Samir Gautam

ATP8B1 Deficiency Disrupts the Bile Canalicular Membrane Bilayer Structure in Hepatocytes, But FXR Expression and Activity Are Maintained

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane. METHODS ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs. RESULTS ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase. CONCLUSIONS ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

Type 2 inositol 1,4,5-trisphosphate receptor modulates bile salt export pump activity in rat hepatocytes.

Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short‐term regulation of Bsep activity. Ca2+ regulates targeting and insertion of transporters in many cell systems, and Ca2+ release near the canalicular membrane is mediated by the type II inositol 1,4,5‐trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido‐fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen‐ and endotoxin‐induced cholestasis. Conclusion: These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis. (HEPATOLOGY 2011;)

Exterior design: strategies for redecorating the bacterial surface with small molecules

Recombinant techniques for expressing heterologous proteins and sugars on the surface of bacteria have been known since the 1980s, and have proven useful in a variety of settings from biocatalysis to vaccinology. The past decade has also seen the emergence of novel methods that allow modification of bacterial surfaces with small non-biological compounds. Such technologies enable researchers to harness the unique properties of synthetic materials on a live bacterial platform, opening the door to an exciting new set of applications. Here we review strategies for bacterial surface display and describe how they have been applied thus far. We believe that chemical surface display holds great potential for advancing research in basic bacteriology and applied fields of biotechnology and biomedicine.

Chemical probes reveal an extraseptal mode of cross-linking in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen and a model organism for studying cell wall synthesis in Gram-positive cocci. The prevailing model of cell wall biogenesis in cocci holds that peptidoglycan synthesis (i.e., transglycosylation and cross-linking) is restricted spatially to the septal cross-wall and temporally to cell division. Previously, we developed a method for visualizing cross-linking in S. aureus using fluorescently tagged mimics of the endogenous substrate of penicillin-binding proteins (PBPs). These probes are incorporated into the cell wall of S. aureus specifically by PBP4, allowing localization of the enzymes cross-linking activity in vivo with precise spatial and temporal resolution. Here, using this methodology, we have discovered that PBP4 is active not only at the septum, but unexpectedly at the peripheral wall as well. These results challenge the long-held belief that peptidoglycan synthesis is restricted to the septum in spherical bacteria, and instead indicate the presence of two spatiotemporally distinct modes of cross-linking in S. aureus: one at the septum during cell division, and another at the peripheral wall between divisions.

Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterial pathogen that produces a range of infections including cellulitis, pneumonia, and septicemia. The principle mechanism in antistaphylococcal host defense is opsonization with antibodies and complement proteins, followed by phagocytic clearance. Here we use a previously developed technique for installing chemical epitopes in the peptidoglycan cell wall to show that surface glycopolymers known as wall teichoic acids conceal cell wall epitopes, preventing their recognition and opsonization by antibodies. Thus, our results reveal a previously unrecognized immunoevasive role for wall teichoic acids in S. aureus: repulsion of peptidoglycan-targeted antibodies.

Constrictive pericarditis with a calcified pericardial band at the level of left ventricle causing mid-ventricular obstruction

An adolescent presented with insidious onset and gradually progressive distension of abdomen associated with bilateral ankle swelling of few months duration. He had one episode of prolonged low-grade self-limiting febrile illness during childhood but had not consulted to doctor and never had been diagnosed as case of tuberculosis or acute pericarditis. A detail clinical evaluation showed raised central venous pressure, ascites and ankle oedema. Systemic examination was not much informative except ejection systolic murmur in third left intercostal space. Echocardiography and CT scan heart showed localised thickened pericardium with calcific band around the left ventricle at mid ventricle level. The band around the heart caused the heart to have a ‘dumbbell’ appearance with ballooning in apical area and a rare mid-ventricular obstruction in the left. A diagnosis of chronic constrictive pericarditis with calcific band was made and the patient was referred to another centre for cardiac surgery.

An Activity-Based Probe for Studying Crosslinking in Live Bacteria†

Penicillin-binding proteins (PBPs) catalyze the crosslinking of peptidoglycan (PG), an essential process for bacterial growth and survival, and a common antibiotic target. Yet, despite its importance, little is known about the spatiotemporal aspects of crosslinking—largely because of a lack of experimental tools for studying the reaction in live bacteria. Here we introduce such a tool: an activity-based probe that enables visualization and relative quantitation of crosslinking in vivo. In Staphylococcus aureus, we show that fluorescent mimics of the natural substrate of PBPs (PG stem peptide) are covalently incorporated into the cell wall, installing fluorophores in place of natural crosslinks. These fluorescent stem peptide mimics (FSPMs) are selectively recognized by a single PBP in S. aureus: PBP4. Thus, we were able to use FSPM pulse-labeling to localize PBP4 activity in live cells, showing that it is recruited to the septum in a manner dependent on wall teichoic acid.

Emergent pacemaker placement in a patient with Lyme carditis-induced complete heart block and ventricular asystole.

We report a case of a 31-year-old man who presented to the emergency department after four episodes of syncope within a 24 h time span. He was found to have symptomatic complete heart block associated with episodes of ventricular asystole lasting 5–6 s. He underwent emergent permanent pacemaker insertion during which he was found to have no underlying rhythm. He was later found to have positive serologies for Lyme disease despite no known exposure to ticks and neither signs nor symptoms of the disease. The pacemaker was ultimately removed due to resolution of his heart block with antibiotic therapy.

A Slick Solution to a Sticky Problem

Samir Gautam,†,‡ Lokesh Sharma,† Charles S. Dela Cruz,† and David Adam Spiegel*,‡ †Internal Medicine, Section of Pulmonary and Critical Care, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, United States ‡Department of Chemistry, Yale University, 225 Prospect Street, P.O. Box 208107, New Haven, Connecticut 06520, United States A Viewpoint for Biochemistry on the article in Cell, “Immunomimetic Designer Cells Protect Mice from MRSA Infection” In his 2002 essay entitled “Can a biologist fix a radio?Or, what I learned while studying apoptosis”, Yuri Lazebnik chides the biology community for its over-reliance on reductionistic experimental techniques such as genetic deletion to describe complex systems. He humorously illustrates his argument by describing how a biologist might attempt to fix a radio, including the obligatory annihilation of each electrical component “at close range with metal particles” to determine its function, an approach analogous to the murine knockout experiments upon which biologists so depend. In contrast, he submits that an engineer would solve the problem handily based on a facility with standardized technical language (electrical diagramming) that fully and unambiguously describes the mechanical device. The rub, of course, is that radios are built by engineers in the first place; cells, much less animals, are not. Indeed, a principle advantage engineers hold over biologists in terms of “handiness” derives from a hundred years of research into the fundamental principles of electromagnetism and another hundred in refining the practice of electrical engineering, which allows them to create electrical devices according to a standardized methodology. A similar position of privilege is enjoyed by modern organic chemists, who have likewise spent centuries designing and synthesizing a huge array of small molecules and natural products, including the chemical therapeutics that serve as the backbone of modern medicine, to establish a robust fundamental and technical appreciation for how molecules behave. Accordingly, it might be stated that the true understanding of a system, as achieved in areas of physics and chemistry, is indicated by the ability to construct one. In recent years, biologists have made strides toward this lofty goal as they have endeavored to create cells, either from scratch in the case of synthetic biology or by modifying existing cells for therapeutic ends. A particularly successful example of the latter comes from immuno-oncologists, who have genetically engineered T-cells to express specific sensors for cancer antigens called chimeric antigen receptors (CARs). Ligation of these receptors elicits robust effector responses (cytokine release by CD4+ cells and direct cytotoxicity by CD8+ cells), leading to the elimination of targeted tumor cells (Figure 1). The clinical success of CAR T-cells in the treatment of advanced hematologic malignancies is unprecedented; for instance, anti-CD19 CAR-T therapy induces complete remission in ∼90% of patients with chemo-resistant B cell leukemias, in whom life expectancy would otherwise be months. Now, Liu et al. apply this engineering principle to develop a novel therapeutic for infectious disease. They target methicillin-resistant Staphylococcus aureus (MRSA), a Grampositive bacterium that is responsible for more deaths in the United States than any other bacterial pathogen, and can infect virtually any tissue, including blood, lung, and skin. A particularly feared form of MRSA infection is establishment of biofilms on implanted hardware, such as prosthetic joints or heart valves, which almost invariably necessitates surgical removal of the foreign material due to resistance of the associated biofilms to antibiotics. To tackle this problem, Liu et al. construct an elegant genetic network consisting of three principal components (Figure 1): (i) a sensing mechanism for constituents of the S. aureus cell wall, namely, lipoproteins and wall teichoic acids, which are detected via the cell-surface pathogen recognition receptors TLR1, TLR2, and TLR6, along with the co-receptor CD14; (ii) a highly optimized signal transduction module based on the immune transcription factors NFκB and AP-1 and the promoter for interferon-β; and (iii) a response element leading to expression of proteins that function as either diagnostics (secreted alkaline phosphatase) or therapeutics

Chapter 4 – Pushing the Bacterial Envelope: Strategies for Re-Engineering Bacterial Surfaces with Heterologous Proteins and Sugars

Over the past three decades, a powerful array of techniques has been developed for expressing heterologous proteins and saccharides on the surface of bacteria. Surface-engineered bacteria, in turn, have proven useful in a variety of settings, including high-throughput screening, biofuel production, and vaccinology. In this chapter, we provide a comprehensive review of methods for displaying polypeptides and sugars on the bacterial cell surface, and discuss the many innovative applications these methods have found to date. While already an important biotechnological tool, we believe bacterial surface display may be further improved through integration with emerging methodology in other fields, such as protein engineering and synthetic chemistry. Ultimately, we envision bacterial display becoming a multidisciplinary platform with the potential to transform basic and applied research in bacteriology, biotechnology, and biomedicine.