Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

Chemo-enzymatic synthesis of the ALG1-CDG biomarker and evaluation of its immunogenicity.

Congenital disorders of glycosylation (CDG) are a growing group diseases that result from defects in genes involved in glycan biosynthesis pathways. One tetrasaccharide, i.e., Neu5Ac-α2, 6-Gal-ß1, 4-GlcNAc-ß1, 4-GlcNAc, was recently reported as the biomarker of ALG1-CDG, the disease caused by ALG1 deficiency. To develop a novel diagnostic method for ALG1-CDG, chemo-enzymatic synthesis of the tetrasaccharide biomarker linked to phytanyl phosphate and the biomarker's immune stimulation were investigated in this study. The immunization study using liposomes bearing phytanyl-linked tetrasaccharide revealed that they stimulated a moderate immune response. The induced antibody showed strong binding specificity for the ALG1-CDG biomarker, indicating its potential in medical applications.

Vaccination with Recombinant Non-transmembrane Domain of Protein Mannosyltransferase 4 Improves Survival during Murine Disseminated Candidiasis.

Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.

genetic determinants of intrinsic colistin tolerance in Acinetobacter baumannii.

Acinetobacter baumannii is a leading cause of multidrug-resistant infections worldwide. This organism poses a particular challenge due to its ability to acquire resistance to new antibiotics through adaptation or mutation. This study was undertaken to determine the mechanisms governing the adaptability of A. baumannii to the antibiotic colistin. Screening of a transposon mutant library identified over 30 genes involved in inducible colistin resistance in A. baumannii. One of the genes identified was lpsB, which encodes a glycosyltransferase involved in lipopolysaccharide (LPS) synthesis. We demonstrate that loss of LpsB function results in increased sensitivity to both colistin and cationic antimicrobial peptides of the innate immune system. Moreover, LpsB is critical for pathogenesis in a pulmonary model of infection. Taken together, these data define bacterial processes required for intrinsic colistin tolerance in A. baumannii and underscore the importance of outer membrane structure in both antibiotic resistance and the pathogenesis of A. baumannii.

Immune response to Candida albicans is preserved despite defect in O-mannosylation of secretory proteins.

The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.

Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis.

We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.

Mannosylphosphodolichol synthase activity is associated with a 32 kDa phosphoprotein.

Failure of actinomycin D to block the activation of Dol-P-Man synthase in isoproterenol-treated capillary endothelial cells, supported that isoproterenol effect was not mediated by active transcription of the Dol-P-Man synthase gene during a short-term beta-adrenoreceptor stimulation. Instead, it was a net effect of protein phosphorylation by cAMP-dependent protein kinase. Using antibody as a probe we have now demonstrated that Dol-P-Man synthase activity is associated with a 32 kDa ER phosphoprotein.

Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae.

The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis. The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band. Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment.