Lysine crotonylation is widespread on proteins of diverse functions and localizations in Toxoplasma gondii.

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.

Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii.

Global Proteomic Analysis of Lysine Malonylation in Toxoplasma gondii.

Lysine malonylation (Kmal) is a new post-translational modification (PTM), which has been reported in several prokaryotic and eukaryotic species. Although Kmal can regulate many and diverse biological processes in various organisms, knowledge about this important PTM in the apicomplexan parasite Toxoplasma gondii is limited. In this study, we performed the first global profiling of malonylated proteins in T. gondii tachyzoites using affinity enrichment and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Three experiments performed in tandem revealed 294, 345, 352 Kmal sites on 203, 236, 230 malonylated proteins, respectively. Computational analysis showed the identified malonylated proteins to be localized in various subcellular compartments and involved in many cellular functions, particularly mitochondrial function. Additionally, one conserved Kmal motif with a strong bias for cysteine was detected. Taken together, these findings provide the first report of Kmal profile in T. gondii and should be an important resource for studying the physiological roles of Kmal in this parasite.

Prevalence and genotypes of Enterocytozoon bieneusi in pigs in southern China.

Enterocytozoon bieneusi is one of the most important causative agents of microsporidiosis, causing diarrhoea the symptoms of enteric disease in humans and animals. Although there is some information on the prevalence and genotypes of E. bieneusi in China, there is still a lack of data in pigs in southern China. In the present study, a total of 396 faecal specimens were collected from pigs in Zhejiang, Guangdong and Yunnan provinces in southern China, and were examined by nested PCR amplification of the ribosomal internal transcribed spacer (ITS) for the prevalence and genotypes of E. bieneusi. The overall prevalence of E. bieneusi in pigs was 31.57% (125/396), forming 15 genotypes, including 9 known genotypes (EbpC, EbpA, D, G, H, PigEBITS5, Henan-IV, KIN-1, CHS5) and 6 novel genotypes (GD1, ZJ1, ZJ2, YN1, YN2 and YN3), which were all clustered into Group 1. Moreover, multilocus sequence typing (MLST) showed that 6, 3, 4 and 5 types were identified in MS1, MS3, MS7 and MS4 loci, respectively, representing four multilocus genotypes (MLGs), designated as MLGs novel-1 to novel-4 in the present study. This is the first detailed study of E. bieneusi using MLST in pigs in southern China, which extended information about the distribution of E. bieneusi genotypes in China.

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression. Baby hamster kidney cells (BHK-21) were transfected with plasmids encoding GRA15 genes of either type I GT1 strain (GRA15I) or type II PRU strain (GRA15II). Gene expression patterns of transfected and nontransfected BHK-21 cells were investigated using RNA-sequencing analysis. GRA15I and GRA15II induced both known and novel transcriptional changes in the transfected BHK-21 cells compared with nontransfected cells. Pathway analysis revealed that GRA15II was mainly involved in the regulation of tumor necrosis factor (TNF), NF-κB, HTLV-I infection, and NOD-like receptor signaling pathways. GRA15I preferentially influenced the synthesis of unsaturated fatty acids in host cells. Our findings support the hypothesis that certain functions of GRA15 protein are strain dependent and that GRA15 modulates the expression of signaling pathways and genes with important roles in T. gondii pathophysiology. A greater understanding of host signaling pathways influenced by T. gondii effectors would allow the development of more efficient anti-T. gondii therapeutic schemes, capitalizing on disrupting parasite virulence factors to advance the treatment of toxoplasmosis.

Identification of two novel host proteins interacting with Toxoplasma gondii 14-3-3 protein by yeast two-hybrid system.

Toxoplasma gondii deploys many effector proteins in order to hijack and manipulate host cell signaling pathways, allowing parasite colonization, subversion of immune responses, and disease progression. T. gondii effector protein 14-3-3 (Tg14-3-3) promotes parasite dissemination inside the body, by enhancing the migratory ability of infected microglia and dendritic cells. Understanding both the mechanism of action and the host targets of Tg14-3-3 effector is important because of their importance to the parasite's virulence. The aim of the present study was to explore the function of Tg14-3-3 by utilizing the yeast two-hybrid system (Y2HS) to identify novel Tg14-3-3 interactors/substrates in host cells. A human cDNA library was screened using Tg14-3-3 as the bait. Tg14-3-3 (RH strain, Type I) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. The bait protein expression was validated by Western blotting analysis, auto-activation, and toxicity investigation compared with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). Two positive Tg14-3-3 interactors identified by this screening, hCG1821272 and eIF5B (eukaryotic translation initiation factor 5B), were isolated and characterized. This approach made it possible to gain a better understanding of the function of Tg14-3-3 in regulating host proteins involved in key cellular processes, such as translational initiation and cell migration.

Occurrence of Enterocytozoon bieneusi in Donkeys (Equus asinus) in China: A Public Health Concern.

Enterocytozoon bieneusi is an important zoonotic parasite. It can infect virtually all animal species and has a global distribution. However, the prevalence of E. bieneusi in donkeys (Equus asinus) has only been reported in Algeria and Spain, and no information is available concerning genotypes of E. bieneusi in donkeys worldwide. In the present study, a total of 301 donkey fecal samples (48 from Jilin Province, 224 from Shandong Province and 29 from Liaoning Province) were collected and examined by PCR amplification of the internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 5.3% (16/301), with 6.3% (3/48) in Jilin Province, 4.9% (11/224) in Shandong Province, and 6.9% (2/29) in Liaoning Province. Prevalence in different age groups ranged from 4.2 to 5.5%. E. bieneusi prevalence in donkeys sampled in different seasons varied from 4.2 to 6.5%. Altogether, four E. bieneusi genotypes were identified in this study, with two known genotypes (J and D) and two novel genotypes (NCD-1and NCD-2). Phylogenetic analysis revealed that genotypes D, NCD-1 and NCD-2 belonged to group 1, while the remaining genotype J was clustered into group 2. These findings revealed the occurrence of E. bieneusi in donkeys in China for the first time. Moreover, the present study also firstly genotyped the E. bieneusi in donkeys worldwide. These findings extend the distribution of E. bieneusi genotypes and provide baseline data for controlling E. bieneusi infection in donkeys, other animals and humans.

Global miRNA expression profiling of domestic cat livers following acute Toxoplasma gondii infection.

Although microRNAs (miRNAs) play an important role in liver homeostasis, the extent to which they can be altered by Toxoplasma gondii infection is unknown. Here, we utilized small RNA sequencing and bioinformatic analyses to characterize miRNA expression profiles in the liver of domestic cats at 7 days after oral infection with T. gondii (Type II) strain. A total of 384 miRNAs were identified and 82 were differentially expressed, of which 33 were up-regulated and 49 down-regulated. Also, 5690 predicted host gene targets for the differentially expressed miRNAs were identified using the bioinformatic algorithm miRanda. Gene ontology analysis revealed that the predicted gene targets of the dysregulated miRNAs were significantly enriched in apoptosis. Kyoto Encyclopedia of Genes and Genomes analysis showed that the predicted gene targets were involved in several pathways, including acute myeloid leukemia, central carbon metabolism in cancer, choline metabolism in cancer, estrogen signaling pathway, fatty acid degradation, lysosome, nucleotide excision repair, progesterone-mediated oocyte maturation, and VEGF signaling pathway. The expression level of 6 upregulated miRNAs (mmu-miR-21a-5p, mmu-miR-20a-5p, mmu-miR-17-5p, mmu-miR-30e-3p, mmu-miR-142a-3p, and mmu-miR-106b-3p) was confirmed by stem-loop quantitative reverse transcription PCR, which yielded results consistent with the sequencing data. These findings expand our understanding of the regulatory mechanisms of miRNAs underlying T. gondii pathogenesis and contribute new database information on cat miRNAs, opening a new perspective on the prevention and treatment of T. gondii infection.

Research advances in interactions related to Toxoplasma gondii microneme proteins.

Toxoplasma gondii microneme proteins (TgMICs), secreted by micronemes upon contact with host cells, are reported to play important roles in multiple stages of the T. gondii life cycle, including parasite motility, invasion, intracellular survival, and egress from host cells. Meanwhile, during these processes, TgMICs participate in many protein-protein and protein-carbohydrate interactions, such as undergoing proteolytic maturation, binding to aldolase, engaging the host cell receptors and forming the moving junction (MJ), relying on different types of ectodomains, transmembrane (TM) domains and cytoplasmic domains (CDs). In this review, we summarize the research advances in protein-protein and protein-carbohydrate interactions related to TgMICs, and their intimate associations with corresponding biological processes during T. gondii infection, which will contribute to an improved understanding of the molecular pathogenesis of T. gondii infection, and provide a basis for developing effective control strategies against T. gondii.

The presence of Giardia intestinalis in donkeys, Equus asinus, in China.

BACKGROUND: Giardia intestinalis is one of the most important zoonotic enteric parasites. As no information regarding prevalence and genotype of G. intestinalis in donkeys (Equus asinus) in China is available, 181 faecal samples from 48 donkeys from Jilin Province, from 104 from Shandong Province and from 29 from Liaoning Province were examined between May and December 2015. FINDINGS: Twenty-eight (15.47%) out of 181 donkey samples were tested G. intestinalis-positive by nested amplification of the triosephosphate isomerase (tpi) gene. The prevalence in different regional groups varied from 10.42 to 18.27%. The prevalence in adult and young donkeys was 14.29 and 22.92%, respectively. Otherwise, the prevalence was 11.69% in summer and 18.27% in winter. However, no statistically significant differences were found in relation to region or age group. Sequence analysis of the tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci identified 4, 1 and 3 subtypes of assemblage B, respectively. Moreover, four novel multilocus genotypes (MLGs novel-1 to novel-4) were identified in assemblage B. CONCLUSIONS: This first report of G. intestinalis in donkeys in China indicates that further studies of nation-wide molecular epidemiology and geographical distribution of Giardia in donkeys are warranted. Effective strategies should be implemented to control G. intestinalis infection in donkeys, other animals and humans.

Identification of host proteins interacting with Toxoplasma gondii GRA15 (TgGRA15) by yeast two-hybrid system.

BACKGROUND: Toxoplasma gondii, an obligate intracellular protozoan parasite, possesses the remarkable ability to co-opt host cell machinery in order to maintain its intracellular survival. This parasite can modulate signaling pathways of its host through the secretion of polymorphic effector proteins localized in the rhoptry and dense granule organelles. One of such effectors is T. gondii type II-specific dense granule protein 15, TgGRA15, which activates NF-κB pathway. The aim of the present study was to identify the host interaction partner proteins of TgGRA15. METHODS: We screened a yeast two-hybrid mouse cDNA library using TgGRA15 as the bait. TgGRA15 (PRU strain, Type II) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. Then, the bait protein expression was validated by western blotting analysis, followed by auto-activation and toxicity tests in comparison with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). RESULTS: This screening led to the identification of mouse Luzp1 and AW209491 as host binding proteins that interact with TgGRA15. Luzp1 contains three nuclear localizing signals and is involved in regulating a subset of host non-coding RNA genes. CONCLUSIONS: These findings reveal, for the first time, new host cell proteins interacting with TgGRA15. The identification of these cellular targets and the understanding of their contribution to the host-pathogen interaction may serve as the foundation for novel therapeutic and prevention strategies against T. gondii infection.

Molecular Detection and Genetic Characterization of Toxoplasma gondii in Farmed Minks (Neovison vison) in Northern China by PCR-RFLP.

Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison). This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. 36 (8.6%) of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

Transcriptional changes of mouse splenocyte organelle components following acute infection with Toxoplasma gondii.

Toxoplasmosis is a globally spread zoonosis. The pathogen Toxoplasma gondii can hijack cellular organelles of host for replication. Although a number of important cellular life events are controlled by cell organelles, very little is known of the transcriptional changes of host cellular organelles after infection with T. gondii. Herein, we performed RNA-sequencing (RNA-seq) and bioinformatics analyses to study the global organelle component changes. It was found that many transcripts of the mouse spleen cellular organelle components were altered by acute T. gondii infection with the RH strain (Type I). Most differentially expressed transcripts of mitochondrial components were downregulated, especially those involved in biosynthetic and metabolic processes. Moreover, mitochondria based apoptosis process was downregulated. In terms of cytoskeleton, most differentially expressed transcript of cytoskeleton components were also downregulated, including septin cytoskeleton, cytoskeleton organization, centrosome and myosin. For endolysosomal system, ion transporters were downregulated at mRNA level, whereas the cytolytic components were increased, such as granzymes, Rab27a and perforin1 (Prf1). The main transcripts of Golgi apparatus components involved in sialylation or vesicle-mediated transportation were downregulated, while immune related components were upregulated. For endoplasmic reticulum (ER), posttranslational modification, drug metabolism and material transportation related transcripts were downregulated. In addition, T. gondii antigen cross-presentation by MHC-I complex could be downregulated by the downregulation of CD76 and ubiquitination related transcripts. The present study, for the first time, described the transcriptional changes of the mouse spleen cellular organelles following acute T. gondii infection, which provides a foundation to study the interaction between T. gondii and host cells at the sub-cellular level.

Molecular characterization of the Haemonchus contortus phosphoinositide-dependent protein kinase-1 gene (Hc-pdk-1).

BACKGROUND: Phosphoinositide-dependent protein kinase-1 (PDK-1), which functions downstream of phosphoinositide 3-kinase (AGE-1) and activates protein kinases of the AGC family, plays critical roles in regulating biology processes, such as metabolism, growth, development and survival. In the free-living nematode Caenorhabditis elegans, PDK-1 is a key component of the insulin-like signalling pathway, regulating the entry into and exit from dauer (arrested development). Although it is proposed that similar molecular mechanisms control the transition from the free-living to the parasitic stages of nematodes, nothing is known about PDK-1 in Haemonchus contortus, a socioeconomically important gastric nematode of ruminants. METHODS: Here, we isolated and characterized the pdk-1 gene (Hc-pdk-1) and its inferred product (Hc-PDK-1) from H. contortus. Using in vitro and in vivo methods, we then studied the transcriptional profiles of Hc-pdk-1 and anatomical gene expression patterns of Hc-PDK-1 in different developmental stages of C. elegans. RESULTS: In silico analysis of Hc-PDK-1 displayed conserved functional domains, such as protein kinase and pleckstrin homology (PH) domains and two predicted phosphorylation sites (Thr226/Tyr229), which are crucial for the phosphorylation of downstream signalling. The Hc-pdk-1 gene is transcribed in all of the main developmental stages of H. contortus, with its highest transcription in the infective third-stage larvae (iL3) compared with other stages. Transgene constructs, in which respective promoters were fused to the coding sequence for green fluorescent protein (GFP), were used to transform C. elegans, and to localize and compare the expression of Hc-pdk-1 and Ce-pdk-1. The expression of GFP under the control of the Hc-pdk-1 promoter was localized to the intestine, and head and tail neurons, contrasting somewhat the profile for the C. elegans ortholog, which is expressed in pharynx, intestine and head and tail neurons. CONCLUSIONS: This is the first characterization of pdk-1/PDK-1 from a trichostrongyloid nematode. Taken together, the findings from this study provide a first glimpse of the involvement of Hc-pdk-1 in the insulin-like signalling pathway in H. contortus.

[Research Progress on Genetic Diversity in Animal Parasitic Nematodes].

The development of molecular genetic markers for parasitic nematodes has significant implications in fundamental and applied research in Veterinary Parasitology. Knowledge on genetic diversity of nematodes would not only provide a theoretical basis for understanding the spread of drug-resistance alleles, but also have implications in the development of nematode control strategies. This review discusses the applications of molecular genetic markers (RFLP, RAPD, PCR-SSCP, AFLP, SSR and mitochondrial DNA) in research on the genetic diversity of parasitic nematodes.

Exploring the role of two interacting phosphoinositide 3-kinases of Haemonchus contortus.

BACKGROUND: Phosphoinositide 3-kinases (PI3Ks) are relatively conserved and important intracellular lipid kinases involved in signalling and other biological pathways. In the free-living nematode Caenorhabditis elegans, the heterodimeric form of PI3K consists of catalytic (AGE-1) and regulatory (AAP-1) subunits. These subunits are key components of the insulin-like signalling pathway and play roles in the regulation of the entry into and exit from dauer. Although, in parasitic nematodes, similar components are proposed to regulate the transition from free-living or arrested stages to parasitic larvae, nothing is known about PI3Ks in relation to the transition of third-stage larvae (L3s) to parasitism in Haemonchus contortus. METHODS: An integrated molecular approach was used to investigate age-1 and aap-1 of H. contortus (Hc-age-1 and Hc-aap-1) in C. elegans. RESULTS: The two genes Hc-age-1 and Hc-aap-1 were transcribed in all life stages, with the highest levels in the egg, infective L3 and adult female of H. contortus. The expression of these genes was localized to the intestine, contrasting the pattern of their orthologues in C. elegans (where they are expressed in both head neurons and the intestine). The yeast two-hybrid analysis demonstrated that the adaptor-binding domain of Hc-AGE-1 interacted strongly with the Hc-AAP-1; however, this complex did not rescue the function of its orthologue in age-1-deficient C. elegans. CONCLUSIONS: This is the first time that the PI3K-encoding genes have been characterized from a strongylid parasitic nematode. The findings provide insights into the role of the PI3K heterodimer represented by Hc-age-1 and Hc-aap-1 in the developmental biology of H. contortus.

Prevalence of coccidian infection in suckling piglets in China.

To determine the prevalence of coccidian infection in suckling piglets in China, fecal samples from 779 litters of suckling piglets were collected on 80 different farms in 17 provinces from September 2009 to December 2010. These samples were examined through saturated saline flotation technique. The prevalences of coccidian infection ranged from 0 to 32.5% among different provinces and the average was 16.7% (130/779). The highest prevalence of 19.9% (69/346) was found in 8-14 day-old litters of suckling piglets. Seven coccidian species were detected in the positive litters of suckling piglets, including Isospora suis (63.9%), Eimeria debliecki (46.9%), Eimeria polita (19.2%), Eimeria suis (20.8%), Eimeria perminuta (13.9%), Eimeria scabra (4.6%), and Eimeria yanglingensis (1.5%). 55.4% of the positive litters of suckling piglet infected more than one coccidian species. The results of this investigation will provide the relevant basic data for control strategies against porcine coccidiosis on pig farms in China.