Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

The fungal pathogen Magnaporthe oryzae can cause rice blast and wheat blast diseases, which threatens worldwide food production. During infection, M. oryzae follows a sequence of distinct developmental stages adapted to survival and invasion of the host environment. M. oryzae attaches onto the host by the conidium, and then develops an appressorium to breach the host cuticle. After penetrating, it forms invasive hyphae to quickly spread in the host cells. Numerous genetic studies have focused on the mechanisms underlying each step in the infection process, but systemic approaches are needed for a broader, integrated understanding of regulatory events during M. oryzae pathogenesis. Many infection-related signaling events are regulated through post-translational protein modifications within the pathogen. N-linked glycosylation, in which a glycan moiety is added to the amide group of an asparagine residue, is an abundant modification known to be essential for M. oryzae infection. In this study, we employed a quantitative proteomics analysis to unravel the overall regulatory mechanisms of N-glycosylation at different developmental stages of M. oryzae. We detected changes in N-glycosylation levels at 559 glycosylated residues (N-glycosites) in 355 proteins during different stages, and determined that the ER quality control system is elaborately regulated by N-glycosylation. The insights gained will help us to better understand the regulatory mechanisms of infection in pathogenic fungi. These findings may be also important for developing novel strategies for fungal disease control. Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis

TLR4 and NKT Cell Synergy in Immunotherapy against Visceral Leishmaniasis

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal β-(1–4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1–4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic

Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus

Light and electron microscopic detection of (3 Gal β 1,4 GlcNAc β 1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

The Datura stramonium lectin recognizes with high affinity the disaccharide N -acetyllactosamine (Gal β 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N -acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47409/1/418_2004_Article_BF00524763.pd