Nikolai V. Khramtsov

Cloning and Analysis of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Cytoplasmic Form Hsp70

ABSTRACT. An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

Molecular and phylogenetic analysis of Cryptosporidium muris from various hosts

Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

Virus‐like, double‐stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double‐stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L‐dsRNA) had homology with viral RNA‐dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S‐dsRNA) had limited similarity with mitogen‐activated c‐June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus‐like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parvum, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Association of RNA Polymerase Complexes of the Parasitic Protozoan Cryptosporidium parvum with Virus-Like Particles: Heterogeneous System

ABSTRACT RNA polymerase complexes were purified from Cryptosporidium parvum, a parasitic protozoan known to infect many species of mammals including humans. Western blot analysis revealed the association of the complexes with two different proteins, encoded by large and small segments of viral double-stranded RNAs. Each complex was found to contain only double-stranded RNA, both double- and single-stranded RNA, or only single-stranded RNA. Maximum RNA-dependent RNA polymerase activity was observed within the complexes containing both double- and single-stranded RNAs. These complexes possessed both transcriptase and replicase polymerase activities. Virus-like particles with a diameter of 31 nm were copurified with RNA polymerase complexes, and buoyant density and polymerase studies suggest that C. parvum harbors a putative double-stranded RNA virus which separately encapsidates the large and small RNA segments. The mechanism of replication and other characteristics of this virus are similar to those of the viruses of the family Partitiviridae, previously identified only in fungi and plants.

The putative acetyl-coa synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-coa synthetases

We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns. The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species. Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs. This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine-binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family.

Sequence of the parasitic protozoan, Cryptosporidium parvum, putative protein disulfide isomerase-encoding DNA

A composite 1876-bp DNA encoding a putative protein disulfide isomerase (PDI) has been constructed from clones isolated from Cryptosporidium parvum (C. parvum) genomic and cDNA libraries and the nucleotide sequence determined. As predicted from the open reading frame (ORF), the protein product has a predicted molecular size of 54 kDa and a high degree of homology to PDIs from other species.

Sequence of the gene encoding hsp90e from Cryptosporidium parvum

A composite 2364 nt DNA sequence with an open reading frame (ORF) encoding an endoplasmic reticulum-associated heat shock protein 90 (CpHsp90e) was determined from clones isolated from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Transcription was verified by isolation of a clone from a cDNA library with a similar restriction map to that observed with genomic DNA. The predicted protein consists of 787 amino acids, has a predicted molecular size of 89.2 kDa, and was found to share strong homology with other endoplasmic reticulum-associated hsp90 proteins.

Cloning and molecular characterization of a gene encoding a Cryptosporidium parvum putative 20S proteasome β1-type subunit.

A DNA sequence composed of 1281 nucleotides (nt) consisting of a single open reading frame (ORF) encoding a putative 20s proteasome β1—type subunit was isolated from clones derived from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Southern blot analysis suggested that the sequenced DNA exists in the C. parvum genome as a single copy; transcription was verified through reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA isolated from C. parvum sporozoites. The predicted protein consists of 210 amino acids (aa), contains characteristic amino acids common to all proteasomal subunits, and shares stronger similarity to the βl-type subunit of yeast than to other types of β-subunits.