Development of a DNA aptamer for screening neisseria meningitidis serogroup B by cell SELEX

Background: Artificial oligonucleotides like DNA or RNA aptamers can be used as biodiagnostic alternatives for antibodies to detect pathogens. Comparing to antibodies, artificial oligonucleotides are produced easily at lower costs and are more stable. Neisseria meningitidis, the causative agent of meningitis, is responsible for about 1% of infections in an epidemic period. Specific DNA aptamers that bind to N. meningitidis serogroup B were identified by whole-cell Systemic Evolution of Ligands by EXponential Enrichment [SELEX]

Methods: The SELEX begins with a library of labeled ssDNA molecules. After six rounds of selection and two rounds of counter-selection, 60 clones were obtained, of which the binding efficiency of 21 aptamers to the aforementioned bacterium was tested by flow cytometry

Results: The aptamers K3 and K4 showed the highest affinity to N. meningitidis serogroup B and no affinity to N. meningitidis serogroups Y, A, and C, or to other meningitis causing bacteria. The dissociation constant [Kd value] for K3 and K4 were calculated as 28.3 +/- 8.9 pM and 39.1 +/- 8.6 pM, respectively. K3 aptamer with the lowest Kd was chosen as the main aptamer. K3 could detect N. meningitidis in patients' cerebrospinal fluid [CSF] samples and in CSF from healthy volunteers inoculated with N. meningitidis serogroup B [ATCC 13090] at 200 and 100 CFU ml[-1], respectively

Conclusion: The findings suggest the application of the developed aptamer in specific detection of N. meningitidis serogroup B amongst a group of meningitis causing bacteria

[Immunogenicity of amino acid region 7601-8140 in biofilm associated protein of acinetobacter baumannii]

Acinetobacter baumannii [A. baumannii] is a major hospital pathogen with a high capacity to resist most common anti-microbial agents. A. baumannii is the etiologic agent for various illnesses including pneumonia, meningitis, and bloodstream infections. Biofilm associated proteins [Bap] are specific cell surface proteins essential for the formation of biofilm and play a main role in its pathogenicity. Previously, we have studied various regions of this protein. Considering different criteria, some regions were introduced as conserved and immunogenic. The immunogenicity of one of those regions pertaining to amino acids 706-1076 previously examined has shown that its expression triggers high antibody levels when injected to mice thereby protecting the animals against the bacterium. The present study examines region 4 of the Bap protein in order to validate the previous bioinformatics studies and its immunogenicity. In order to obtain immunity against this pathogen, a 1620 bp gene from Bap was amplified and cloned in pET32a. This region from Bap was cloned, expressed and verified by monoclonal antibodies. BALB/c mice were immunized by subcutaneous injection of the pure recombinant protein. Mice immune response was determined by ELISA. High titer of raised antibodies implied that the recombinant protein was a strong antigen and immunogen. The results indicate that this protein can be a suitable choice for developing a new recombinant vaccine against A. baumannii

[Unveiling a specific immunogen protein in acinetobacter baumannii]

Acinetobacter baumannii [A. baumannii] is a Gram-negative, non-motile aerobic bacterium which is known as a nosocomial pathogen that is often resistant to a broad range of antibiotics. The pathogen is a serious agent of mortality and morbidity in hospitals, particularly among immunocompromised patients. Treatment and control of its infections is complicated owing to its high antibiotic resistance, survival in various environmental conditions and utilization of wide range of nutrient sources. Early detection of the pathogen in established infections is pivotal for infection control. Culture and biochemical tests are current methods for detection of the bacterium, which take approximately 2-5 days. Hence, a new, rapid, specific and affordable diagnostic test is needed. Development of such test depends on a suitable biomarker that lacks crossreactivity with other bacteria. This study intends to unveil a 34.4 kDa outer membrane protein [OMP] introduced by Islam et al. in A. baumannii ATCC19606. We harnessed various bioinformatic servers to screen the entire proteome of this bacterium. Properties critical to the screening included molecular weight, localization, topology, homology, antigenicity and allergenicity of proteins. Three proteins were found as suitable candidate molecular weights as well as localization points of view. BLAST searches, antigen probability predictions and other analyses led to the selection of one protein as the best specific antigen of A. baumannii. The in silico analyses unveiled the best candidate protein vide accession number ZP_05827218.1

[Role of two-partner secretion system dependent FhaB1 and fhab2 proteins in adhesion of acinetobacter baumannii ATCC19606 to human epithelial cells]

Acinetobacter baumannii [A. baumannii] has a good potential to colonize on various surfaces. As a virulence factor, adhesion to surfaces is the first step in colonization. The Two-Partner Secretion System [TPS] proteins are key factors for bacterial attachment. The purpose of this study is to identify and study the role of this family of proteins in adhesion of A. baumannii to human epithelial cells. Gene homologues that encoded the TPS were analyzed by bioinformatics tools and the primers were designed accordingly. The constructs synthesized in the pET22b vector were transferred to BL21[DE3]. The transformed cells were named FhaB1 and FhaB2. The protein expression on the cell membrane was studied in addition to bacterial adhesion and biofilm formation by recombinant strains, A. baumannii and E.coli BL21[DE3]. Bioinformatic studies showed the bacterial potential of producing two exoproteins [FhaB1 and FhaB2]. Expression of the recombinant proteins on the outer membrane was confirmed by Western Blot Analysis and whole cell ELISA. The results revealed an association between the recombinant cells and bacterial adhesion and biofilm formation. FhaB1, FhaB2 and A. baumannii exhibited enhanced adherence to human lung epithelial cells compared to E.coli BL21[DE3] TPS in A.baumannii is of adherence and colonization factors and is one of the bacterial virulence factors

DNA vaccine against escherichia coli o157:H7

Infection with Escherichia coli O157:H7 rarely leads to bloody diarrhea and causes hemolytic uremic syndrome with renal failure that can be deadly dangerous. Intimin, translocated Intimin receptor [Tir], and enterohemorrhagic E. coli [EHEC] secreted protein A [EspA] proteins are the virulence factors expressed by locus of enterocyte effacement locus of EHEC. This bacterium needs EspA as a conduit for Tir delivery into the host cell and the surface arrayed Intimin, which docks the bacterium to the translocated Tir. Here we used triplet synthetic gene [eit] which was designed from three genes: espA coding EspA 120 lacking 36 amino acids from the N-terminal of the protein, eae coding Intimin constructed of 282 amino acids from the C-terminal and tir coding Tir 103, residues 258-361 which interacts with Intimin. The multimeric gene was cloned in two eukaryotic vectors pAAV-multiple cloning site-green fluorescent protein and pCI-neo. The pAAV was used for gene expression assay in cell line 293T and pCI-neo-EIT [EspA, Intimin, Tir] was used as DNA vaccine in mice. Test groups were injected intramuscularly with pCI-neo-EIT four times and mice control group was injected under the same conditions with PBS or pCI-neo vector. The titration of serums showed that BALB/c mice were successfully immunized with DNA vaccine compared to control groups and also they were protected against challenges of live oral using E. coli O157:H7. Conclusion: The results suggest that the DNA vaccine could induce protective immunity either alone or in combination with purified antigens to reduce EHEC infection

Chemical composition and antifungal activity of cuminum cyminum l essential oil from alborz mountain against aspergillus species

Aflatoxin Bj [AFBj] is a highly toxic and hepatocarcinogenic metabolite produced by Aspergillus species. Some natural products are known to kill fungi and destroy toxins and toxin-producing agents. The purpose of this study is to provide experimental data on the antifungal activity of cumin oils and their components that could be considered suitable for application in foods and drugs. The essential oil [EO] of Cuminum cyminum L. collected from Alborz Mountain, Iran, was obtained by hydro-distillation. The oil was analyzed by gas chromatography [GC] and chromatography/mass spectrophotometry [GC/MS]. The antifungal activity of the oil was studied with regard to the inhibition of the growth of Aspergillus flavus PICC-AF39 .Aspergillusflavus PlCC-AF24,Aspergillusparasiticus NRRL-2999 and Aspergillus niger. The minimal inhibitory [MIC] and minimal fungicidal [MFC] concentrations of the oil were determined. a-Pinene [29.2%], limonene [21.7%], 1,8-cineole [18.1%], linalool [10.5%], linalyl acetate [4.8%], and a-terpineole [3.17%]were the major components of the essential oil from C. cyminum L, and the oil showed a strong inhibitory effect on fungal growth. Essential oils could be safely used as preservatives in pharmaceuticals as well as health and food products to protect them against toxigenic fungal infections

[Antimicrobial, nitric oxide radical scavenging and cytotoxic properties of Thymus daenensis essential oil]

Despite toxic effects of some essential oils, their use is not under control. With a view to increasing trend of utilisation of herbal products, some biological aspects of Thymus daenensis are repoted here for the first time. Antimicrobial properties using disk diffusion and dilution tests, nitric oxide radical scavenging by Marcocci et al method and cytotoxic properties employing dimethylthiazolyl diphenyltetrazolium bromide reduction test were carried out with Thymus daenensis and commercial Thyme essential oils and their main chemical compound, thymol. The microbial sensitivity to the oils were in Candida albicans>E. coli>S. aureus>P. aeruginosa order. The minimum inhibitory and microbicidal concentrations were in the range of 0.04-10mg/ml. Nitric oxide radical scavenging was dose dependent with an IC50 of 5, 75, 863 micro g, and total phenolics of 644.07 +/- 6.79, 16.94 +/- 2.55, 10.33 +/- 2.31 micro g Gallic acid equivalent per mg sample and total flavonoid content of 73.51 +/- 1.34, 0.56 +/- 0.02, 0.21 +/- 0.09 mg Catechin equivalent per gram T. daenensis oil, commercial thyme oil and thymol respectively. The concentrations from T. daenensis oil, commercial thyme oil and thymol required to exert 50% fatal effect [IC50] on healthy human normal lymphocytes and Hela cells were 1455, 12.10, 2867 and 4.95, 3.61, 1730 micro g respectively. T. daenensis with its good antimicrobial property can prevent formation of toxic reactive oxygen species and as a good antioxidant, it can directly scavenge NO and O2-. With a view to cancerous cells killing properties of the oils at their lowest concentrations without fatal effect on normal healthy cells, feasibility of their application in combating cancerous cells may be promising

Immunogenic and protective potentials of recombinant receptor binding domain and a C-terminal fragment of Clostridium botulinum neurotoxin type E

Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain as the N-terminal half and the binding domain as the Cterminal half [Hc]. One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc [the full length binding domain] and the c-terminal quarter of binding domain [HcQ], were cloned in pET-28a vector and over-expressed in E. coli BL21 [DE3] cells. These recombinant proteins were purified by affinity Ni-NTA column [under native condition]. Mice were vaccinated with 2 microg of purified proteins, respectively; at step one with complete adjuvant, steps two and three with incomplete adjuvant and step four only with phosphate buffered saline [PBS]. Enzyme-linked immunosorbent assay [ELISA] has been performed with mice serum samples 14 days following their third and final vaccination. Binding activity of the purified proteins to ganglioside and synaptotagmin II was analyzed by ELISA. The results showed that HcQ and Hc could bind with ganglioside. Based on challenge experiments it was revealed that HcQ, Hc and BoNT/E toxoid could give protections in mice challenged with 10[2], 10[4] and 10[5] minimum lethal dose [MLD] dose of BoNT/E

Elicitation of IgY in chicken egg yolk by recombinant fragments of UreC and its efficacy against Helicobacter pylori

Helicobacter pylori multiplies and causes infection in human gastric mucosal layer. New approaches have focused on using specific treatments, such as immunotherapy, to limit this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. In order to prepare recombinant proteins, the synthetic genes for total ureC protein [UreCt] and its N [ureCn] and C [ureCc] terminal fragments were ligated into pET28a. The recombinant proteins were expressed in E. coli BL21[DE3]. White leghorn hens were injected with the purified recombinant proteins. IgY recovered from egg yolk, using PEG precipitation. Finally, urease neutralizing ability of the antibodies was evaluated by urease activity assay in presence of the purified IgY. SDS-PAGE analysis revealed a good expression and purification of the recombinant proteins. Indirect ELISA observation demonstrated high antibody titer in sera and egg yolks and high ability of IgY Anti-UreCt and IgY Anti-UreCc antibodies in recognition of urease subunit C. Anti-UreCT and Anti-UreCc IgYs were more potential H. pylori urease inhibitors than Anti-UreCn. While all three UreC fragments induce prophylactic responses. UreCt and UreCc possess almost equal responses. Anti-UreCc IgY has advantage of smaller size and is preferred for its activity and easier protein recovery and purification process. These features emphasize on importance of simpler, easier and cost effective antibody production

Antibody production against enterobacteriacea using recombinant Ferric enterobactin protein [FepA]

Escherichia coll O157:H7 is a gram-negative rod-shaped bacterium. E coll O157:H7 is an enterohemorrhagic [EHEC] strain of the bacterium Escherichia coli and a cause of foodborne illness. Infection often leads to bloody diarrhea by producing a toxin called Shiga toxin, which damages the intestines, and occasionally leads to kidney failure, especially in young children and elderly people. A 2241 bpfepA gene of E. coli O157:H7 codes for production of a ferric enterobactin binding membrane protein that is essential for iron uptake by the bacterium. Inhibition of iron uptake can protect invasion of host by the bacterium. In this study we attempted to evaluate immunogenicity of the membrane protein, FepA. In order to produce recombinant FepA protein, the genomic, fepA gene of 2241 bp long was amplified by PCR from E coli O157:H7. The PCR product was ligated to pET28a. The recombinant protein was then expressed in E. coli BL21DE3 by IPTG induction. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to Balb/C mice in order to induce immunity. Antibody titer was determined by ELISA. The recombinant protein of 85 KD was produced and purified. Immunogenicity of the recombinant protein was determined by injecting Balb/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein, thus heaving mice tolerance of 10[6] LD[50]. With a view to the significant recognition by the antibody of ferric enterobactin binding protein, the notion of its application in restriction of enterobacteriacea propagation could be feasible

Simultaneous detection of Escherichia coli O157:H7, toxigenic Vibrio cholera, and Salmonella typhimurium by multiplex PCR

Escherichia coli O157:H7, Vibrio cholerae, and Salmonella typhimurium are pathogenic bacteria found inn contaminated water and food. No assay method is currently available on simultaneous detection or identification of all the three pathogens. Our aim was to develop a rapid and reliable method for this purpose. A protocol for sample collection, and a PCR procedure was designed specifically for the assay. Selected fragments of 239 bp, 432 bp, and 360 bp for E. coli O157 lipopolysaccharide [LPS] gene [rfbE], V. cholerae toxin gene [ctx], and Salmonella typhimurium putative cytoplasmic protein gene [STM4497], respectively, were amplified from the extracted bacterial DNA samples in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. All unknown samples were verifiably identified. The assay was sensitive enough to detect and identify as few as 100 cells of E. coli O157:H7, V. cholerae and Salmonella typhimurium. The presence of other bacteria did not interfere with the analysis. This assay is a specific and reliable tool which allows cost-effective detection o all three bacterial pathogens in one reaction tube