the protein-sequence data at each stage of the analysis are diagrammed.

Figure 2

Cryptosporidium parvum genomic Southern blot. C. parvum genomic DNA, 5 μg per lane. Lanes were probed for the following genes: (1) aminopeptidase N; (2) glucose-6-phosphate isomerase; (3) leucine aminopeptidase; (4) pteridine transporter (BT-1); and (5) glutamine synthetase. Lanes (1-4) were restricted with BamH1 and lane (5) with EcoR1. The ladder is shown in 1 kb increments. See Additional data file 1 for probes and methods.

Figure 3

Region of leucine aminopeptidase multiple sequence alignment that illustrates several characters uniting apicomplexan sequences with plant and cyanobacterial sequences. The red box denotes an indel shared between apicomplexans, plants and cyanobacteria. The number preceeding each sequence is the position in the individual sequence at which this stretch of similarity begins. GenBank GI numbers for each sequence are as indicated in Additional data file 1. Colored boxes preceeding the alignment indicate the taxonomic group for the organisms named to the left. Red, apicomplexan; green, plant and cyanobacterial; blue, eubacterial; lavender, other protists and eukaryotes.

Figure 4

Phylogenetic analyses. (a) Leucine aminopeptidase; (b) glucose-6-phosphate isomerase; (c) tryptophan synthetase β subunit. Numbers above the branches (where space permits) show the puzzle frequency (with TREE-PUZZLE) and bootstrap support for both maximum parsimony and neighbor-joining analyses respectively. Asterisks indicate that support for this branch is below 50%. The scale is as indicated. GI accession numbers and alignments are provided in Additional data file 1.

Figure 5

Phylogenetic analyses of 1,4-α-glucan branching enzyme. Numbers above the branches (where space permits) show the puzzle frequency (TREE-PUZZLE) and bootstrap support for both maximum parsimony and neighbor-joining analyses respectively; Asterisks indicate that support for this branch is below 50%. The scale is as indicated. GI accession numbers and alignment are provided in Additional data file 1.

Figure 6

Expression profiles of select genes in C. parvum-infected HCT-8 cells. The expression level of each gene is calculated as the ratio of its RT-PCR product to that of C. parvum 18s rRNA. (a) glucose-6-phospate isomerase; (b) leucine aminopeptidase; (c) pteridine transporter (BT-1); (d) tyrosyl-tRNA synthetase; (e) calcium-dependent protein kinase; (f) dihydrofolate reductase-thymidine synthetase (DHFR-TS). The genes examined in (a-c, e) represent transferred genes of different origins; (d, f) represent non-transferred references. Error bars show the standard deviation of the mean of six independent time-course experiments.