Potjanee Srimanote

Characterization of Saa, a Novel Autoagglutinating Adhesin Produced by Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli Strains That Are Virulent for Humans

ABSTRACT The capacity of Shiga toxigenic Escherichia coli(STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of saacloned in pBC results in a 9.7-fold increase in adherence of E. coli JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of saa in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of saa were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of Yersinia enterocolitica and Eib, a recently described phage-encoded immunoglobulin binding protein fromE. coli. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.

A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.

Proteome Analyses of Cellular Proteins in Methicillin-Resistant Staphylococcus aureus Treated with Rhodomyrtone, a Novel Antibiotic Candidate

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25–62.5 µg/ml, and the minimal bactericidal concentration (MBC) was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5–125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.

Human monoclonal ScFv neutralize lethal Thai cobra, Naja kaouthia, neurotoxin.

Animal derived anti-Naja. kaouthia (Thai cobra) venom is used for specific treatment of the snake bitten victims. Many recipients develop allergic reaction or anti-isotype response which causes serum sickness. A better therapeutic antibody is needed. In this study, long alpha-neurotoxin was purified from the N. kaouthia holovenom and verified by 2D-LC/MS-MS. The toxin was used as antigen in a phage bio-panning to select phage clones displaying human single chain variable antibody fragments (HuScFv) from a phage display antibody library constructed from immunoglobulin genes of non-immunized Thai blood donors. HuScFv that specifically bound to the neurotoxin were produced from huscfv-phagemid transformed E. coli clones and affinity purified. The HuScFv could neutralize toxicity of the N. kaouthia neurotoxin and rescued the envenomized mice from the neurotoxin mediated lethality. Peptide mimotope of the neutralizing HuScFv matched with an amino acid sequence (epitope) located in the loop-3 of the N. kaouthia long alpha-neurotoxin which functions in acetylcholine receptor binding. The mimotope is also similar to peptide sequences found on other snake venom neurotoxins implying a possibility of the HuScFv to exert pan-neutralizing activity against multiple snake neurotoxins.

Characterization of a Novel Type IV Pilus Locus Encoded on the Large Plasmid of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli Strains That Are Virulent for Humans

ABSTRACT The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.

Interleukin-25 (IL-25) Promotes Efficient Protective Immunity against Trichinella spiralis Infection by Enhancing the Antigen-Specific IL-9 Response

ABSTRACT Mammalian hosts often develop distinct immune response against the diverse parasitic helminths that have evolved for immune evasion. Interleukin-25 (IL-25), an IL-17 cytokine family member, plays a key role in initiating the protective immunity against several parasitic helminths; however, the involvement and underlying mechanisms by which IL-25 mediates immune response against Trichinella spiralis infection have not been investigated. Here we showed that IL-25 functions in promoting protective immunity against T. spiralis infection. Mice treated with IL-25 exhibited a lower worm burden and fewer muscle larvae in the later stage of T. spiralis infection. In contrast, mice treated with neutralizing antibody against IL-25 failed to expel T. spiralis effectively. During T. spiralis infection, intestinal IL-25 expression was rapidly elevated before the onset of IL-4 and IL-9 induction. While antigen-specific Th2 and Th9 immune responses were both developed during T. spiralis infection, an antigen-specific Th9 response appeared to be transiently induced in the early stage of infection. Mice into which antigen-specific T cells deficient in IL-9 were transferred were less effective in worm clearance than those given wild-type T cells. The strength of the antigen-specific Th9 immune response against T. spiralis could be enhanced or attenuated after treatment with IL-25 or neutralizing antibody against IL-25, respectively, correlating positively with the levels of intestinal mastocytosis and the expression of IL-9-regulated genes, including mast cell- and Paneth cell-specific genes. Thus, our study demonstrates that intestinal IL-25 promotes protective immunity against T. spiralis infection by inducing antigen-specific Th9 immune response.

A Human Single Chain Transbody Specific to Matrix Protein (M1) Interferes with the Replication of Influenza A Virus

A cell penetrating format of human single chain antibody (HuScFv) specific to matrix protein (M1) of influenza A virus was produced by molecular linking of the gene sequence encoding the HuScFv (huscfv) to a protein transduction domain, i.e., penetratin (PEN) of the Drosophila homeodomain. DNA of a recombinant phagemid vector carrying the huscfv was used as a platform template in a three-step PCR for generating a nucleotide sequence encoding a 16 amino acid PEN peptide. The PEN-HuScFv had negligible cytotoxicity on living MDCK cells. They were readily translocated across the cell membrane and bound to native M1 in the A/H5N1-infected cells as revealed by immunofluorescent confocal microscopy. The PEN-HuScFv, when used to treat the influenza virus infected cells, reduced the number of viruses released from the cells. In conclusion, the cell penetrating M1-specific HuScFv, a transbody, produced in this study affected the influenza A virus life cycle in living mammalian cells. While the molecular mechanisms of the PEN-HuScFv need more investigation, the reagent warrants further testing in animals before developing it into a human immunotherapeutic anti-influenza formula.

Toxic Marine Puffer Fish in Thailand Seas and Tetrodotoxin They Contained

A total of 155 puffers caught from two of Thailand’s seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.

Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein

The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test.

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/VHH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/VHH from a humanized-camel VH/VHH display library. VH/VHH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed VHH hallmark and were designated VHH6 and VHH24; other clones were conventional VH, designated VH9 and VH13. All VH/VHH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, VHH6 and VHH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/VHH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.