Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

Drug treatment for toxoplasmosis is problematic, because current drugs cannot eradicate latent infection with Toxoplasma gondii and can cause bone marrow toxicity. Because latent infection remains after treatment, relapse of infection is a problem in both infections in immunocompromised patients and in congenitally infected patients. To identify lead compounds for novel drugs against Toxoplasma gondii, we screened a chemical compound library for anti-Toxoplasma activity, host cell cytotoxicity, and effect on bradyzoites. Of 878 compounds screened, 83 demonstrated >90% parasite growth inhibition. After excluding compounds that affected host cell viability, we further characterized two compounds, tanshinone IIA and hydroxyzine, which had IC50 values for parasite growth of 2.5 μM and 1.0 μM, respectively, and had no effect on host cell viability at 25 μM. Both tanshinone IIA and hydroxyzine inhibited parasite replication after invasion and both reduced the number of in vitro-induced bradyzoites, whereas, pyrimethamine, the current therapy, had no effect on bradyzoites. Both tanshinone IIA and hydroxyzine are potent lead compounds for further medicinal chemistry. The method presented for evaluating compounds for bradyzoite efficacy represents a new approach to the development of anti-Toxoplasma drugs to eliminate latency and treat acute infection.

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.

IFN-gamma (IFN-gamma) has been shown to activate astrocytes to acquire immune functions. In this study the effect of IFN-gamma on murine astrocytes was investigated via microarray analysis. The activating effect of IFN-gamma on the astrocyte transcriptome showed predominance toward pathways involved in adaptive immunity, initiation of the immune response and innate immunity. Previously unknown astrocytic genes expressed included members of the p47 GTPases and guanine nucleotide binding protein (GBP) families. Down-regulatory effects of IFN-gamma stimulation were confined to pathways involved in growth regulation, cell differentiation and cell adhesion. This data supports the notion that astrocytes are an important immunocompetant cell in the brain and indicate that astrocytes may have a significant role in various infectious diseases such as Toxoplasmic Encephalitis and neurological diseases with an immunological component such as Alzheimer's and autoimmune disorders.

The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism.

Upon the osteoporotic condition, sluggish osteogenesis, excessive bone resorption, and chronic inflammation make the osseointegration of bioinert titanium (Ti) implants with surrounding bone tissues difficult, often lead to prosthesis loosening, bone collapse, and implant failure. In this study, we firstly designed clickable mussel-inspired peptides (DOPA-N3) and grafted them onto the surfaces of Ti materials through robust catechol-TiO2 coordinative interactions. Then, two dibenzylcyclooctyne (DBCO)-capped bioactive peptides RGD and BMP-2 bioactive domain (BMP-2) were clicked onto the DOPA-N3-coated Ti material surfaces via bio-orthogonal reaction. We characterized the surface morphology and biocompatibility of the Ti substrates and optimized the osteogenic capacity of Ti surfaces through adjusting the ideal ratios of BMP-2/RGD at 3:1. In vitro, the dual-functionalized Ti substrates exhibited excellent promotion on adhesion and osteogenesis of mesenchymal stem cells (MSCs), and conspicuous immunopolarization-regulation to shift macrophages to alternative (M2) phenotypes and inhibit inflammation, as well as enhancement of osseointegration and mechanical stability in osteoporotic rats. In summary, our biomimetic surface modification strategy by bio-orthogonal reaction provided a convenient and feasible method to resolve the bioinertia and clinical complications of Ti-based implants, which was conducive to the long-term success of Ti implants, especially in the osteoporotic or inflammatory conditions.

Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

Nosema bombycis is a pathogen of the silkworm that belongs to the microsporidia, a group of obligate intracellular parasites related to fungi. N. bombycis infection causes the disease pébrine in silkworms. Insects utilize hemolymph melanization as part of the innate immune response to fight against pathogens, and melanization relies on a serine protease-mediated prophenoloxidase (PPO) activation cascade that is tightly regulated by serine protease inhibitors (serpins). Previous studies showed that N. bombycis infection suppressed silkworm hemolymph melanization, however the mechanism has not been elucidated. We hypothesize that N. bombycis can secret serpins (NbSPNs) to inhibit host serine proteases in the PPO activation cascade, thus suppressing phenoloxidase (PO) activity and the consequent melanization. We demonstrated in this study that N. bombycis infection suppressed silkworm PO activity and melanization and we identified the expression of N. bombycis serpin 6 (NbSPN6) in the hemolymph of the infected host. When recombinant NbSPN6 was added to normal hemolymph, PO activity was inhibited in a dose-dependent manner. Moreover, in vivo analysis by RNA interference technology showed that when NbSPN6 expression is blocked, the inhibitory effects on PO activity can be reversed and the proliferation of N. bombycis within host can be suppressed. These results demonstrated the indispensable role of NbSPN6 in successful pathogen infection. To further elucidate the molecular basis of NbSPN6 suppressing host defense, we determined that the host serine protease prophenoloxidase-activating enzyme (PPAE) is the direct target of NbSPN6 inhibition. Taken together, our novel study is the first to elucidate the molecular mechanism of pathogen-derived serpin inhibiting hemolymph melanization and, thus, regulating host innate immune responses. This study may also provide novel strategies for preventing microsporidia infection.

Polyetheretherketone (PEEK) has been widely used as orthopedic and dental materials due to excellent mechanical and physicochemical tolerance. However, its biological inertness, poor osteoinduction, and weak antibacterial activity make the clinical applications in a dilemma. Inspired by the mussel adhesion mechanism, here we reported a biomimetic surface strategy for rational integration and optimization of anti-infectivity and osteo-inductivity onto PEEK surfaces using a mussel foot proteins (Mfps)-mimic peptide with clickable azido terminal. The peptide enables mussel-like adhesion on PEEK biomaterial surfaces, leaving azido groups for the further steps of biofunctionalizations. In this study, antimicrobial peptide (AMP) and osteogenic growth peptide (OGP) were bioorthogonally clicked on the azido-modified PEEK biomaterials to obtain a dual-effect of host defense and tissue repair. Since bioorthogonal clicking allows precise collocation between AMP and OGP through changing their feeding molar ratios, an optimal PEEK surface was finally obtained in this research, which could long-term inhibit bacterial growth, stabilize bone homeostasis and facilitate interfacial bone regeneration. In a word, this upgraded mussel surface strategy proposed in this study is promising for the surface bioengineering of inert medical implants, in particular, achieving rational integration of multiple biofunctions to match clinical requirements.

Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δmag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1β (IL-1β) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1β release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1β release) in Δmag1 parasites completely inhibited all IL-1β release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection.IMPORTANCEToxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system.

Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods.

Microsporidia are intracellular fungus-like pathogens and the infection symptoms include recurrent diarrhea and systematic inflammations. The major infection route of microsporidia is the digestive tract. Since microsporidia are hard to fully eliminate, the interactions and persistence of the pathogen within epithelium may modulate host susceptibility to digestive disorders. In this study, both in vitro and in vivo infection models were applied. The alterations of epithelial barrier integrity, permeability, and tight junction proteins after microsporidia infection were assessed on MDCK/Caco-2 monolayers. The fecal intestinal microbiota and tissue alterations after microsporidia infection were assessed on C57BL/6 mice. Moreover, the susceptibility to develop dextran sulfate sodium (DSS)-induced inflammatory bowel diseases (IBDs) was also analyzed by the murine infection model. The results demonstrated that microsporidia infection increased epithelium permeability, weakened wound healing capability, and destructed tight junction protein zonula occludens-1. Microsporidia infection also dysregulates intestinal microbiota. These impairing effects of microsporidia increased host vulnerability to develop enteritis as shown by the murine model of DSS-induced IBD. Our study is the first to elucidate molecular mechanisms of the damaging effects of microsporidia on host epithelium and pointed out the cryptic threats of latent microsporidia infection to public health as reflected by the increased chances of developing more severe diseases.IMPORTANCEMicrosporidia are widely present in nature and usually cause latent and persistent infections in hosts. Given the fact that the digestive tract is the major infection route, it is of great importance to explore the consequences of microsporidia infection on the intestinal epithelial barrier and the risks to the host. In this study, we demonstrated the destructing effects of microsporidium infection on epithelial barriers manifested as increased epithelial permeability, weakened wound healing ability, and disrupted tight junctions. Moreover, microsporidia made the host more susceptible to dextran sulfate sodium-induced inflammatory bowel disease. These findings provide new evidence for us to better understand and develop novel strategies for microsporidia prevention and disease control.

The requirement for the favorable therapeutics against ulcerative colitis (UC) is that anti-inflammatory drugs can be specifically internalized by macrophages and subsequently be on-demand released to suppress inflammation. Herein, we developed a type of multi-bioresponsive anti-inflammatory drug (curcumin, CUR)-loaded nanoparticles (NPs) that were derived from natural silk fibroin and followed by surface functionalization with chondroitin sulfate (CS). The generated CS-CUR-NPs had a desired average particle size (175.4 nm), a uniform size distribution and negative surface charge (-35.5 mV). Strikingly, these NPs exhibited excellent bioresponsibility when triggered with the intrinsic stimuli (acidity, glutathione and reactive oxygen species) within activated macrophages, indicating that they could conceivably confer the on-demand intracellular drug release. Furthermore, we found that CS functionalization yielded notably targeted drug delivery to macrophages, and thereby enhanced the anti-inflammatory activities of NPs. Most importantly, animal experiments revealed that these nanotherapeutics could remarkably alleviate the symptoms of UC, maintain the homeostasis of intestinal microbiota and improve the survival rate of mice with UC through the route of oral administration or intravenous injection. Our results suggest that these facilely fabricated CS-CUR-NPs, which exhibit excellent biocompatibility, multi-bioresponsive drug release and macrophage-targeted capacity, could be exploited as a promising therapeutic platform for clinical UC treatment.

Toxoplasma gondii is an obligate intracellular parasite that chronically infects up to a third of the human population. The parasites persist in the form of cysts in the central nervous system and serve as a reservoir for the reactivation of toxoplasmic encephalitis. The cyst wall is known to have abundant O-linked N-acetylgalactosamine glycans, but the existing metabolic labeling methods do not allow selective labeling of intracellular parasite glycoproteins without labeling of host glycans. In this study, we have integrated Cu(I)-catalyzed bioorthogonal click chemistry with a specific esterase-ester pair system in order to selectively deliver azidosugars to the intracellular parasites. We demonstrated that α-cyclopropyl modified GalNAz was cleaved by porcine liver esterase produced in the parasites but not in the host cells. Our proof-of-concept study demonstrates the feasibility and potential of this esterase-ester click chemistry approach for the selective delivery of small molecules in a stage-specific manner.IMPORTANCE Selective delivery of small molecules into intracellular parasites is particularly problematic due to the presence of multiple membranes and surrounding host cells. We have devised a method that can deliver caged molecules into an intracellular parasite, Toxoplasma gondii, that express an uncaging enzyme in a stage-specific manner without affecting host cell biology. This system provides a valuable tool for studying many intracellular parasites.

The microsporidian Nosema bombycis is an obligate intracellular parasite of Bombyx mori, that lost its intact tricarboxylic acid cycle and mitochondria during evolution but retained its intact glycolysis pathway. N. bombycis hexokinase (NbHK) is not only a rate-limiting enzyme of glycolysis but also a secretory protein. Indirect immunofluorescence assays and recombinant HK overexpressed in BmN cells showed that NbHK localized in the nucleus and cytoplasm of host cell during the meront stage. When N. bombycis matured, NbHK tended to concentrate at the nuclei of host cells. Furthermore, the transcriptional profile of NbHK implied it functioned during N. bombycis' proliferation stages. A knock-down of NbHK effectively suppressed the proliferation of N. bombycis indicating that NbHK is an important protein for parasite to control its host.

The association between dietary carbohydrate intake and colorectal cancer (CRC) risk remains controversial. We therefore conducted this meta-analysis to assess the relationship between them. A literature search from the databases of PubMed, Embase, Web of Science and Medline was performed for available articles published in English (up to September 2016). Pooled relative risk (RR) with 95% confidence interval (CI) was calculated to evaluate the association between dietary carbohydrate intake and CRC risk. The random-effect model (REM) was selected as the pooling method. Publication bias was estimated using Egger's regression asymmetry test and funnel plot. A total of 17 articles involving 14402 CRC patients and 846004 participants were eligible with the inclusion criteria in this meta-analysis. The pooled RR with 95% CI of dietary carbohydrate intake for CRC, colon cancer and rectum cancer risk were 1.08 (95% CI =0.93-1.23, I2 =68.3%, Pheterogeneity<0.001), 1.09 (95% CI =0.95-1.25, I2 =48.3%) and 1.17 (95% CI =0.98-1.39, I2 =17.8%) respectively. When we conducted the subgroup analysis by gender, the significant association was found in men's populations (summary RR =1.23, 95% CI =1.01-1.57), but not in the women's populations. In the further subgroup analyses for study design and geographic locations, we did not find any association between dietary carbohydrate intake and CRC risk in the subgroup results respectively. No significant publication bias was found either by the Egger's regression asymmetry test or by the funnel plot. This meta-analysis suggested that higher dietary carbohydrate intake may be an increased factor for CRC risk in men populations. Further studies are wanted to confirm this relationship.

Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori.

Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis.

Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A(2) (TXA(2)). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA(2) is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA(2) accounts for up to 90% of the circulating levels of TXA(2) in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA(2) receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA(2) synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA(2) in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA(2) may result in novel therapeutic targets for a disease with limited treatment options.

Postmenopausal osteoporosis (OP) is one of the most common bone diseases that affects millions of aging women. Reduced osteogenesis and increased oxidative stress have been implicated in bone marrow mesenchymal stem cells (BMMSCs) derived from OP patients. Melatonin has shown positive effects on osteoblast differentiation and bone formation; however, it was unknown whether melatonin could restore OP-impaired osteogenic potential of BMMSCs and what the underlying mechanisms entailed. The objective of this study is to investigate (1) whether melatonin can restore the impaired osteogenic potential of OP BMMSCs by preserving their antioxidant functions, and if so, (2) whether intravenous administration of melatonin can prevent OP-induced bone loss in ovariectomized (OVX) rats. Ovariectomies were performed in female rats and BMMSCs were isolated from the osteoporotic rats 3 months later. In vitro treatment with melatonin successfully improved the osteogenic differentiation of OP BMMSCs, as evidenced by increased levels of matrix mineralization and osteoblast-specific genes. In melatonin-treated OP BMMSCs, intracellular oxidative stress was significantly attenuated, while levels of intracellular antioxidant enzymes were noticeably up-regulated - particularly superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1). Silent information regulator type 1 (SIRT1) was involved in the melatonin-mediated recovery of osteogenesis and antioxidant functions. Meanwhile, in vivo injections of melatonin via the tail vein successfully ameliorated the bone micro-architecture in ovariectomized rat femurs. Further experiments confirmed that BMMSCs derived from melatonin-treated OVX rats exerted well-preserved antioxidant properties and osteogenic potential. Our findings demonstrate that the administration of melatonin is a promising strategy for treating patients with postmenopausal OP by preserving the antioxidant properties and osteogenic potential of their BMMSCs.