Imaging Tumor-Targeting Bacteria Using 18F-Fluorodeoxysorbitol Positron Emission Tomography.

BACKGROUND: Microbial-based cancer treatments are an emerging field, with multiple bacterial species evaluated in animal models and some advancing to clinical trials. Noninvasive bacteria-specific imaging approaches can potentially support the development and clinical translation of bacteria-based cancer treatments by assessing the tumor and off-target bacterial colonization. METHODS: 18F-Fluorodeoxysorbitol (18F-FDS) positron emission tomography (PET), a bacteria-specific imaging approach, was used to visualize an attenuated strain of Yersinia enterocolitica, currently in clinical trials as a microbial-based cancer treatment, in murine models of breast cancer. RESULTS: Y. enterocolitica demonstrated excellent 18F-FDS uptake in in vitro assays. Whole-body 18F-FDS PET demonstrated a significantly higher PET signal in tumors with Y. enterocolitica colonization compared to those not colonized, in murine models utilizing direct intratumor or intravenous administration of bacteria, which were confirmed using ex vivo gamma counting. Conversely, 18F-fluorodeoxyglucose (18F-FDG) PET signal was not different in Y. enterocolitica colonized versus uncolonized tumors. CONCLUSIONS: Given that PET is widely used for the management of cancer patients, 18F-FDS PET could be utilized as a complementary approach supporting the development and clinical translation of Y. enterocolitica-based tumor-targeting bacterial therapeutics.

Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A.

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.

NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus.

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.

Chemical synthesis of Burkholderia Lipid†A modified with glycosyl phosphodiester-linked 4-amino-4-deoxy-ß-L-arabinose and its immunomodulatory potential.

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-ß-L-arabinose (ß-L-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by ß-L-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the ß-L-Ara4N H-phosphonate to α-lactol of ß(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1'-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, ß-L-Ara4N-substituted Burkholderia Lipid A. The ß-L-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A.

The lipopolysaccharide from Capnocytophaga canimorsus reveals an unexpected role of the core-oligosaccharide in MD-2 binding.

Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a "hybrid backbone" lacking the 4' phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 - lipid A complex in case the 4' phosphate is not present.

Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)-a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids-disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible ß(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.

Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.

Development of αGlcN(1↔1)αMan-based lipid A mimetics as a novel class of potent Toll-like receptor 4 agonists.

The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4·MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible ßGlcN(1→6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like αGlcN(1↔1)αMan scaffold resembling the molecular shape of TLR4·MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected αGlcN(1↔1)αMan disaccharide has been elaborated. The α,α-(1↔1) linkage was attained by the glycosylation of 2-N-carbamate-protected α-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. αGlcN(1↔1)αMan-based Lipid A mimetics (α,α-GM-LAM) induced potent activation of NF-κB signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the "agonistic" conformation attained by a rigid αGlcN(1↔1)αMan scaffold represents an efficient approach toward powerful and adjustable TLR4 activation.

Conformationally constrained lipid A mimetics for exploration of structural basis of TLR4/MD-2 activation by lipopolysaccharide.

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on ß,α-trehalose-type scaffold. Replacement of the flexible three-bond ß(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond ß,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a ß-GlcN(1↔1)α-GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.