A Cell Culture Platform for the Cultivation of Cryptosporidium parvum.

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer-derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc.

Experimental determination of codon usage-dependent selective pressure on high copy-number genes in Saccharomyces cerevisiae.

One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes, particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here, we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required, optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings deepen our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.

Past and future trends of Cryptosporidium in vitro research.

Cryptosporidium is a genus of single celled parasites capable of infecting a wide range of animals including humans. Cryptosporidium species are members of the phylum apicomplexa, which includes well-known genera such as Plasmodium and Toxoplasma. Cryptosporidium parasites cause a severe gastro-intestinal disease known as cryptosporidiosis. They are one of the most common causes of childhood diarrhoea worldwide, and infection can have prolonged detrimental effects on the development of children, but also can be life threatening to HIV/AIDS patients and transplant recipients. A variety of hosts can act as reservoirs, and Cryptosporidium can persist in the environment for prolonged times as oocysts. While there has been substantial interest in these parasites, there is very little progress in terms of treatment development and understanding the majority of the life cycle of this unusual organism. In this review, we will provide an overview on the existing knowledge of the biology of the parasite and the current progress in developing in vitro cultivation systems. We will then describe a synopsis of current and next generation approaches that could spearhead further research in combating the parasite.

Application of microRNA Targeted 3'UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools.

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

Localization of Fe-S Biosynthesis Machinery in Cryptosporidium parvum Mitosome.

Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron-sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron-sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology.

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

Human TorsinA can function in the yeast cytosol as a molecular chaperone.

TorsinA (TorA) is an AAA+ (ATPases associated with diverse cellular activities) ATPase linked to dystonia type 1 (DYT1), a neurological disorder that leads to uncontrollable muscular movements. Although DYT1 is linked to a 3 bp deletion in the C-terminus of TorA, the biological function of TorA remains to be established. Here, we use the yeast Saccharomyces cerevisiae as a tractable in vivo model to explore TorA function. We demonstrate that TorA can protect yeast cells against different forms of environmental stress and show that in the absence of the molecular disaggregase Hsp104, TorA can refold heat-denatured luciferase in vivo in an ATP-dependent manner. However, this activity requires TorA to be translocated to the cytoplasm from the endoplasmic reticulum in order to access and process cytoplasmic protein aggregates. Furthermore, mutational or chemical inactivation of the ATPase activity of TorA blocks this activity. We also find that TorA can inhibit the propagation of certain conformational variants of [PSI+], the aggregated prion form of the endogenous Sup35 protein. Finally, we show that while cellular localisation remains unchanged in the dystonia-linked TorA mutant ΔE302-303, the ability of this mutant form of TorA to protect against cellular stress and to facilitate protein refolding is impaired, consistent with it being a loss-of-function mutation.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions.

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive 'toolbox' available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP (Sc) . Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.

Engineering the chaperone network of CHO cells for optimal recombinant protein production and authenticity.

All proteins fold into a defined three-dimensional shape that is compatible with the cellular role and/or biological activity of those proteins. Molecular chaperones are a family of proteins whose role is to assist the folding and targeting of proteins in both normal and stressed cells. The rational manipulation of chaperone levels in a cell line engineered to produce a defined recombinant protein (rP) can significantly improve both the achievable steady-state levels and authenticity of a wide range of recombinant proteins. Here, we describe the methodology associated with expressing a variety of molecular chaperones in Chinese hamster ovary (CHO) lines in order to improve their recombinant protein production capacity. These chaperones include both those that facilitate the folding of the polypeptide chain (i.e. Hsp70, Hsp40) and those that can re-fold proteins that have misfolded in the cell (i.e. ClpB/Hsp104). This latter property is particularly important given the propensity for highly expressed recombinant proteins to misfold in the "foreign" cellular environment.

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

BACKGROUND: Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in ß-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of ß-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. METHODOLOGY/PRINCIPAL FINDINGS: As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and ß tubulin subunits, microtubule disassembly and cell death. In contrast, ß-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. CONCLUSIONS: Our results support the hypothesis that PhLP3 is important for the maintenance of ß-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.

Transcriptomic and phenotypic analysis of the effects of T-2 toxin on Saccharomyces cerevisiae: evidence of mitochondrial involvement.

At 5 µg mL(-1) , T-2 toxin significantly upregulated the transcription of 281 genes and downregulated 86. Strongly upregulated genes included those involved in redox activity, mitochondrial functions, the response to oxidative stress, and cytoplasmic rRNA transcription and processing. Highly repressed genes have roles in mitochondrial biogenesis, and the expression and stability of cytoplasmic rRNAs. T-2 toxin inhibition of growth was greater in a medium requiring respiration, and was antagonized by antioxidants. T-2 toxin treatment induced reactive oxygen species, caused nucleolytic damage to DNA, probably mitochondrial, and externalization of phosphatidylserine. Deletion mutations causing respiratory deficiency substantially increased toxin tolerance, and deletion of some TOR (target of rapamycin) pathway genes altered T-2 toxin sensitivity. Deletion of FMS1, which plays an indirect role in cytoplasmic protein synthesis, markedly increased toxin tolerance. Overall, the findings suggest that T-2 toxin targets mitochondria, generating oxy-radicals and repressing mitochondrial biogenesis genes, thus inducing oxidative stress and redox enzyme genes, and triggering changes associated with apoptosis. The large transcriptional changes in genes needed for rRNA transcription and expression and the effects of deletion of FMS1 are also consistent with T-2 toxin damage to the cytoplasmic translational mechanism, although it is unclear how this relates to the mitochondrial effects.

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells.

Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast 'protein disaggregase' Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70/40 chaperones. Over-expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5-fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over-expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over-expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells.

Development of a novel yeast cell-based system for studying the aggregation of Alzheimer's disease-associated Abeta peptides in vivo.

Alzheimer's disease is the most common neurodegenerative disease, affecting approximately 50% of humans by age 85. The disease process is associated with aggregation of the Abeta peptides, short 39-43 residue peptides generated through endoproteolytic cleavage of the Alzheimer's precursor protein. While the process of aggregation of purified Abeta peptides in vitro is beginning to be well understood, little is known about this process in vivo. In the present study, we use the yeast Saccharomyces cerevisiae as a model system for studying Abeta-mediated aggregation in an organism in vivo. One of this yeast's endogenous prions, Sup35/[PSI+], loses the ability to aggregate when the prion-forming domain of this protein is deleted. We show that insertion of Abeta peptide sequences in place of the original prion domain of this protein restores its ability to aggregate. However, the aggregates are qualitatively different from [PSI+] prions in their sensitivity to detergents and in their requirements on trans-acting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of Abeta peptides in an organism in vivo.

Fission yeast Dss1 associates with the proteasome and is required for efficient ubiquitin-dependent proteolysis.

Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.