Schistosoma mansoni infection alters the host pre-vaccination environment resulting in blunted Hepatitis B vaccination immune responses.

Schistosomiasis is a disease caused by parasitic flatworms of the Schistosoma spp., and is increasingly recognized to alter the immune system, and the potential to respond to vaccines. The impact of endemic infections on protective immunity is critical to inform vaccination strategies globally. We assessed the influence of Schistosoma mansoni worm burden on multiple host vaccine-related immune parameters in a Ugandan fishing cohort (n = 75) given three doses of a Hepatitis B (HepB) vaccine at baseline and multiple timepoints post-vaccination. We observed distinct differences in immune responses in instances of higher worm burden, compared to low worm burden or non-infected. Concentrations of pre-vaccination serum schistosome-specific circulating anodic antigen (CAA), linked to worm burden, showed a significant bimodal distribution associated with HepB titers, which was lower in individuals with higher CAA values at month 7 post-vaccination (M7). Comparative chemokine/cytokine responses revealed significant upregulation of CCL19, CXCL9 and CCL17 known to be involved in T cell activation and recruitment, in higher CAA individuals, and CCL17 correlated negatively with HepB titers at month 12 post-vaccination. We show that HepB-specific CD4+ T cell memory responses correlated positively with HepB titers at M7. We further established that those participants with high CAA had significantly lower frequencies of circulating T follicular helper (cTfh) subpopulations pre- and post-vaccination, but higher regulatory T cells (Tregs) post-vaccination, suggesting changes in the immune microenvironment in high CAA could favor Treg recruitment and activation. Additionally, we found that changes in the levels of innate-related cytokines/chemokines CXCL10, IL-1ß, and CCL26, involved in driving T helper responses, were associated with increasing CAA concentration. This study provides further insight on pre-vaccination host responses to Schistosoma worm burden which will support our understanding of vaccine responses altered by pathogenic host immune mechanisms and memory function and explain abrogated vaccine responses in communities with endemic infections.

Schistosoma mansoni infection alters the host pre-vaccination environment resulting in blunted Hepatitis B vaccination immune responses.

The impact of endemic infections on protective immunity is critical to inform vaccination strategies. In this study, we assessed the influence of Schistosoma mansoni infection on host responses in a Ugandan fishing cohort given a Hepatitis B (HepB) vaccine. Concentrations of schistosome-specific circulating anodic antigen (CAA) pre-vaccination showed a significant bimodal distribution associated with HepB titers, which were lower in individuals with high CAA. We established that participants with high CAA had significantly lower frequencies of circulating T follicular helper (cTfh) subpopulations pre- and post-vaccination and higher regulatory T cells (Tregs) post-vaccination. Polarization towards higher frequencies of Tregs: cTfh cells can be mediated by changes in the cytokine environment favoring Treg differentiation. In fact, we observed higher levels of CCL17 and soluble IL-2R pre-vaccination (important for Treg recruitment and development), in individuals with high CAA that negatively associated with HepB titers. Additionally, alterations in pre-vaccination monocyte function correlated with HepB titers, and changes in innate-related cytokines/chemokine production were associated with increasing CAA concentration. We report, that by influencing the immune landscape, schistosomiasis has the potential to modulate immune responses to HepB vaccination. These findings highlight multiple Schistosoma -related immune associations that could explain abrogated vaccine responses in communities with endemic infections. Author Summary: Schistosomiasis drives host immune responses for optimal pathogen survival, potentially altering host responses to vaccine-related antigen. Chronic schistosomiasis and co-infection with hepatotropic viruses are common in countries where schistosomiasis is endemic. We explored the impact of Schistosoma mansoni ( S. mansoni ) infection on Hepatitis B (HepB) vaccination of individuals from a fishing community in Uganda. We demonstrate that high schistosome-specific antigen (circulating anodic antigen, CAA) concentration pre-vaccination, is associated with lower HepB antibody titers post-vaccination. We show higher pre-vaccination levels of cellular and soluble factors in instances of high CAA that are negatively associated with HepB antibody titers post-vaccination, which coincided with lower frequencies of circulating T follicular helper cell populations (cTfh), proliferating antibody secreting cells (ASCs), and higher frequencies of regulatory T cells (Tregs). We also show that monocyte function is important in HepB vaccine responses, and that high CAA is associated with alterations in the early innate cytokine/chemokine microenvironment. Our findings suggest that in individuals with high CAA and likely high worm burden, schistosomiasis creates and sustains an environment that is polarized against optimal host immune responses to the vaccine, which puts many endemic communities at risk for infection against HepB and other diseases that are preventable by vaccines.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-ß. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV.

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.

Human Monocyte Subsets Are Transcriptionally and Functionally Altered in Aging in Response to Pattern Recognition Receptor Agonists.

Age-related alterations in immunity have been linked to increased incidence of infections and decreased responses to vaccines in the aging population. Human peripheral blood monocytes are known to promote Ag presentation and antiviral activities; however, the impact of aging on monocyte functions remains an open question. We present an in-depth global analysis examining the impact of aging on classical (CD14+CD16-), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) monocytes. Monocytes sorted from nonfrail healthy adults (21-40 y) and old (≥65 y) individuals were analyzed after stimulation with TLR4, TLR7/8, and retinoic acid-inducible gene I agonists. Our data showed that under nonstimulated conditions, monocyte subsets did not reveal significant age-related alternations; however, agonist stimulated-monocytes from adults and old subjects did show differences at the transcriptional and functional levels. These alternations in many immune-related transcripts and biological processes resulted in reduced production of IFN-α, IFN-γ, IL-1ß, CCL20, and CCL8, and higher expression of CX3CR1 in monocytes from old subjects. Our findings represent a comprehensive analysis of the influence of human aging on pattern recognition receptors signaling and monocyte functions, and have implications for strategies to enhance the immune response in the context of infection and immunization.

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection.

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection.

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.

Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors.

Aging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG-I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro-inflammatory and antiviral cytokines and chemokines including TNFα, IL-6, IL-1ß, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up-regulation of PD-L1 on monocytes and T cells, and increased expression of PD-L2 and B7-H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR-stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T-cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age-associated changes in cytokine, chemokine and interferon production, as well as co-stimulatory protein expression could contribute to the blunted memory B- and T-cell immune responses to vaccines and infections.

Inadequate T follicular cell help impairs B cell immunity during HIV infection.

The majority of HIV-infected individuals fail to produce protective antibodies and have diminished responses to new immunizations. We report here that even though there is an expansion of follicular helper T (TFH) cells in HIV-infected individuals, the cells are unable to provide adequate B cell help. We found a higher frequency of programmed cell death ligand 1 (PD-L1)(+) germinal center B cells from lymph nodes of HIV-infected individuals suggesting a potential role for PD-1-PD-L1 interaction in regulating TFH cell function. In fact, we show that engagement of PD-1 on TFH cells leads to a reduction in cell proliferation, activation, inducible T-cell co-stimulator (ICOS) expression and interleukin-21 (IL-21) cytokine secretion. Blocking PD-1 signaling enhances HIV-specific immunoglobulin production in vitro. We further show that at least part of this defect involves IL-21, as addition of this cytokine rescues antibody responses and plasma cell generation in vitro. Our results suggest that deregulation of TFH cell-mediated B cell help diminishes B cell responses during HIV infection and may be related to PD-1 triggering on TFH cells. These results demonstrate a role for TFH cell impairment in HIV pathogenesis and suggest that enhancing their function could have a major impact on the outcome and control of HIV infection, preventing future infections and improving immune responses to vaccinations.

Recruitment and retention of B cells in the central nervous system in response to alphavirus encephalomyelitis.

Sindbis virus (SINV) infection of neurons results in nonfatal viral encephalomyelitis and provides a model system for understanding recovery from virus infection of the central nervous system (CNS). Infection is followed by clearance of infectious virus, a gradual decrease in viral RNA, and then long-term maintenance of low levels of viral RNA. Antibody to the E2 glycoprotein is important for virus clearance, and B cells enter the CNS along with CD4(+) and CD8(+) T cells during the early clearance phase. Antibody-secreting cells (ASCs) are present in the CNS and become enriched for SINV-specific ASCs. We have evaluated the factors within the CNS that facilitate continued local antibody production after infection. Expression of CXCL9, CXCL10, CCL1, CCL2, and CCL5 chemokine mRNAs increased early, and infiltrating B cells expressed CXCR3, CXCR5, and CCR7. The mRNAs for IL-10 and IL-21, cytokines important for B cell proliferation and differentiation, rose rapidly and remained elevated long after clearance of infectious virus. Active proliferation of B cells, as indicated by Ki-67 expression, continued for months. Bromodeoxyuridine (BrdU) labeling of proliferating cells showed that ASCs produced in the draining cervical lymph nodes during the early germinal center response were preferentially retained in the CNS. Sustained increase in B-cell-activating factor (BAFF) mRNA in the CNS and BAFF receptor expression by B cells coincided with the long-term maintenance of SINV-specific ASCs in the brain. We conclude that multiple changes in the brain microenvironment facilitate B-cell entry and support proliferation and differentiation and long-term survival of antiviral ASCs during recovery from alphaviral encephalomyelitis.

Foxo3a: an integrator of immune dysfunction during HIV infection.

Chronic HIV infection, which is primarily characterized by the progressive depletion of total CD4(+) T cells, also causes persistent inflammation and immune activation. This is followed by profound changes in cellular and tissue microenvironments that often lead to prolonged immune dysfunction. The global nature of this immune dysfunction suggests that factors that are involved in immune cell survival, proliferation, differentiation and maturation are all affected. Of particular interest is the transcriptional factor Foxo3a that regulates a number of genes that are critical in the development and the maintenance of T and B cells, dendritic cells (DCs) and macrophages. Alterations in the microenvironment mediated by HIV infection cause significant increase in the transcriptional activity of Foxo3a; this has major impact on T cell and B cell immunity. In fact, recent findings from HIV infected individuals highlight three important points: (1) the alteration of Foxo3a signaling during HIV infection deregulates innate and adaptive immune responses; (2) Foxo3a-mediated effects are reversible and could be restored by interfering with the Foxo3a pathway; and (3) down-regulation of Foxo3a transcriptional activity in elite controllers (ECs) represents a molecular signature, or a correlate of immunity, associated with natural protection and lack of disease progression. In this review, we will discuss how HIV-infection altered microenvironments could result in impaired immune responses via the Foxo3a signaling pathway. Defining precisely the molecular mechanisms of how persistent inflammation and immune activation are able to influence the Foxo3a pathway could ultimately help in the development of novel approaches to improve immune responses in HIV infected subjects.

Clearance of virus infection from the CNS.

Viruses that cause encephalomyelitis infect neurons and recovery from infection requires noncytolytic clearance of virus from the nervous system to avoid damaging these irreplaceable cells. Several murine model systems of virus infection have been used to identify clearance mechanisms. Quantitative analysis of Sindbis virus clearance over 6 months shows three phases: day 5-7, clearance of infectious virus, but continued presence of viral RNA; day 8-60, decreasing levels of viral RNA; day 60-180, maintenance of viral RNA at low levels. Antiviral antibody and interferon-γ have major roles in clearance with a likely role for IgM as well as IgG antibody. Long-term residence of virus-specific immune cells in the nervous system is necessary to prevent virus reactivation.

Alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system.

Sindbis virus (SINV) infection of the central nervous system (CNS) provides a model for understanding the role of the immune response in recovery from alphavirus infection of neurons. Virus clearance occurred in three phases: clearance of infectious virus (days 3 to 7), clearance of viral RNA (days 8 to 60), and maintenance of low levels of viral RNA (>day 60). The antiviral immune response was initiated in the cervical lymph nodes with rapid extrafollicular production of plasmablasts secreting IgM, followed by germinal center production of IgG-secreting and memory B cells. The earliest inflammatory cells to enter the brain were CD8(+) T cells, followed by CD4(+) T cells and CD19(+) B cells. During the clearance of infectious virus, effector lymphocytes in the CNS were primarily CD8(+) T cells and IgM antibody-secreting cells (ASCs). During the clearance of viral RNA, there were more CD4(+) than CD8(+) T cells, and B cells included IgG and IgA ASCs. At late times after infection, ASCs in the CNS were primarily CD19(+) CD38(+) CD138(-) Blimp-1(+) plasmablasts, with few fully differentiated CD38(-) CD138(+) Blimp-1(+) plasma cells. CD19(+) CD38(+) surface Ig(+) memory B cells were also present. The level of antibody to SINV increased in the brain over time, and the proportion of SINV-specific ASCs increased from 15% of total ASCs at day 14 to 90% at 4 to 6 months, suggesting specific retention in the CNS during viral RNA persistence. B cells in the CNS continued to differentiate, as evidenced by accumulation of IgA ASCs not present in peripheral lymphoid tissue and downregulation of major histocompatibility complex (MHC) class II expression on plasmablasts. However, there was no evidence of germinal center activity or IgG avidity maturation within the CNS.

Dependence of stress resistance on a spore coat heteropolysaccharide in Dictyostelium.

In Dictyostelium, sporulation occurs synchronously as prespore cells approach the apex of the aerial stalk during culmination. Each prespore cell becomes surrounded by its own coat comprised of a core of crystalline cellulose and a branched heteropolysaccharide sandwiched between heterogeneous cysteine-rich glycoproteins. The function of the heteropolysaccharide, which consists of galactose and N-acetylgalactosamine, is unknown. Two glycosyltransferase-like genes encoding multifunctional proteins, each with predicted features of a heteropolysaccharide synthase, were identified in the Dictyostelium discoideum genome. pgtB and pgtC transcripts were modestly upregulated during early development, and pgtB was further intensely upregulated at the time of heteropolysaccharide accumulation. Disruption of either gene reduced synthase-like activity and blocked heteropolysaccharide formation, based on loss of cytological labeling with a lectin and absence of component sugars after acid hydrolysis. Cell mixing experiments showed that heteropolysaccharide expression is spore cell autonomous, suggesting a physical association with other coat molecules during assembly. Mutant coats expressed reduced levels of crystalline cellulose based on chemical analysis after acid degradation, and cellulose was heterogeneously affected based on flow cytometry and electron microscopy. Mutant coats also contained elevated levels of selected coat proteins but not others and were sensitive to shear. Mutant spores were unusually susceptible to hypertonic collapse and damage by detergent or hypertonic stress. Thus, the heteropolysaccharide is essential for spore integrity, which can be explained by a role in the formation of crystalline cellulose and regulation of the protein content of the coat.

Role of SP65 in assembly of the Dictyostelium discoideum spore coat.

Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.

Initiation of mucin-type O-glycosylation in dictyostelium is homologous to the corresponding step in animals and is important for spore coat function.

Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by alpha-linked GlcNAc rather than alpha-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide alpha-N-acetylglucosaminyltransferase (pp alpha-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp alpha-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp alpha-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp alpha-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp alpha-GlcNAc-T2 attaches GlcNAc in alpha-linkage to the Thr residues of all the synthetic mucin repeats. pp alpha-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp alpha-GlcNAc-T2 and the animal pp alpha-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.

Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.