Duodenal ulcer is caused in the proximal duodenum of humans. It causes erosion of cell layers at the beginning of the small intestine. The major cause for this disease is attributed to infections caused by bacterium known as Helicobacter Pylori. Helicobacter pylori (H. pylori) is a micro-aerophilic spiral-shaped Gram-negative bacterium that colonizes the stomach for almost the entire lifetime of the host [1].H. pylori affects a majority of people of the world through gastro duodenal diseases, such as peptic ulcer disease and gastric adenocarcinoma. The interesting aspect about the helicobacter pylori is that not all people affected by this bacterium developed cancer. This encourages us to investigate more about the significant virulence factors related to the Helicobacter Pylori infections. The specific aims are thus designed in accordance to that.
Aim 1: Identification of DU (Duodenal Ulcer) related antigens from H. Pylori
Every bacterium has a number of proteins present in it, which determines its activities within a host body. Each of those proteins performs different functions within the host body. It is therefore necessary to identify those proteins, present in H. Pylori which causes duodenal. Hence we will collect serum samples from patients who are already affected with duodenal ulcer, and understand the activities of the bacterium which would help us understand the disease process, by undertaking the research methods that have been chosen for this purpose, namely Two Dimensional Electrophoresis and Immunoblotting. This would help us identify the proteins, which are the virulence factors for the duodenal ulcer.
Aim 2: Characterization of identified immunogenic protein
Upon identification of the immunogenic proteins present in the Helicobacter pylori, their functions and activities will be studied. This would help understand how the bacterium escapes the effects of the human immune system, initiated against it on its invasion of host cells. This can be accomplished by carrying out the appropriate research methods that have been chosen.
Aim 3: Characterization of their roles in the development of DU
On understanding the characterization of the identified immunogenic proteins, it would clear to us on what are the proteins that cause the duodenal ulcer. Hence their roles in the development to DU would be characterized. This would be accomplished by studying the roles of the identified virulence factor in IL-8 induction in the infected cells and in the cytokine level in the cells.
BACKGROUND AND SIGNIFICANCE:
RESEARCH DESIGN AND METHODS:
Activity 1:
Experimental Procedures:
I. Patients and Serum Samples:
Serum samples will be collected from 100 individuals who are suffering from Duodenal Ulcer and other gastric disorders. Presence of H. pylori will be determined by culture and histological examination of gastric biopsy specimens. Patients who undergo gastrectomy and hence diagnosed with DU can be enrolled for sampling. For the non-ulcerous group, subjects from health clinics who receive a gastrointestinal endoscopic examination and showed no lesions of DU will be used. This serves as the control group for comparison against the experimental group when the experimental results are evaluated.
II. Bacterial Strain and Culture Conditions:
Helicobacter pylori strains will be isolated from endoscopic biopsy samples from the stomach of patients with Duodenal Ulcer. The bacteria will be cultured on a BBL Stacker plate (BD Biosciences) at micro-aerobic conditions. Liquid cultures will be grown in flasks containing Brucella broth (Difco) supplemented with 10% FBS (fetal bovine serum) with constant agitation. The culture medium will be centrifuged for 10 min and pelleted. The supernatant will be filtered to eliminate intact bacterial cells.
III. Two Dimensional Electrophoresis and Immunoblotting:
Cell surface proteins will be extracted from H. Pylori using acid-glycine extraction procedure. The extract will be precipitated using 20% TCA (tricarboxylic acid). Two dimensional electrophoresis will be used to separate the proteins. The first dimension of the 2D gel will be run on IPG strips and the second dimension on SDS-polysacrylamide gels.
For Immunodetection, the proteins on the 2D gel will be transferred to a PVDF membrane, after which the membrane will be blocked by incubation for 1 hr in blocking buffer. Then we incubate it with serum samples from DU patients.
IV. Protein Identification:
The individual protein spots will be from the 2D gel and subjected to in-gel tryptic digestion. Then the spots will be destained and dried in a SpeedVAc concentrator. The resulting peptides are extracted sequentially and the combined extracts are lyophilized and analyzed using a mass spectrometer (Applied Biosystems, MA). Peak lists of spectra will be created using Mascot Search in Analyst QS 1.1(Applied Biosystems). Then the peak lists are uploaded to Mascot MS/MS Ion Search program (Mascot Version 2.0) on the Matrix Science website, and protein identification is performed against the National Centre for Biotechnology Information non redundant database.
The ion scores for the matching proteins are collected. From the list, the protein names and locus tags in H. pylori strain are selected and listed in a table for convenient reference.
Expected Outcome:
Of all the identified proteins, the highly frequent proteins in most of the patient samples analyzed will be chosen. The most common proteins will be compared with the 100 patient samples and the most frequently occurring protein(s) will be identified. The identified immuno protein will be referred to as Protein A from here on.
Potential Pitfall:
Using 2D electrophoresis, it might be possible that we may not be able to identify new virulence factors. In that case, highly sophisticated methods such as, ______. (Yet to figure out)
Activity 2:
I. Cloning and Expression of Recombinant protein A:
H. Pylori will be lysed followed by RNase treatment and the genomic DNA will further be purified using phenol chloroform and precipitated with 70% ethanol. Then, PCR (polymerase chain reaction) will be performed using gene A specific primers. The amplified gene fragment will be cloned into expression vector pQE30 with 6X Histidine tag at its C-terminal and transferred into E.coli strain.
For expression of recombinant proteins, cells grown at 25 °C to an O.D of 1.0 (A600) will be induced using 1mM IPTG. The extracted recombinant proteins will be purified using Ni-NTA column and dialyzed against PBS, and the endotoxin content will be measured using a QCL-1000 kit (BioWhittaker, MD)
II. Preparation of Polyclonal Anti-protein 'A' Antibodies:
White rabbits will be injected intradermally with 500 micrograms of purified recombinant protein A in 1ml of PBS emulsified with 1ml of Freund's incomplete adjuvant (Difco). Boosters of 500micrograms in 1ml of PBS emulsified with 1ml of Freund's incomplete adjuvant will be given intradermally at weeks 3 and 6, then the rabbit will be bled 10 days after the last boost, and the serum will be used for Immunoblotting experiments.
III. Serologic Stud to compare native and recombinant protein A to identify its properties:
Recombinant protein A will be electrophoresed on a SDS gel and transferred to a PVDF membrane and Immunoblotting will be performed. Serum samples from patients with DU, gastritis, GC or normal controls diluted to 1:1000 will be screened for reactivity with protein A for Immunoblotting. Also, we intend to use rabbit anti protein A antibody as primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG anti-body (Invitrogen) as secondary antibody. Bound antibody will be detected followed by exposure to x-ray film (Eastman Kodak Co.).
Purified, recombinant protein A will be separated by 12.5% SDS-PAGE and then stained with Coomassie Blue or immunoblotted with the primary antibodies (as mentioned above). Reactivity of protein A with primary antibodies will be tested. 2D immunoblots
of acid-glycine extract from H. pylori will be probed with serum from a DU patient or with anti-protein A antibodies.
Western blot analysis will also be carried out to analyse the location of immunogenic protein expression by H.pylori, by comparing the total cell surface proteins and secreted proteins.
Expected outcome: Have to decide if we need this part
Potential Pitfall: Have to decide if we need this part
Activity 3:
i. Characterization of role of protein A in development of DU:
IL-8 Levels From Gastric Cancer Cells Co cultured With H pylori:
In vitro IL-8 secretion from gastric epithelial cells will be examined. Briefly, gastric epithelial cell line, MKN45 cells (Japanese Cancer Research Resource Bank, Tsukuba, Japan) will be plated into 24-well plates and cultured for 2 days. Experiments will be performed in duplicate for clinical samples and will be performed at least 3 times for wild type and its isogenic mutants. H pylori (multiplicity of infection [MOI] of 100) or brain heart infusion (BHI) broth (control) will be added to the cultured cells for 20 hours, and IL-8 in the supernatant will be assayed by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN) in duplicate.
ii. Role of identified virulence factor in cytokine level in cells:
Cytokines are proposed to be critical in the pathogenesis of H. pylori-associated GC. In addition to eliciting humoral immune responses, H. pylori infection strongly up-regulates cytokine production by monocytes/ macrophages. Cells will be incubated for 24 h with protein A, and then the supernatants will be collected and stored at 80 °C until assayed for cytokine production. Levels of cytokines in the culture supernatants will be measured using Quantikine ELISA assay kit (R&D Systems, Minneapolis, MN) for IL-8, IL-6, IL-1 , TNF. All experiments will be performed in triplicate. Furthermore to verify that the cytokine release from cells was due to protein A and not the contaminating LPS, protein A and LPS will be digested with proteinase K (PK/substrate molar ratio of 1:10) for 1 h at 37 °C, and then the PK was inactivated by heating at 100 °C for 10 min. PK-treated protein A and LPS will be then used to treat cells as described above.
Expected outcome:
Results will be presented as the percentage of non treated cells after subtracting the blank values (medium only). After the proteins are tested for cell proliferation, their functional and structural properties will be studies by examining their cell structures.
Potential Pitfall: Have to decide if we need this part
