Yeongjin Hong

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.

Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI‐MT‐I) have different substrate specificities and are targets of species‐ specific inhibitors of GPI biosynthesis. GPI‐MT‐I, however, has not been molecularly characterized. Characterization of GPI‐MT‐I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI‐MT‐I and the gene responsible, PIG‐M. PIG‐M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG‐M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals.

We describe a protocol for imaging bacterial luciferase (Lux)-expressing bacteria in small living animals. In this protocol, light emitted by Lux-expressing bacteria is detected and monitored by a cooled charge-coupled device detector. When these bacteria are administered to animals, it provides a potentially valuable approach to generate sensitive whole-body images with extremely low background. This imaging technology should enable the real-time monitoring of bacterial migration into both primary and metastatic tumors in several different mouse tumor models at a strong quantification power.

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin

Aerolysin of the Gram‐negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The α‐toxin of Gram‐positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)‐anchored proteins and generate pores in the cells plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI‐anchored proteins, but nevertheless bound aerolysin poorly and was 10‐fold less sensitive than wild‐type cells. A cDNA of N‐acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N‐glycan is involved in the binding. Removal of mannoses by α‐mannosidase II was important for the binding of aerolysin. In contrast, the α‐toxin killed GnTI‐deficient and wild‐type CHO cells equally, indicating that its binding to GPI‐anchored proteins is independent of N‐glycan. Because SL bound to wild‐type but not to GnTI‐deficient cells, and because a hybrid toxin consisting of SL and the α‐toxin killed wild‐type cells 10‐fold more efficiently than GnTI‐ deficient cells, SL with its binding site for N‐glycan contributes to the high binding affinity of aerolysin.

New paradigm for tumor theranostic methodology using bacteria-based microrobot.

We propose a bacteria-based microrobot (bacteriobot) based on a new fusion paradigm for theranostic activities against solid tumors. We develop a bacteriobot using the strong attachment of bacteria to Cy5.5-coated polystyrene microbeads due to the high-affinity interaction between biotin and streptavidin. The chemotactic responses of the bacteria and the bacteriobots to the concentration gradients of lysates or spheroids of solid tumors can be detected as the migration of the bacteria and/or the bacteriobots out of the central region toward the side regions in a chemotactic microfluidic chamber. The bacteriobots showed higher migration velocity toward tumor cell lysates or spheroids than toward normal cells. In addition, when only the bacteriobots were injected to the CT-26 tumor mouse model, Cy5.5 signal was detected from the tumor site of the mouse model. In-vitro and in-vivo tests verified that the bacteriobots had chemotactic motility and tumor targeting ability. The new microrobot paradigm in which bacteria act as microactuators and microsensors to deliver microstructures to tumors can be considered a new theranostic methodology for targeting and treating solid tumors.

Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol

Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases.Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutantPig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen inPig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F.

Inhibition of Tumor Growth and Metastasis by a Combination of Escherichia coli–mediated Cytolytic Therapy and Radiotherapy

We have reported that Escherichia coli K-12 colonizes hypoxic and necrotic tumor regions after intravenous injection into tumor-bearing mice. In this study, we established a novel strategy for cancer therapy using engineered bacteria to enhance the therapeutic effects of radiation. E. coli strain K-12 was engineered to produce cytolysin A (ClyA), and its effects on tumor growth in primary and metastatic tumor models were evaluated. A single treatment with E. coli-expressing ClyA significantly decreased tumor growth rates initially (9 days after treatment); however, the tumors tended to grow thereafter. With only radiotherapy (RT; 21 Gy), the tumor growth rates were retarded, but not the tumor sizes. A combination of therapy with E. coli-expressing ClyA and radiation [a total of 5 x 10(7) colony-forming units (CFU) and 21 Gy] resulted in significant tumor shrinkage and even complete disappearance of tumors in mice with tumors derived from murine CT26 colon cancer. Furthermore, treatment with E. coli-expressing ClyA markedly suppressed metastatic tumor growth and prolonged the survival time in mice. The results described here indicate that therapy with engineered E. coli could significantly improve the results of RT, and could exert a striking inhibitory effect on the development of lung metastasis.

Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin

Engineered Salmonella secreting heterologous bacterial flagellin suppress tumor growth by activating intratumoral macrophages. Two bacteria can be better than one In some cases, injecting tumors with specific bacteria can help eradicate the tumors by stimulating inflammation and triggering an antitumor immune response. A classic example of this is injection of bladder cancer with bacillus Calmette-Guérin, but more recent approaches have used bacteria such as Clostridium and Salmonella species. Building on the idea of antitumor bacterial therapy, Zheng et al. engineered a weakened strain of Salmonella typhimurium to produce the flagellin B protein from another bacterium, Vibrio vulnificus. The engineered bacteria induced an effective antitumor immune response, successfully treating tumors in several different mouse models with no evidence of toxicity. We report a method of cancer immunotherapy using an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)–negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered Salmonella appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonella resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

The glycan core of GPI-anchored proteins modulates aerolysin binding but is not sufficient: the polypeptide moiety is required for the toxin-receptor interaction.

Sensitivity of mammalian cells to the bacterial toxin aerolysin is due to the presence at their surface of glycosylphosphatidyl inositol (GPI)‐anchored proteins which act as receptors. Using a panel of mutants that are affected in the GPI biosynthetic pathway and Trypanosoma brucei variant surface glycoproteins, we show that addition of an ethanolamine phosphate residue on the first mannose of the glycan core does not affect binding. In contrast, the addition of a side chain of up to four galactose residues at position 3 of this same mannose leads to an increase in binding. However, protein free GPIs, which accumulate in mutant cells deficient in the transamidase that transfers the protein to the pre‐formed GPI‐anchor, were unable to bind the toxin indicating a requirement for the polypeptide moiety, the nature and size of which seem of little importance although two exceptions have been identified.

GPI1 Stabilizes an Enzyme Essential in the First Step of Glycosylphosphatidylinositol Biosynthesis

Attachment of glycosylphosphatidylinositol (GPI) is essential for the surface expression of many proteins. Biosynthesis of glycosylphosphatidylinositol is initiated by the transfer ofN-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol. In mammalian cells, this reaction is mediated by a complex of PIG-A, PIG-H, PIG-C, and GPI1. This complexity may be relevant for regulation and for usage of a particular phosphatidylinositol. However, the functions of the respective components have been unclear. Here we cloned the mouse GPI1 gene and disrupted it in F9 embryonal carcinoma cells. Disruption of the GPI1 gene caused a severe but not complete defect in the generation of glycosylphosphatidylinositol-anchored proteins, indicating some residual biosynthetic activity. A complex of PIG-A, PIG-H, and PIG-C decreased to a nearly undetectable level, whereas a complex of PIG-A and PIG-H was easily detected. A lack of GPI1 also caused partial decreases of PIG-C and PIG-H. Therefore, GPI1 stabilizes the enzyme by tying up PIG-C with a complex of PIG-A and PIG-H.

Dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves

SummaryAlthough winter dysentery (WD), which is caused by the bovine coronavirus (BCoV) is characterized by the sudden onset of diarrhea in many adult cattle in a herd, the pathogenesis of the WD-BCoV is not completely understood. In this study, colostrum-deprived calves were experimentally infected with a Korean WD-BCoV strain and examined for viremia, enteric and nasal virus shedding as well as for viral antigen expression and virus-associated lesions in the small and large intestines and the upper and lower respiratory tract from 1 to 8 days after an oral infection. The WD-BCoV-inoculated calves showed gradual villous atrophy in the small intestine and a gradual increase in the crypt depth of the large intestine. The WD-BCoV-infected animals showed epithelial damage in nasal turbinates, trachea and lungs, and interstitial pneumonia. The WD-BCoV antigen was detected in the epithelium of the small and large intestines, nasal turbinates, trachea and lungs. WD-BCoV RNA was detected in the serum from post-inoculation day 3. These results show that the WD-BCoV has dual tropism and induces pathological changes in both the digestive and respiratory tracts of calves. To our knowledge, this is the first detailed report of dual enteric and respiratory tropisms of WD-BCoV in calves. Comprehensive studies of the dual tissue pathogenesis of the BCoV might contribute to an increased understanding of similar pneumoenteric CoV infections in humans.