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  • In summary we investigated the fluoride sensitivity


    In summary, we investigated the fluoride sensitivity of different S. mutans strains in terms of enolase activity. Lower enolase activity was not always associated with lower S. mutans growth in cultures with NaF. Gene analysis showed that UA130 and NCH105 both have enolase point mutations. Unique amino RO 4929097 substitutions may have allowed for enolase activity even in the presence of fluoride, and might be causally related to fluoride resistance in NCH105.
    Acknowledgments This study was partly supported by a Grant-in-Aid for Scientific Research B (22390391539) from JSPS, Japan.
    Introduction Taenia multiceps is a taeniid cestode with an adult stage that lives in the small intestine of dogs and other canids. The larval stage (Coenurus cerebralis) of the parasite is usually found in the central nervous system of sheep, goats and some other ruminants, which is responsible for the cerebral coenurosis. The disease in sheep can usually cause neurological symptoms, or sometimes even death from starvation after several weeks (Scala et al., 2007). C. cerebralis infections in sheep and goats are thus of great economic importance in many parts of the world (Sharma and Chauhan, 2006, Varcasia et al., 2011). Humans can acquire the parasite via ingestion of T. multiceps eggs that excreted by definitive hosts, and causing human coenurosis. The metacestode that lives in muscle results in intra-muscular mass (Benifla et al., 2007), and brain infestation causing headaches, vomiting, pappiloedema and even hemiparesis (El-On et al., 2008, Ambekar et al., 2013). So, cerebral coenurosis is not only an important veterinary problem but also a potential zoonotic risk to human health. Chemotherapy against coenurosis is considered little effect on the formation of C. cerebralis cysts, and removal of the coenurus by surgical is not available for large scale treatment under field conditions (Sharma and Chauhan, 2006, Manunta et al., 2012). More recently, the recombinant antigens Tm16 and Tm18 were prepared from the onchosphere of T. multiceps and were used successfully to protect sheep against infection by this parasite (Gauci et al., 2008, Varcasia et al., 2009). Nevertheless, identification of more target antigens will improve our understanding of the biology of T. multiceps. Enolase, a kind of ubiquitous enzyme, has been widely identified in Pseudomonas aeruginosa (Ceremuga et al., 2014), Leishmania mexicana (Vanegas et al., 2007), Fasciola hepatica (Bernal et al., 2004) and Ornithodoros moubata (Díaz-Martín et al., 2013). Enolase is a multifunctional protein that is characterized as catalyzing the reversible dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate (PEP) and plasminogen receptor on the surface of several pathogens (Cayir et al., 2014, Ramajo-Hernández et al., 2007). Furthermore, the protein could also induce host-protective immunity and is considered potential vaccine candidate that has been evaluated in several helminths (Yang et al., 2010, Chen et al., 2012). In our previous study, an enolase-like gene/protein in T. multiceps was selected from two-dimensional (2-D) electrophoresis and mass spectrometry (Li et al., 2014). However, no information regarding the biological functions and the immunogenicity of enolase in T. multiceps could be available until now. In the present study, we identified the enolase gene in T. multiceps by analysis of the gene expression from different developmental stages, expression of the recombinant T. multiceps enolase (rTmENO) protein and then detected its enzymatic activity and plasminogen-binding activity, and further evaluation of the protective efficacy induced by rTmENO in sheep, which provided foundation for our understanding of the biological functions of TmENO and its application in development of vaccines against T. multiceps infection.
    Material and methods
    Discussion In this study, we amplified the TmENO gene from genomic DNA extracted from T. multiceps to obtain the complete enolase coding sequence. After the sequence analysis and removal of the introns, all the rare codons were replaced with bias codons, and the optimized TmENO ORF sequence was artificially synthesized. A previous study obtained EgEnolase gene from E. granulosus through removing introns of its genomic DNA by two turns of asymmetric PCR (Gan et al., 2010), and the full length enolase cDNA was obtained by rapid amplification of the cDNA ends (RACE) based on the Haemonchus contortus expression sequence tag (Han et al., 2012). For the strategy for obtaining target gene, our experimental approach was simple and practicable. After cloning and sequencing, the T. multiceps gene encoded a deduced polypeptide of 433 amino acids. Based on the multiple sequence alignment of enolases, the predicted amino acid sequence of TmENO had a high sequence identity with enolases from other helminths, ranging from 98% (T. asiatica) and 94% (E. granulosus) to 67% (Onchocerca volvulus). This finding also confirmed the high degree of enolase conservation (Avilán et al., 2011).