A noncytolytic α toxin recombinant protein protects turkeys against Clostridium septicum challenge.

Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial species, has been implicated as the causative agent of gangrenous dermatitis. A recombinant noncytolytic C. septicum α toxin (NCAT) peptide was developed for use as a vaccine and demonstrated to be safe at concentrations as high as 1 mg/ml. NCAT, used as a purified antigen, partially purified antigen, or in combination with native antigens, was compared to salt-fractionated α toxin combined with denatured C septicum bacteria (native) in a vaccination trial. Three-day-old poults were placed into one of five groups and received two, 0.2-ml vaccinations 5 wk apart. Subcutaneous challenge with 3.2 x 10(7) log phase C. septicum resulted in 78% to 95% of the vaccinated birds surviving challenge compared to 48% of sham-injected controls. By ELISA analysis on NCAT-coated plates, birds receiving vaccines containing the recombinant NCAT peptide showed significantly higher blood serum antibody concentrations than did birds receiving vaccines containing native antigens or alum controls. Additionally, high levels of maternally transferred antibodies reactive to NCAT-purified antigens found in the pre-immune sera from naive 3-day-old poults suggest that the tertiary structure of the NCAT peptide has a high homology to the native protein structure. In conclusion, our study showed that the use of a vaccine comprised of a noncytolytic recombinant α toxin peptide antigen provided clinical protection equal to the use of vaccines formulated with inactivated native proteins at a reduced overall cost.

Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

Altered expression of antimicrobial peptide genes in the skin of dogs with atopic dermatitis and other inflammatory skin conditions.

BACKGROUND: Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced production of several skin antimicrobial peptides, but more recent data have called those results into question. HYPOTHESIS/OBJECTIVES: To compare the mRNA expression of seven antimicrobial peptide genes in lesional and adjacent nonlesional skin biopsy specimens from dogs with AD with those from normal dogs and from dogs experiencing other inflammatory skin conditions. ANIMALS: Normal dogs and patients with AD or other inflammatory skin conditions were enrolled with owner permission and approval of the Institutional Animal Care and Use Committee. METHODS: Transcripts were measured by quantitative RT-PCR using a standard curve assessment. RESULTS: Normal transcript levels for all seven antimicrobial peptides varied depending on the body site assessed. Transcripts for secretory leukocyte proteinase inhibitor (SLPI) and skin-derived antileucoproteinase (SKALP; also known as elafin) were typically ~10-fold greater in number than transcripts for the canine ß-defensins (CBD)-1, -102, -103, -122 and -124. Transcripts for SKALP, SLPI, CBD-1, CBD-103 and CBD-122 were lower in both lesional and adjacent nonlesional skin from dogs with AD in comparison to normal skin. Transcripts were reduced to a similar extent versus normal dogs in skin of dogs with inflammatory skin conditions from both lesional and nonlesional biopsies, except for CBD-122, which was reduced only in lesional skin. Compared with normal dog skin, transcripts for CBD-102 and CBD-124 were unaffected in dogs with AD. CONCLUSIONS AND CLINICAL IMPORTANCE: Both SKALP and SLPI may be important contributors to skin innate immunity, but their decreased expression in AD patients does not account for increased skin infections compared with other skin conditions.

The Cryptosporidium parvum transcriptome during in vitro development.

Cryptosporidiosis is caused by an obligate intracellular parasite that has eluded global transcriptional or proteomic analysis of the intracellular developmental stages. The transcript abundance for 3,302 genes (87%) of the Cryptosporidium parvum protein coding genome was elucidated over a 72 hr infection within HCT8 cells using Real Time-PCR. The parasite had detectable transcription of all genes in vitro within at least one time point tested, and adjacent genes were not co-regulated. Five genes were not detected within the first 24 hr of infection, one containing two AP2 domains. The fewest genes detected were at 2 hr post infection, while 30% (985) of the genes have their highest expression at 48 and/or 72 hr. Nine expression clusters were formed over the entire 72 hr time course and indicate patterns of transcriptional increases at each of the 7 time points collected except 36 hr, including genes paralleling parasite 18S rRNA transcript levels. Clustering within only the first 24 hr of infection indicates spikes in expression at each of the 4 time points, a group paralleling 18S rRNA transcript levels, and a cluster with peaks at both 6 and 24 hr. All genes were classified into 18 functional categories, which were unequally distributed across clusters. Expression of metabolic, ribosomal and proteasome proteins did not parallel 18S rRNA levels indicating distinct biochemical profiles during developmental stage progression. Proteins involved in translation are over-represented at 6 hr, while structural proteins are over-represented at 12 hr. Standardization methods identified 107 genes with <80% at a single of its total expression at a single time point over 72 hr. This comprehensive transcriptome of the intracellular stages of C. parvum provides insight for understanding its complex development following parasitization of intestinal epithelial cells.

Expression and regulation of IL-22 by bovine peripheral blood gamma/delta T cells.

IL-22 is a novel T and NK cell cytokine that belongs to the IL-10 cytokine family. Here we report the identification of a bovine IL-22 ortholog that is expressed by mitogen activated bovine peripheral blood gamma/delta T cells. The full-length bovine IL-22 cDNA contained a 68 bp 5'-untranslated region (UTR), a 570-bp open reading frame, and a 480-bp 3'-UTR. The deduced pre-IL-22 has 190 amino acid residues containing a secretory signal peptide from amino acids 1-33 and several potential N-glycosylation sites. The mature protein is predicted to be a secreted, alpha-helical molecule. The bovine IL-22 gene is approximately 7.5 kb in length and is comprised of five introns and six exons, and the first exon is non-coding. Computer analysis and gel shift assay showed that the -1132 and -879 region in the 5' upstream gene sequence contained putative transcription factor binding sites for STATx, Sox-5/9, Sp1, Ik-1, and AREB6. Mutagenesis of STATx and Sox5/9 binding sites decreased promoter functionality by approximately 50%, suggesting their importance in transcription regulation of IL-22. Expression of IL-22 transcripts induced by various mitogens indicated existence of two regulatory control pathways in gamma/delta T cells; IL-2 or PMA treatment induced a slow accumulation of IL-22 mRNA without affecting the maximum induction pathway, whereas ConA treatment rapidly induced a limited amount of IL-22 transcripts. Similar maximal levels of IL-22 transcripts could be induced in gamma/delta T cells and alpha/beta T cells. We conclude that bovine gamma/delta T cells are important sources of IL-22 and suggest a role for this cytokine in regulating immune responses at mucosal surfaces, including the gut.

A genome-sequence survey for Ascogregarina taiwanensis supports evolutionary affiliation but metabolic diversity between a Gregarine and Cryptosporidium.

We have performed a whole-genome-sequence survey for the gregarine, Ascogregarina taiwanensis and herein describe both features unique to this early diverging apicomplexan and properties that unite it with Cryptosporidium, the Coccidia, and the Apicomplexa. Phylogenetic trees inferred from a concatenated protein sequence comprised of 10,750 amino acid positions, as well as the large subunit rRNA genes, robustly support phylogenetic affinity of Ascogregarina with Cryptosporidium at the base of the apicomplexan clade. Unlike Cryptosporidium, Ascogregarina possesses numerous mitochondrion-associated pathways and proteins, including enzymes within the Krebs cycle and a cytochrome-based respiratory chain. Ascogregarina further differs in the capacity for de novo synthesis of pyrimidines and amino acids. Ascogregarina shares with Cryptosporidium a Type I fatty acid synthase and likely a polyketide synthase. Cryptosporidium and Ascogregarina possess a large repertoire of multidomain surface proteins that align it with Toxoplasma and are proposed to be involved in coccidian-like functions. Four families of retrotransposable elements were identified, and thus, retroelements are present in Ascogregarina and Eimeria but not in other apicomplexans that have been analyzed. The sum observations suggest that Ascogregarina and Cryptosporidium share numerous molecular similarities, not only including coccidian-like features to the exclusion of Haemosporidia and Piroplasmida but also differ from each other significantly in their metabolic capacity.

Characterization of a Cryptosporidium parvum protein that binds single-stranded G-strand telomeric DNA.

We have initiated a project to characterize telomere-associated proteins of Cryptosporidium parvum. Searching public databases with C. parvum expressed sequence tag (EST) sequences revealed one EST sequence that is highly similar to Gbp1p of Chlamydomonas reinhardtii (Cr Gbp1p), a protein that binds single-stranded telomeric DNA. This EST was used to clone a gene encoding a 198 amino acids long protein (CpGbp). Sequence analysis suggested that CpGbp contains two RNA recognition motif (RRMs) domains linked with a short hinge region. RT-PCR analysis showed that the mRNA expression of CpGbp was up- and down-regulated significantly comparing to that of CpDNAPol, suggesting a potential role of CpGbp playing in the parasite's life cycle. In Western blot analysis, monoclonal antibody against recombinant CpGbp identified one band (approximately 23kDa) specifically from cell extracts of C. parvum sporozoites. Confocal microscopy analysis with anti-CpGbp antibody localized CpGbp proteins to the nucleus, consistent with its potential role in telomere length regulation. In electrophoretic mobility shift assays (EMSAs), recombinant CpGbp bound oligonucleotide TG3 that bears three copies of C. parvum telomeric DNA G-strand repeat "TTTAGG", but not C-strand or double-stranded telomeric DNA sequences. To map the binding domain and to define the binding site of CpGbp, we constructed four CpGbp deletion mutants and synthesized ten TG3 mutants and tested their binding affinities by EMSAs. We found that only the RRM domain at N-terminus has oligonucleotide-binding ability in vitro. And the minimal sequence necessary for CpGbp's binding is "GTTTAGGTTTAG". These data support the notion that CpGbp represents a C. parvum single-stranded telomeric DNA binding protein.

Biphasic modulation of apoptotic pathways in Cryptosporidium parvum-infected human intestinal epithelial cells.

The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

Inhibition of apoptosis in Cryptosporidium parvum-infected intestinal epithelial cells is dependent on survivin.

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing severe diarrheal disease in a wide variety of mammals, including humans. C. parvum infection has been associated with induction of apoptosis in exposed epithelial cells, and we now demonstrate that apoptosis is restricted to a subset of cells actively infected with C. parvum. Approximately 20% of the infected cells underwent apoptosis within 48 h of infection, suggesting that the majority of the infected cells are rescued from apoptosis. C. parvum infection resulted in low-level activation of multiple members of the caspase family, including caspase-2, -3, -4, -6, -8, and -9. The kinetics of caspase activation correlated with apoptosis over a 48-h time course. Pan caspase inhibitors reduced apoptosis of epithelial cells infected by C. parvum. Furthermore, C. parvum infection inhibited staurosporine-induced apoptosis and caspase-3/7 activation at 24 h and 48 h. Infection with C. parvum led to upregulation of genes encoding inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2, XIAP, and survivin. Knockdown of survivin gene expression, but not that of c-IAP1, c-IAP2, or XIAP expression, increased caspase-3/7 activity as well as apoptosis of infected cells and decreased C. parvum 18S rRNA levels. These data suggest that the apoptotic response of infected intestinal epithelial cells is actively suppressed by C. parvum via upregulation of survivin, favoring parasite infection.

Identification of putative cis-regulatory elements in Cryptosporidium parvum by de novo pattern finding.

BACKGROUND: Cryptosporidium parvum is a unicellular eukaryote in the phylum Apicomplexa. It is an obligate intracellular parasite that causes diarrhea and is a significant AIDS-related pathogen. Cryptosporidium parvum is not amenable to long-term laboratory cultivation or classical molecular genetic analysis. The parasite exhibits a complex life cycle, a broad host range, and fundamental mechanisms of gene regulation remain unknown. We have used data from the recently sequenced genome of this organism to uncover clues about gene regulation in C. parvum. We have applied two pattern finding algorithms MEME and AlignACE to identify conserved, over-represented motifs in the 5' upstream regions of genes in C. parvum. To support our findings, we have established comparative real-time -PCR expression profiles for the groups of genes examined computationally. RESULTS: We find that groups of genes that share a function or belong to a common pathway share upstream motifs. Different motifs are conserved upstream of different groups of genes. Comparative real-time PCR studies show co-expression of genes within each group (in sub-sets) during the life cycle of the parasite, suggesting co-regulation of these genes may be driven by the use of conserved upstream motifs. CONCLUSION: This is one of the first attempts to characterize cis-regulatory elements in the absence of any previously characterized elements and with very limited expression data (seven genes only). Using de novo pattern finding algorithms, we have identified specific DNA motifs that are conserved upstream of genes belonging to the same metabolic pathway or gene family. We have demonstrated the co-expression of these genes (often in subsets) using comparative real-time-PCR experiments thus establishing evidence for these conserved motifs as putative cis-regulatory elements. Given the lack of prior information concerning expression patterns and organization of promoters in C. parvum we present one of the first investigations of gene regulation in this important human pathogen.

Regulation of apoptotic pathways in bovine gamma/delta T cells.

T lymphocytes bearing gamma/delta TCRs are a major population of T cells in neonatal calves and discrete subsets of gamma/delta T cells display tissue-specific accumulation and responsiveness to infection. To enhance our understanding of the immunobiology of gamma/delta T cells, we characterized the gene expression profile of circulating bovine gamma/delta T cells following stimulation with recombinant human IL-2 and ConA. Statistical analysis of microarray data identified 108 genes with significantly altered expression, including four genes associated with apoptosis. Real-time reverse transcription-PCR (RT-PCR) analysis of 15 genes related to apoptotic pathways showed that both the Fas-mediated and the mitochondrial apoptotic pathways were repressed in circulating bovine gamma/delta T cells in response to mitogen activation, indicating that stimulated peripheral bovine gamma/delta T cells are resistant to activation-induced apoptosis.

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

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.

Intron-containing beta-tubulin transcripts in Cryptosporidium parvum cultured in vitro.

The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the beta-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the beta-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced beta-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed beta-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing beta-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of beta-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in beta-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

Cryptosporidium parvum regulation of human epithelial cell gene expression.

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening diarrhoeal disease in immunocompromised individuals. Efforts to develop novel therapeutic strategies have been hampered by the lack of understanding of the pathogenesis of infection. To better understand the host response to C. parvum infection, gene expression profiles of infected human ileocecal adenocarcinoma cells were analysed by using Affymetrix oligonucleotide microarrays containing probe sets for 12,600 human genes. Statistical analysis of expression data from three independent experiments identified 223 genes whose expression was reproducibly regulated by C. parvum infection at 24 h post-inoculation (125 up-regulated and 98 down-regulated), 13 of which were validated by quantitative reverse transcriptase polymerase chain reaction analysis. This analysis revealed the consistent up-regulation of host heat-shock genes and genes for pro-inflammatory chemokines IL-8, RANTES, and SCYB5. Multiple genes for host actin and tubulin genes were up-regulated whereas genes for actin binding proteins were down-regulated, confirming previous observations of host cytoskeleton rearrangement in response to C. parvum infection. In addition, host genes associated with cell proliferation and apoptosis were differentially regulated, reflecting the complexity of host-parasite interaction. Together, this study demonstrated that C. parvum infection results in significant changes in host biochemical pathways and provides new insights into specific biological processes of infectious disease caused by an intracellular protozoan parasite.

The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma.

Coccidian parasites are transmitted via a fecal oocyst stage that is exceptionally resistant to environmental stress and harsh chemical treatments, which allows parasites to stably persist outside a host. Because of its oocyst durability Cryptosporidium parvum is a significant water- and food-borne pathogen of humans, as well as animals of agricultural importance. To date, only one apicomplexan oocyst membrane protein has been identified, Cryptosporidium oocyst wall protein 1 (COWP1). COWP1 has a highly cysteine-rich periodicity due to arrays of two apicomplexan-specific motifs, designated the type I and type II domains. In this study, exhaustive BLAST screening of a complete C. parvum genome sequence database resulted in identification of eight additional genes encoding similar arrays of cysteine-rich type I and/or type II domains. Transcript expression analysis revealed that all COWP genes are abundantly expressed at a time when developing oocysts are observed, roughly 48 to 72 h after inoculation of in vitro cultures. A monoclonal antibody recognizing COWP8 specifically localized to the C. parvum oocyst wall, supporting the hypothesis that multiple COWPs play a role in the oocyst wall structure. BLAST screening of the Toxoplasma gondii genome sequence database resulted in identification of a gene encoding at least one COWP homolog (TgOWP1), and this multiexon sequence information was used to isolate a full-length cDNA. Exhaustive screening of Plasmodium sp. genome sequence databases by using COWP genes as BLAST queries failed to detect similar proteins in PLASMODIUM: We therefore propose that the COWP family of proteins have a structural role in apicomplexan species that produce durable shed cysts capable of surviving environmental stress.