Luz P. Blanco

Diversity, biogenesis and function of microbial amyloids

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimers, Parkinsons and Huntingtons diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Background Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations. Methodology and Principal Findings Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/−17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached ≥106 titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-γ and TNF-α cytokine production and elevated levels of IgG2 subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4°C, 6 months at 25°C and 6 weeks at 40°C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models. Conclusions Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy.

Iron induces bimodal population development by Escherichia coli.

Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air–biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H2O2 toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

The Role of Ganglioside GM1 in Cellular Internalization Mechanisms of Poly(amidoamine) Dendrimers

Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by (1)H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH(2)) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM(1)-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH(2) dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH(2) was not ganglioside GM(1) dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM(1) in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.

Prior Helicobacter pylori infection ameliorates Salmonella typhimurium‐induced colitis: Mucosal crosstalk between stomach and distal intestine

Background: Helicobacter pylori infection is associated with a lower risk of chronic autoimmune diseases including inflammatory bowel disease (IBD). H. pylori modulates the gastric immune response, decreasing the local inflammatory response to itself. In mice, chronic Salmonella typhimurium infection induces colitis similar to Crohns disease, characterized by inflammation, which progresses toward fibrosis. The aim of this study was to determine whether prior H. pylori infection acts at a distance to modulate the immune response of S. typhimurium‐induced colitis. Methods: Mice were infected with the mouse‐adapted strain of H. pylori (SS1), followed by infection with S. typhimurium. The effect of H. pylori on colitis was determined by gross pathology, histopathology, cytokine response, and development of fibrosis in the cecum. Gastritis and systemic immune response was measured in response to infection. Results: H. pylori suppresses the Th17 response to S. typhimurium infection in the mouse cecum, but does not alter the Th2 or T‐regulatory response or the development of fibrosis. H. pylori infection induces IL‐10 in the mesenteric lymph nodes, suggesting an extragastric mechanism for immunomodulation. H. pylori / S. typhimurium coinfection decreases inflammation in both the cecum and the stomach. Conclusions: This study demonstrates a potential mechanism for the negative association between H. pylori and IBD in humans. H. pylori represses the lower gastrointestinal tract Th17 response to bacterially induced colitis via extragastric immunomodulatory effects, illustrating immunological crosstalk between the upper and lower gastrointestinal tract. (Inflamm Bowel Dis 2011;)

Bacterial‐associated cholera toxin and GM1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M‐like cells in a Caco‐2 monolayer. Interspersed among the epithelial monolayer of Caco‐2 cells we detect cells that display M‐like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre‐incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT‐dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.

Nanoemulsion mucosal adjuvant uniquely activates cytokine production by nasal ciliated epithelium and induces dendritic cell trafficking

While the nasal mucosa is a potentially useful site for human immunization, toxin‐based nasal adjuvants are generally unsafe and less effective in humans. Safe mucosal adjuvants that activate protective immunity via mucosal administration are highly dependent on barrier antigen sampling by epithelial and DCs. Here, we demonstrate that protein antigens formulated in unique oil‐in‐water nanoemulsions (NEs) result in distinctive transcellular antigen uptake in ciliated nasal epithelial cells, leading to delivery into nasal associated lymphoid tissue. NE formulation also enhances MHC class II expression in epithelial cells and DC activation/trafficking to regional lymphoid tissues in mice. These materials appear to induce local epithelial cell apoptosis and heterogeneous cytokine production by mucosal epithelial cells and mixed nasal tissues, including G‐CSF, GM‐CSF, IL‐1a, IL‐1b, IL‐5, IL‐6, IL‐12, IP‐10, KC, MIP‐1a, TGF‐β, and TSLP. This is the first observation of a nasal adjuvant that activates calreticulin‐associated apoptosis of ciliated nasal epithelial cells to generate broad cytokine/chemokine responses in mucosal tissue.

Distinct Pathways of Humoral and Cellular Immunity Induced with the Mucosal Administration of a Nanoemulsion Adjuvant

Nasal administration of an oil-in-water nanoemulsion (NE) adjuvant W805EC produces potent systemic and mucosal, Th-1– and Th-17–balanced cellular responses. However, its molecular mechanism of action has not been fully characterized and is of particular interest because NE does not contain specific ligands for innate immune receptors. In these studies, we demonstrate that W805EC NE adjuvant activates innate immunity, induces specific gene transcription, and modulates NF-κB activity via TLR2 and TLR4 by a mechanism that appears to be distinct from typical TLR agonists. Nasal immunization with NE-based vaccine showed that the TLR2, TLR4, and MyD88 pathways and IL-12 and IL-12Rβ1 expression are not required for an Ab response, but they are essential for the induction of balanced Th-1 polarization and Th-17 cellular immunity. NE adjuvant induces MHC class II, CD80, and CD86 costimulatory molecule expression and dendritic cell maturation. Further, upon immunization with NE, adjuvant mice deficient in the CD86 receptor had normal Ab responses but significantly reduced Th-1 cellular responses, whereas animals deficient in both CD80 and CD86 or lacking CD40 failed to produce either humoral or cellular immunity. Overall, our data show that intranasal administration of Ag with NE induces TLR2 and TLR4 activation along with a MyD88-independent Ab response and a MyD88-dependent Th-1 and Th-17 cell–mediated immune response. These findings suggest that the unique properties of NE adjuvant may offer novel opportunities for understanding previously unrecognized mechanisms of immune activation important for generating effective mucosal and systemic immune responses.

Antibodies Enhance Interaction of Vibrio cholerae with Intestinal M-Like Cells

ABSTRACT Intestinal M cells bear a receptor for secretory immunoglobulin A (IgA) (sIgA) facing the lumen of the epithelial surfaces. Cells bearing this receptor are also found throughout an experimental monolayer consisting of polarized Caco-2 cells, a colon adenocarcinoma cell line. The presence of antibodies (mainly sIgA) in the lumen of the small intestine led us to explore the participation of the sIgA receptor and antibodies in the interaction of Caco-2-associated M-like cells with the mucosal pathogen Vibrio cholerae. Here, we demonstrate that sIgA antibodies isolated from pooled healthy human colostrums, as well as IgG from pooled healthy human serum, can recognize V. cholerae. Furthermore, opsonization enhances M-like-cell transcytosis of V. cholerae strains. We also show that the cholera toxin (CT) receptor ganglioside GM1 colocalizes with the sIgA receptor in cells of the epithelial monolayer. Both sIgA and IgG antibodies compete for the attachment of soluble CT subunit B to immobilized GM1. Our results indicate that in this in vitro model system of intestinal epithelia, human sIgA and IgG contribute to the uptake of V. cholerae by M-like cells, probably through an interaction with GM1. Our results support previous findings of others showing that sIgA can act as an endogenous adjuvant and that sIgA is important for the antigen-sampling function of M cells.

Transient or persistent norovirus infection does not alter the pathology of Salmonella typhimurium induced intestinal inflammation and fibrosis in mice

Murine noroviruses (MNV) are currently the most prevalent viruses infecting mouse research colonies. Concurrent infection of research mice with these viruses can dramatically alter the experimental outcome in some research models, but not others. In this report, we investigated the effect of MNV1 and MNV4 on a murine model of intestinal inflammation and fibrosis induced by Salmonella typhimurium infection in C57BL/6 mice. Subsequent co-infection of these mice with MNV1 or MNV4 did not lead to major changes in histopathology, the inflammatory response, or the fibrotic response. Thus, MNV does not substantially alter all gastrointestinal research models, highlighting the importance of investigating potential alterations in the research outcome by MNV on an individual basis. We hypothesize that this is particularly important in cases of research models that use immunocompromised mice, which could be more sensitive to MNV infection-induced changes.