Symbiotic relationship between gut bacteria and humans

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Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR). Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR)

IgA-enhancing effects of membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC15893

Immunoglobulin (Ig) A in the mucus of the intestinal tract plays an important role in preventing the invasion of pathogenic microorganisms and regulating the composition of the gut microbiota. Several strains of probiotic lactic acid bacteria (LAB) are known to promote intestinal IgA production. Bacteria are also known to naturally release spherical membrane vesicles (MVs) that are involved in various biological functions such as quorum sensing, pathogenesis, and host immunomodulation. However, the production of MVs by LAB and their effects on host immunity remain poorly understood. In this study, we investigated the MV production by Lactobacillus sakei subsp. sakei NBRC15893 isolated from kimoto, the traditional seed mash used for brewing sake. MVs were separated from the culture broth of L. sakei NBRC15893 through filtration and density gradient ultracentrifugation and were observed by transmission electron microscopy. The MVs showed a spherical morphology, with a diameter of 30–400 nm, and contained proteins and nucleic acids. In addition, both the LAB cells and purified MVs promoted IgA production by murine Peyer’s patch cells. This MV- and cell-induced IgA production was suppressed by neutralization of Toll-like receptor (TLR) 2, which recognizes cell wall components of gram-positive bacteria, using an anti-TLR2 antibody. Collectively, our results indicate that MVs released from L. sakei NBRC15893 enhance IgA production by activating host TLR2 signaling through its cell wall components. Thus, it is important to consider novel interactions between gut microbiota and hosts via MVs, and MVs derived from probiotic bacteria could have promising applications as safe adjuvants.Japan Society for the Promotion of Science (JSPS) KAKENHI grant (Nos. 16K18302 and 18K04857 [to S.Y.Y]; 15H05790, 16H01373, 17H04134, and 26293111 [to J.K.]

アフリカトリパノソーマ発育段階特異的細胞表面タンパク質に関する研究

博士(獣医学)岐阜大学(Gifu University

Expression and characterization of cathepsin B from tsetse (Glossina morsitans morsitans)

Digestive enzymes in tsetse fly midgut are thought to modulate the development of African trypanosome which is a causative agent of trypanosomosis in human and animal. Cathepsin B is induced after the first blood meal ingestion and being higher in trypanosome infected flies. A DNA fragment encoding pro-cathepsin B (930 bp) (Accession No. AF329480_1) was cloned and expressed in E. coli and P. pastoris protein expression systems. An active recombinant cathepsin B (rGmcathB) produced by P. pastoris was migrating from 35 to 45 kDa under reducing condition. rGmcathB exhibited the highest proteolytic activity at pH 4.0 with wide range temperature 25-30oC, also degraded bovine hemoglobin and serum albumin. rGmcathB exhibited hydrolysis preference for Z-Arg-Arg-MCA (Kcat/KM 7.58 mM-1sec-1) and bovine hemoglobin (Kcat/KM 3.77x103 mM-1sec-1). The proteolytic activity of rGmcathB was inhibited by specific cysteine protease inhibitor (E-64) confirmed belonging to papain-like cysteine protease family. These results indicated that rGmcathB shows the activity of cathepsin B and have high affinity with blood protein referring a role in blood meal digestion. In this study, the recombinant protein expressed by E. coli expression system was not enzymatically active as shown in the recombinant protein expressed by P. pastoris expression system. This finding implies that P. pastoris expression system is more suitable for expressing enzymatically active recombinant proteases than E. coli expression system

Characterization and gene cloning of l-xylulose reductase involved in l-arabinose catabolism from the pentose-fermenting fungus <i>Rhizomucor pusillus</i>

<p>l-Xylulose reductase (LXR) catalyzes the reduction of l-xylulose to xylitol in the fungal l-arabinose catabolic pathway. LXR (<i>Rp</i>LXR) was purified from the pentose-fermenting zygomycetous fungus <i>Rhizomucor pusillus</i> NBRC 4578. The native <i>Rp</i>LXR is a homotetramer composed of 29 kDa subunits and preferred NADPH as a coenzyme. The <i>K</i>_m values were 8.71 mM for l-xylulose and 3.89 mM for dihydroxyacetone. The <i>lxr3</i> (<i>Rplxr3</i>) gene encoding <i>Rp</i>LXR consists of 792 bp and encodes a putative 263 amino acid protein (<i>M</i>_r = 28,341). The amino acid sequence of <i>Rp</i>LXR showed high similarity to 3-oxoacyl-(acyl-carrier-protein) reductase. The <i>Rplxr3</i> gene was expressed in <i>Escherichia coli</i> and the recombinant <i>Rp</i>LXR exhibited properties similar to those of native <i>Rp</i>LXR. Transcription of the <i>Rplxr3</i> gene in <i>R. pusillus</i> NBRC 4578 was induced in the presence of l-arabinose and inhibited in the presence of d-glucose, d-xylose, and d-mannitol, indicating that <i>Rp</i>LXR is involved in the l-arabinose catabolic pathway.</p

The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains. Keywords: Colorimetric assay, Drug screening system, Liquid culture, Luciferase assay, Trypanosoma equiperdu