Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of 65kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interaction

Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions

Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes

Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma

The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

Background: Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol. Methods: Two to four month-old mice lacking MIST1 (Mist1 2/2) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36 % of total kcal from ethanol and fat), a control liquid diet (36 % kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR). Results: Ethanol-fed Mist1 2/2 mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1 2/2 mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2a and spliced XBP1. These increases were not observed in Mist1 2/2 pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1 2/2 mice. Conclusions: Our findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated wit

Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover

Anti-angiogenic alternatives to VEGF blockade

Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy

Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast to T. gondii, N. caninum represents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused by N. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, by analogy with T. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle