The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism.

Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.

Characterization of subtilase protease in Cryptosporidium parvum and C. hominis.

Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.

Analysis of the beta-tubulin genes from Enterocytozoon bieneusi isolates from a human and rhesus macaque.

Enterocytozoon bieneusi is the most common and clinically significant microsporidium associated with chronic diarrhea and wasting in immunocompromised humans. Albendazole, which is effective against several helminths, protozoa, and microsporidia, is relatively ineffective against infections due to E. bieneusi. A likely explanation for the observed clinical resistance to albendazole was discovered from sequence analysis of the E. bieneusibeta-tubulin from isolates from an infected human and a naturally infected rhesus macaque. The beta-tubulin of E. bieneusi has a substitution at Glu(198), which is one of six amino acids reported to be associated with benzimidazole sensitivity.

Host cell tropism underlies species restriction of human and bovine Cryptosporidium parvum genotypes.

It has been recognized recently that human cryptosporidiosis is usually caused by Cryptosporidium parvum genotype I ("human" C. parvum), which is not found in animals. Compared to C. parvum genotype II, little is known of the biology of invasion of the human-restricted C. parvum genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among C. parvum genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of C. parvum genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem. C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes. C. parvum genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast, C. parvum genotype I invaded only human small-bowel cells. The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C parvum genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.

Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target.

The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.