Antigen-Specific Memory B-cell Responses to Enterotoxigenic Escherichia coli Infection in Bangladeshi Adults

Background: Multiple infections with diverse enterotoxigenic E. coli (ETEC) strains lead to broad spectrum protection against ETEC diarrhea. However, the precise mechanism of protection against ETEC infection is still unknown. Therefore, memory B cell responses and affinity maturation of antibodies to the specific ETEC antigens might be important to understand the mechanism of protection. Methodology In this study, we investigated the heat labile toxin B subunit (LTB) and colonization factor antigens (CFA/I and CS6) specific IgA and IgG memory B cell responses in Bangladeshi adults (n = 52) who were infected with ETEC. We also investigated the avidity of IgA and IgG antibodies that developed after infection to these antigens. Principal Findings Patients infected with ETEC expressing LT or LT+heat stable toxin (ST) and CFA/I group or CS6 colonization factors developed LTB, CFA/I or CS6 specific memory B cell responses at day 30 after infection. Similarly, these patients developed high avidity IgA and IgG antibodies to LTB, CFA/I or CS6 at day 7 that remained significantly elevated at day 30 when compared to the avidity of these specific antibodies at the acute stage of infection (day 2). The memory B cell responses, antibody avidity and other immune responses to CFA/I not only developed in patients infected with ETEC expressing CFA/I but also in those infected with ETEC expressing CFA/I cross-reacting epitopes. We also detected a significant positive correlation of LTB, CFA/I and CS6 specific memory B cell responses with the corresponding increase in antibody avidity. Conclusion: This study demonstrates that natural infection with ETEC induces memory B cells and high avidity antibodies to LTB and colonization factor CFA/I and CS6 antigens that could mediate anamnestic responses on re-exposure to ETEC and may help in understanding the requirements to design an effective vaccination strategies

Roles for cell surface glycans in guiding human pluripotent stem cell fate

Cell-surface polysaccharides are a fundamental component of the stem cell microenvironment. They are known to modulate developmental signals- critical for pluripotency and differentiation. Nevertheless, the architecture of the cellular glycocalyx and how these structures direct the fate of human pluripotent stem (hPS) cells have not been fully explored. I addressed this gap with a focus on a critical glycan of the hPS cells niche, heparan sulfate (HS). HS is a heterogeneous long-chain cell-surface polysaccharide. The spatial distribution and ultrastructure of this information-rich, signaling polysaccharides are poorly defined. In this work, I aim to understand the interplay between the HS organization and the developmental signal transduction in the hPS cells’ microenvironment. We discovered that HS of hPS cells has a dynamic ultrastructure that undergoes changes during lineage-specific differentiation. These changes also correlate with the cells’ ability to bind specific growth factor. While variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our findings indicate a role for HS ultrastructure in its ability to recruit growth factors in stem cell niche. To advance the understanding of its roles in human development, next we engineered a HS-deficient cell line derived from hPS cells. Parallelly, I set out to develop a synthetic, modular surface-based cell separation strategy that can isolate or enrich cells of interest in a rapid and label-free way. I applied this strategy to isolate genetically engineered HS-deficient hPS cells after a CRISPR modification by engaging the cell surface HS with a small peptide- presenting synthetic surface. These HS-deficient hPS cells aid the investigation further to understand the role of HS in human development. We showed that the multi-lineage commitment of hPS cells depends on HS. Moreover, lack of HS hinders the proper neuronal projections and synaptic vesicle formation in hPS cell-derived neurons, suggesting a specific role of HS in human neural development. Taken together, these results indicate that HS has a highly dynamic ultrastructure that modulates cell fate choices of hPS cells, specifically neuronal connection formation. This work paves the way to a better understanding of HS’s role in early human development.Ph.D

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding

Demographic, clinical and microbiological characteristics of patients.

<p>Demographic, clinical and microbiological characteristics of patients.</p

Anti-LTB (A and D), CFA/I (B and E) and CS6 (C and F) specific IgA and IgG antibody responses in plasma in Bangladeshi adults infected with ETEC.

<p>The columns indicate mean responses and the error bars represent standard errors of the mean (SEM). An asterisk denotes a statistically significant difference (<i>P</i><0.05) from the acute stage of infection (day 2). Mean fold changes and responder frequencies compared to day 2 levels are also indicated.</p

Avidity indices of LTB (A and D), CFA/I (B and E) and CS6 (C and F) specific IgA and IgG antibodies in plasma in Bangladeshi adults infected with ETEC.

<p>Columns indicate mean avidity indices, and error bars represent standard errors of the mean (SEM). An asterisk denotes a statistically significant difference (<i>P</i><0.05) from the acute stage of infection (day 2).</p

Correlation analyses^* of antibody avidity index (AI) and memory B cell (MBC) responses in patients infected with ETEC.

<p>*Spearman's test was used for correlation analyses.</p