Glycans in host-pathogen interactions : an integrated biochemical investigation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.The epithelial cell-extracellular matrix interface primarily comprises of complex glycans and glycoconjugates. The widespread distribution of these glycans on the epithelial cell surface makes them ideal targets for interaction with microbial pathogens. In this thesis, a framework of integrated approaches was developed to characterize the structure-function relationships of host cell surface glycans and examine their role in mediating hostpathogen interactions. The first part of the thesis involves a study of the effect of secreted bacterial sphingomyelinases on the epithelial cell surface proteoglycan (a large glycan- protein conjugate), syndecan-1 and on epithelial tight junctions. The findings presented in this work suggest mechanisms by which sphingomyelinases could enhance bacterial virulence by regulating epithelial cell function. The second part of the thesis investigates the glycan binding requirements that govern the human adaptation and transmission of influenza A viruses by characterizing the molecular interactions between sialylated glycan-receptors and viral hemagglutinin (HA). The study puts forth the concept that the topology or shape (going beyond the chemical c2-3 versus a2-6 sialic acid linkage) adopted by the sialylated glycans is the critical determinant for efficient human adaptation of these viruses. In conclusion, this thesis provides insights into the molecular mechanisms of host-pathogen interactions and enables development of improved strategies for targeted antimicrobial therapies.by Aarthi Chandrasekaran.Ph.D

Decoding the Distribution of Glycan Receptors for Human-Adapted Influenza A Viruses in Ferret Respiratory Tract

Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface. It is generally known that upper respiratory tract of humans and ferrets predominantly express α2→6 sialylated glycan receptors. However much less is known about the fine structure of these glycan receptors and their distribution in different regions of the ferret respiratory tract. In this study, we characterize distribution of glycan receptors going beyond terminal sialic acid linkage in the cranial and caudal regions of the ferret trachea (upper respiratory tract) and lung hilar region (lower respiratory tract) by multiplexing use of various plant lectins and human-adapted HAs to stain these tissue sections. Our findings show that the sialylated glycan receptors recognized by human-adapted HAs are predominantly distributed in submucosal gland of lung hilar region as a part of O-linked glycans. Our study has implications in understanding influenza A viral pathogenesis in ferrets and also in employing ferrets as animal models for developing therapeutic strategies against influenza.Singapore-MIT Alliance for Research and Technolog

Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.National Institute of General Medical Sciences (U.S.) (GM57073)National Institute of General Medical Sciences (U.S.) (U54 GM62116)Singapore. Agency for Science, Technology and ResearchSingapore-MIT Alliance for Research and Technolog

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.National Institute of General Medical Sciences (U.S.) (Glue Grant U54 GM62116)National Institutes of Health (U.S.) (Grant GM57073)Singapore-MIT Alliance for Research and Technolog

Association of ADH1C and ALDH2 genes with alcohol dependence in south Indian Tamilian population: A case control approach

Objectives: Alcohol dependence (AD) poses a serious medical problem and significant public health issue contributing to morbidity and mortality throughout the world. The aim of the study was to establish the allele and genotype frequencies and to test the association of rs698 (ADH1C) and rs671(ALDH2) with the risk of alcohol dependence in south Indian Tamilian population. Methodology: A total of 150 alcohol dependent cases aged between 18- and 65-years fulfilling DSM-V criteria were recruited from the de-addiction centre. Blood donors (n=150) who had a history of alcohol intake with AUDIT score of less than eight were selected for the control group. The alleles were genotyped using TaqMan SNP genotyping assays by quantitative PCR. Association with alcohol dependence was evaluated with various genetic models using the Chi-square test. Multiple logistic regression analysis was performed to explore the effect of covariates. Results: The observed genotype frequency distributions of rs698 and rs671 were in agreement with Hardy Weinberg equilibrium (p>0.05).The dominant and allelic genetic model of ALDH2, rs671 between cases and controls showed a statistically significant association of the genetic variant with AD.&nbsp

Microfluidic assays for biofuels research

We are developing next-generation assays for biofuels research using microfluidic technologies to provide significant improvement over conventional platforms in throughput, sensitivity, multiplexing and speed of analysis. To develop these microscale assays we utilize technologies covering a wide spectrum of complexity based on the requirements of the assays. For example, using off-the-shelf components we have developed a microtiter plate-based assay for screening cellulases against heterogeneous substrates. This approach relies on in-situ biomass regeneration in micro-volumes to volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for screening activities of novel IL-tolerant cellulases. We have also developed more sophisticated platforms for screening of lignocellulolytic enzymes using microfluidic chip for electrophoretic analysis of glycans. These chips are being used to screen genetically-engineered enzymes, assess performance of pretreatment processes, and for discovering cellulase activities in microbial communities. We are also developing high throughput microchips to perform combinatorial DNA assembly for optimization of biofuel synthesis pathways

Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin

A switch in specificity of avian influenza A viruses' hemagglutinin (HA) from avian-like (α2-3 sialylated glycans) to human-like (α2-6 sialylated glycans) receptors is believed to be associated with their adaptation to infect humans1, 2, 3, 4. We show that a characteristic structural topology-and not the α2-6 linkage itself-enables specific binding of HA to α2-6 sialylated glycans and that recognition of this topology may be critical for adaptation of HA to bind glycans in the upper respiratory tract of humans. An integrated biochemical, analytical and data mining approach demonstrates that HAs from the human-adapted H1N1 and H3N2 viruses, but not H5N1 (bird flu) viruses, specifically bind to long α2-6 sialylated glycans with this topology. This could explain why H5N1 viruses have not yet gained a foothold in the human population5, 6. Our findings will enable the development of additional strategies for effective surveillance and potential therapeutic interventions for H5N1 and possibly other influenza A viruses

α2–3 linked glycan distribution in ferret respiratory tract.

<p>MAL-II lectin (<i>green</i>) was used to stain ferret cranial, caudal and lung hilar regions. As seen from the images, MAL-II stained the submucosal glands, the underlying mucosa and some goblet cells (marked as *) in the caudal region. There was no staining of the lung hilar region indicating an absence of α2–3 glycans. The nuclei were stained with PI (<i>red</i>). The apical surface is marked with a <i>white</i> arrow.</p