Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Unraveling unique structure and biosynthesis pathway of N-linked glycans in human fungal pathogen Cryptococcus neoformans by glycomics analysis.

The encapsulated fungal pathogen Cryptococcus neoformans causes cryptococcosis in immunocompromised individuals. Although cell surface mannoproteins have been implicated in C. neoformans pathogenicity, the structure of N-linked glycans assembled on mannoproteins has not yet been elucidated. By analyzing oligosaccharide profiles combined with exoglycosidase treatment, we report here that C. neoformans has serotype-specific high mannose-type N-glycans with or without a ß1,2-xylose residue, which is attached to the trimannosyl core of N-glycans. Interestingly, the neutral N-glycans of serotypes A and D were shown to contain a xylose residue, whereas those of serotype B appeared to be much shorter and devoid of a xylose residue. Moreover, analysis of the C. neoformans uxs1Δ mutant demonstrated that UDP-xylose is utilized as a donor sugar in N-glycan biosynthesis. We also constructed and analyzed a set of C. neoformans mutant strains lacking genes putatively assigned to the reconstructed N-glycan biosynthesis pathway. It was shown that the outer chain of N-glycan is initiated by CnOch1p with addition of an α1,6-mannose residue and then subsequently extended by CnMnn2p with multiple additions of α1,2-mannose residues. Finally, comparative analysis of acidic N-glycans from wild-type, Cnoch1Δ, Cnmnn2Δ, and Cnuxs1Δ strains strongly indicated the presence of xylose phosphate attached to mannose residues in the core and outer region of N-glycans. Our data present the first report on the unique structure and biosynthesis pathway of N-glycans in C. neoformans.

Functional and molecular characterization of novel Hansenula polymorpha genes, HpPMT5 and HpPMT6, encoding protein O-mannosyltransferases.

The genome of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding protein O-mannosyltransferases. Here, we report on the systematic characterization of HpPMT5 and HpPMT6, encoding novel PMT1 and PMT2 subfamily members, respectively. Although no apparent growth defects were detected in the Hppmt5Δ and Hppmt6Δ single mutants, the single mutants showed dramatic sensitivity to the Pmt1p inhibitor, and the Hppmt1pmt5Δ and Hppmt1pmt6Δ double mutants displayed increased susceptibility to cell wall-disturbing reagents. Activation of the cell wall integrity signaling pathway in the double mutant strains was further indicated by the markedly induced phosphorylation of MAP kinases, such as HpMpk1p and HpHog1p. Noticeably, O-mannosylation of the surface glycoproteins HpWsc1p and HpMid2p became severely defective only in the double mutants, supporting the involvement of HpPmt5p and HpPmt6p in O-mannosylation of these sensor proteins. On the other hand, co-immunoprecipitation experiments revealed only marginal interaction between HpPmt5p and HpPmt2p, even in the absence of HpPmt1p. Taken together, our results suggest that the functions of HpPmt5p and HpPmt6p are minor but become crucial upon the loss of HpPmt1p for protein O-mannosylation, which is essential for cell growth, cell wall integrity, and stress resistance in H. polymorpha.

Essential role of YlMPO1, a novel Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN4, in mannosylphosphorylation of N- and O-linked glycans.

Mannosylphosphorylation of N- and O-glycans, which confers negative charges on the surfaces of cells, requires the functions of both MNN4 and MNN6 in Saccharomyces cerevisiae. To identify genes relevant to mannosylphosphorylation in the dimorphic yeast Yarrowia lipolytica, the molecular functions of five Y. lipolytica genes showing significant sequence homology with S. cerevisiae MNN4 and MNN6 were investigated. A set of mutant strains in which Y. lipolytica MNN4 and MNN6 homologues were deleted underwent glycan structure analysis. In contrast to S. cerevisiae MNN4 (ScMNN4), the Y. lipolytica MNN4 homologue, MPO1 (YlMPO1), encodes a protein that lacks the long KKKKEEEE repeat domain at its C terminus. Moreover, just a single disruption of YlMPO1 resulted in complete disappearance of the acidic sugar moiety in both the N- and O-linked glycan profiles. In contrast, even quadruple disruption of all ScMNN6 homologues, designated YlKTR1, YlKTR2, YlKTR3, and YlKTR4, resulted in no apparent reduction in acidic sugar moieties. These findings strongly indicate that YlMpo1p performs a significant role in mannosylphosphorylation in Y. lipolytica with no involvement of the Mnn6p homologues. Mutant strains harboring the YlMPO1 gene disruption may serve as useful platforms for engineering Y. lipolytica glycosylation pathways for humanized glycans without any yeast-specific acidic modifications.

New selectable host-marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha.

Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively.

Construction of stabilized and tagged foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine.

Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.

Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability.

Antigenic properties and virulence of foot-and-mouth disease virus rescued from full-length cDNA clone of serotype O, typical vaccine strain.

We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica.

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.

Protection to homologous and heterologous challenge in pigs immunized with vaccine against foot-and-mouth disease type O caused an epidemic in East Asia during 2010/2011.

Foot-and-mouth disease (FMD) is a highly contagious infectious disease, and the use of vaccines is known to be effective for its prevention. In 2010/2011, there was an epidemic of the South East Asia (SEA) topotype in East Asian countries. We adapted the SEA topotype virus isolated in November 2010 in Korea in cells to analyze the characteristics of the virus and evaluate its possibility as a vaccine. After cell culture adaptation, the FMD virus particle 146S was purified to develop an inactivated oil vaccine for SEA or other topotypes. To measure its immunogenicity, pigs were inoculated with the experimental vaccine at different concentrations of the antigen. The results indicated that the groups immunized with at least 7.5 µg antigen were protected from homologous challenge. The immunized pigs were also protected against heterologous virus (ME-SA topotype) challenge. The genetic variations between the two field isolates and the adapted vaccine strains were identified in six amino acids by complete genome sequencing.

Construction of a bovine enterovirus-based vector expressing a foot-and-mouth disease virus epitope.

A recombinant infectious bovine enterovirus (BEV) vector was constructed to express a foot-and-mouth disease virus (FMDV) capsid protein (VP1) epitope. Sequences encoding the VP1 epitope (amino acid residues 141-160) of FMDV (vaccine strain O1/Manisa/Turkey/69) were inserted into pBLUBEV at the VP1/2A junction. The growth characteristics of the parental virus and viruses derived from recombinant plasmids (pBLUBEV, pBLUBEV-Manisa-epi) were determined by plaque assay and one-step growth curve analysis. There were no significant differences in the growth kinetics and plaque morphologies between transfectant viruses and their parental virus. The expressed VP1 epitope was detected successfully by using indirect immunofluorescence assay with a polyclonal antibody against the FMDV VP1 epitope from Madin Darby bovine kidney (MDBK) cells infected with BEV-Manisa-epi transfectant virus. This study demonstrated a novel alternative live viral vector that may be utilized as a candidate vaccine vector for veterinary applications.

Functional analysis of a Hansenula polymorpha MNN2-2 homologue encoding a putative UDP-N-acetylglucosamine transporter localized in the endoplasmic reticulum.

The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.

Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans.

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.

Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems.

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 muM Cd for 2 h. The putative promoters, about 1,000-bp upstream fragments, of these genes were fused with the yeast-enhanced green fluorescence protein (GFP) gene. The resultant reporter cassettes were introduced into H. polymorpha to evaluate promoter strength and specificity. The promoter derived from the H. polymorpha SEO1 gene (HpSEO1) was shown to drive most strongly the expression of GFP upon Cd treatment among the tested promoters. The Cd-inducible activity was retained in the 500-bp deletion fragment of the HpSEO1 promoter but was abolished in the further truncated 250-bp fragment. The 500-bp HpSEO1 promoter directed specific expression of GFP upon exposure to Cd in a dose-dependent manner, with Cd detection ranging from 1 to 900 muM. Comparative analysis of the Saccharomyces cerevisiae SEO1 (ScSEO1) promoter revealed that the ScSEO1 promoter has a broader specificity for heavy metals and is responsive to arsenic and mercury in addition to Cd. Our data demonstrate the potential use of the HpSEO1 promoter as a bioelement in whole-cell biosensors to monitor heavy metal contamination, particularly Cd.

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis alpha-amylase gene.

The gene encoding Schwanniomyces occidentalis alpha-amylase (AMY) was introduced into the chromosomal delta sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an delta-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The alpha-amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.