Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion

Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles

The role of clathrin in post-golgi trafficking in toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle

Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance

Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation

Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors

<p>Abstract</p> <p>Background</p> <p>Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown.</p> <p>Methods</p> <p>An erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity <it>in vitro </it>were assessed in erlotinib resistant H1650-ER1 cells.</p> <p>Results</p> <p>The erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib.</p> <p>Conclusions</p> <p>Our findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.</p

High prevalence of vitamin D insufficiency and its association with BMI-for-age among primary school children in Kuala Lumpur, Malaysia

<p>Abstract</p> <p>Background</p> <p>Deficiencies of micronutrients can affect the growth and development of children. There is increasing evidence of vitamin D deficiency world-wide resulting in nutritional rickets in children and osteoporosis in adulthood. Data on the micronutrient status of children in Malaysia is limited. The aim of this study was to determine the anthropometric and micronutrient status of primary school children in the capital city of Kuala Lumpur.</p> <p>Methods</p> <p>A cross sectional study of primary aged school children was undertaken in 2008. A total of 402 boys and girls aged 7-12 years, attending primary schools in Kuala Lumpur participated in the study. Fasting blood samples were taken to assess vitamin D [as 25(OH)D], vitamin B₁₂, folate, zinc, iron, and ferritin and haemoglobin concentrations. Height-for-age and body mass index for age (BMI-for-age) of the children were computed.</p> <p>Results</p> <p>Most of the children had normal height-for-age (96.5%) while slightly over half (58.0%) had normal BMI-for-age. A total of 17.9% were overweight and 16.4% obese. Prevalence of obesity was significantly higher among the boys (25%) than in the girls (9.5%) (χ²= 22.949; <it>P </it>< .001). Most children had adequate concentrations of haemoglobin, serum ferritin, zinc, folate and vitamin B₁₂. In contrast, 35.3% of the children had serum 25(OH)D concentrations indicative of vitamin D deficiency(≤37.5 nmol/L) and a further 37.1% had insufficiency concentrations (> 37.5-≤50 nmol/L). Among the boys, a significant inverse association was found between serum vitamin D status and BMI-for-age (χ²= 5.958; <it>P </it>= .016).</p> <p>Conclusions</p> <p>This study highlights the presence of a high prevalence of sub-optimal vitamin D status among urban primary school children in a tropical country. In light of the growing problem of obesity in Malaysian children, these findings emphasize the important need for appropriate interventions to address both problems of obesity and poor vitamin D status in children.</p

The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, But Not the Human, Interferon-Gamma Response

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species

Breast cancer stem cells: implications for therapy of breast cancer

The concept of cancer stem cells responsible for tumour origin, maintenance, and resistance to treatment has gained prominence in the field of breast cancer research. The therapeutic targeting of these cells has the potential to eliminate residual disease and may become an important component of a multimodality treatment. Recent improvements in immunotherapy targeting of tumour-associated antigens have advanced the prospect of targeting breast cancer stem cells, an approach that might lead to more meaningful clinical remissions. Here, we review the role of stem cells in the healthy breast, the role of breast cancer stem cells in disease, and the potential to target these cells

Wnt pathway activity in breast cancer sub-types and stem-like cells

INTRODUCTION: Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear. METHODS: We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro. RESULTS: Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres. CONCLUSIONS: Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers