Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

Antibiotic susceptibility and plasmid profiles of shigella species in Sudan

This study was carried out to determine the antibiotic susceptibility, plasmid profile and conjugative abilities of Shigella species isolated from different towns in Sudan during 2005-2007. Stool specimens were collected in Carry Blair transport medium from patients presenting with diarrhea from different sites in Sudan between the years 2005-2007. All specimens were inoculated on Mac Conkey' s agar and Xylose Lysine Dioxycholate [XLD] [Mast group Ltd. Merseyside UK]. Bacteria was isolated and subjected to different antibiotics to detect sensitivity and transference of resistance. One hundred and fourteen Shigella isolates were included in the study. Eighty [70.1%] were Shigella flexeneri representing the dominant isolate, followed by 20 [17.5%] isolates of Shigella dysenteriae, 9 [7.9%] Shigella sonnei and 5 [4.5%] Shigella boydii. Most of the isolates showed resistance to streptomycin [70%], tetracycline [52%] and co-trimoxazole [43%]. They were highly sensitive to norfioxacin [97%], nalidixic acid [95%], gentamicin [89%] and chloramphenicol [77%]. Multi-drug resistance to two or more antibiotics was apparent in most of the isolates [64, 56.1%]. Fifty nine of the resistant Shigella isolates were studied for their ability to transfer resistance to the donor E. coli K[12] by conjugation. Of these, six were able to transfer resistance to streptomycin, tetracycline and co-trimoxazole. Extraction of the plasmid DNA from both donors and trans-conjugants showed a single type of plasmid with a molecular weight of 4.6 Kb. The transfer of multi-drug resistant plasmids and the emergence of antibiotic Shigella and other bacterial species should raise the awareness and the seriousness of the uncontrolled [unsupervised] use of antibiotics in the medical practice

ARC: automated resource classifier for agglomerative functional classification of prokaryotic proteins using annotation texts.

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general,automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters (C), Cell division (D), Information (I), Translocation (L), Metabolism (M), Stress(R), Signal and communication (S) and 2 ancillary classes: Others (O) and Hypothetical (H).The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps,this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.