ULCER
Acid Resistance
The main goal of this part is; making the E. coli, which we used for eradicating H. Pylori, acid resistant for living in gastric juice’s low pH (pH: 2) microenvironment. Wild-type E. coli already has couple systems to show acid resistance until a certain pH level (pH: 5). Among those systems, the most stable-working and efficient one is known as Glutamate Dependent Acid Resistance(GDAR) system. The most important protein of Gad system is GadE, which controls the synthesis of all other proteins. By overexpressing this protein, we aim to have our bacteria resistant enough to live in gastric juice for a while.
Background
Escherchia coli Natural Acid Resistance System
E. coli possesses four phenotypically distinct systems of acid resistance. These systems:
1. Glutamate dependent acid resistance system (GDAR)
2. Arginine dependent acid resistance system (ADAR)
3. Lysine dependent acid resistance system (LDAR)
4. Ornithine dependent acid resistance system (ODAR)
The most effective one of these systems is the glutamate dependent system so we decided to focus on glutamate dependent system (GDAR).
The GDAR system has in the acid resistance complex pathways. We decided to do the certain pathways that how to do acid resistance with our results of research.
EvgA is the most effective positive regulator of GDAR. (Efficiency: evgA>ydeO>gadE) But evgA takes part in countless number different genes regulation and cellular processes (2,6,7) and also most of this cellular processes are unclear. We cannot predict the results of the overexpression of evgA so we chose gadE and using by overexpression of gadE we can induce glutamate depended acid resistance system. Briefly talk about the impact of the GadE on the mechanism.
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The central activator is the LuxR-family member GadE (formerly known as YhiE). GadE binds to a 20-bp sequence called the gad box, which is located 63-bp upstream of the transcriptional start sites of gadA and gadBC. (4)GadE and GadBox are an important point for GadA and GadBC. This means that GadE and GadBox are an important for acid resistance. The basis section of acid resistance is GadE’s regulation. At least 10 different regulatory take part in this regulation.
GadE have three main activation mechanisms. The first of these is performed by evgA and ydeO. About GadE activation phase: 1. EvgS (sensor kinase) activate EvgA (response regulator) 2. YdeO and evgA are independently from each other and GadE takes an active role in transcription activation. The second GadE activation circuit includes CRP,RpoS and two AraC-like regulators, GadXand GadW. These steps are as follows: 1. GadX and GadW, are located downstream of gadA also GadX and GadW directly activate transcription of gadE 2. GadX and GadW also bind to the gadA and gadBC gad box sequences and seem to repress the gadA and gadBC promoters. (4) GadW inhibits this GadX’s repression Also GadX and GadW regulate indirectly GadA/BC transcriptional functions. 3. The balance of power in this circuit is influenced by cAMP and CRP,which together inhibit the synthesis of RpoS. Growth under acidic conditions reduces the concentration of cAMP in the cell. RpoS increases GadX’s transcriptional function The increase in GadX then stimulates transcription of GadE and also down regulates GadW. The third activation of GadE contains TrmE and glucose. These steps are as follows: 1. The function of TrmE in the cell is not fully defined but it does have a clear effect on tRNA modification 2. TrmE and glucose increase independently GadE’s transcriptional function We discourse GadE’s regulation and then now we will discourse GadE’s effect mechanisms. GadA/BC’s effect mechanisms to acid resistance are as follows: 1. The external pH is normally neutral but if external pH turns to acidic pH, internal pH begins to become acidic pH with HCl diffusion. 2. If the external pH=2,5 and internal pH begins pH=4.2 ± 0.1. GDAR system will activate for acid resistance. 3. GadC is a transmembrane protein. The external pH begins to change from neutral pH to acidic pH, C-plug (is GadC’s subunit) will open. Then glutamate will take inside.
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4. Glutamate in cells convert to GABA by GadB/C that’s glutamate decarboxylase isozymes These steps are as follows: *This step contains pyridoxal phosphate-containing enzymes that replace the α-carboxyl groups of their amino acid substrates with a proton that is recruited from the cytoplasm. *HCl that diffusion from outside dissociates H+ and Cl-. This H+ is used and CO² is released by the agency of GadA/B isozymes than this is the last stage of glutamate changes to GABA. Besides this is the most important and the last step for acid resistance 5. If H+ leaves HCl it is staying back Cl and it will be export from the transport of chlorine channel. 6. The emitted CO² is taken out by diffusion. 7. Finally, the uncovered GABA is thrown out from the transmembrane protein GadC.
In this section, we targeted to overexpress GadE protein. For this purpose, while we’re desinging the gene part, we chose to combine GadE sequence with T7 promoter, a promoter which has a high rate of transcription. In addition to this, to have a controllable system, LacI protein attachable Lac operator was added between T7 and GadE gene sequence. We searched to find a vector which can lay down all those conditions and found out that peT-45 expression vector is useful. When we ordered our genes, we put RFC10 prefix site on 3’ end of GadE and also BamHI restriction enzyme recognition site for cloning to peT-45. We added RFC10 suffix site on 5’ end of the same gene sequence and also XhoI restriction enzyme recognition site for cloning to peT-45 vector, again.
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As shown above,we planned to clone the ordered GadE gene into peT-45 vector by using BamHI and XhoI enzymes. Final construct after this cloning is, respectively T7 promoter-Lac operator(LacO)-HisTag-GadE. Besides, a constitutive promoter called LacI promoter and LacI protein sequence in front of that, found on peT45-b vector’s another part. It is obvious that this part is IPTG-dependent, so Western Blot can be performed easily with the help of His Tag.
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GadE-PSB1C3 CLONNING
We cloned IDT G-Blocks GadE gene into PSB1C3 vector in order to make it ready to be submitted and have many copies of it. For this purpose we digested PSB1C3 vector and GadE G-Blocks with ECoRI and PstI restriction enzymes. Then we ligated these cut genes into the plasmid by using T4 DNA Ligase . Ocurring products were transformed into BL21 competent cell strain. To check if the cloning is correct, a colony PCR was perfomed with Verify Forward and Verify Reverse primers. If the cloning isn’t made properly t the band should be 314 bp long, but if colony PCR worked, then bands should be 858 bp long. As the result of colony PCR, the possible right cloned colonies were incubated in liquid culture for 16 hours. After this incubation, we isolated plasmid DNA from this bacteria culture by miniprep plasmid isolation method. We controlled obtained colonies with cut-check for a second cloning control. We used EcorI and PstI restriction enzymes for cut-check.
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GadE-pET45-b CLONNING
We cloned GadE into PSB1C3 vector successfully, then moved on cloning it into the expression vector pET45-b. For cloning into this plasmid, we removed our gene from PSB1C3-GadE plasmid with BamHI and XhoI enzymes. Then we ligated the cut gene with pET45-b which was cut with the same enzymes. We transformed ligated products into BL21 bacteria strain that we know it has T7 RNA polymerase.
To check if the cloning is correct, a colony PCR was performed with T7 Promoter Forward and T7 Terminator Reverse primers. If the cloning isn’t made properly the band should be 360 bp long, but if colony PCR worked, then bands should be 837 bp long. As the result of colony PCR, the possible right cloned colonies were incubated in liquid culture for 16 hours. After this incubation, we isolated plasmid DNA from this bacteria culture by miniprep plasmid isolation method. We controlled obtained colonies with cut-check for a second cloning control. We used BamHI and XhoI restriction enzymes for cut-check.
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WESTERN BLOTTING
After cloning GadE into pET45-b successfully, we did Western Blot experiment through N-terminal located His Tag in proteins, so we managed to show the production of required proteins.
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FUNCTIONAL ASSAY
To understand if the proteins we produced are functional, we designed and performed a functional assay. We incubated these two types of bacteria in liquid culture for 13 hours at 37 C; pET45-GadE plasmid containing BL21 bacteria and another BL21 bacteria which contains only pET45-b plasmid for negative control. At 13. our, we added 100 mM IPTG into these liquid cultures. By doing this, we removed the suppression on GadE protein expression. After adding IPTG, we incubated 3 hours more at 37 C. We prepared LB mediums with different pH values to show produced GadE proteins’ functionality. These LB mediums’ pH values are respectively 7, 5, 3,5; 2,5 and 2. We added thebacteria whichis incubated for 16 hours into LB mediums at the rate of 1:9. This means, for each pH value we added 0.5 ml liquid culture into 4.5 ml LB medium. We also added 1.5 mM Glutamat in each mix and incubated the final mix at 37 C. We made spectrophotometric measurement in 600 nm periodically for the samples that we incubated. Thus we observed how long the bacteria survives in different pH values. The measurement results are shown below.
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1.5 mMGlutamate / 600 nm OD / 3h 100 mM IPTG
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When the given data checked, it is obvious that the produced GadE is functional and makes E. coli survive in the acidic microenvironment in comparison to negative control results. Based on the data given, the E. coli with overexpressed GadE can survive in pH 2 gastric juice for 5-9 hours.
Acid Repellency
The main objective of this part of our project is to direct resistant E. Coli into the internal mucus layer where Helicobacter Pylori are abundant for a length of time. We will direct our E. Coli using the hydrogen ion difference between the stomach fluid and mucus layer. The PH of gastric juice is 2.0 whereas the pH of gastric mucus layer is 7.0. The chemoreceptor tlpb naturally found in the inner membrane of helicobacter pylori enable these bacteria to move away from acidic condition to a more neutral and basic environment. Therefore, we decided to use the TIpB protein and designed a biobrick for our E. Coli in order to ensure that our Bacteria reach the region where the helicobacter pylori are.
Background
Chemotaxis, movement toward or away from chemicals, is a universal attribute of motile cells and organisms. E. coli cells swim toward amino acids (serine and aspartic acid), sugars (maltose, ribose, galactose, glucose), dipeptides, pyrimidines and electron acceptors (oxygen, nitrate, fumarate).
E. coli's optimal foraging strategy
In isotropic chemical environments, E. coli swims in a random walk pattern produced by alternating episodes of counter-clockwise (CCW) and clockwise (CW) flagellar rotation (Fig. 3, left panel). In an attractant or repellent gradient, the cells monitor chemoeffector concentration changes as they move about and use that information to modulate the probability of the next tumbling event (Fig. 3, right panel. These locomotor responses extend runs that take the cells in favorable directions (toward attractants and away from repellents), resulting in net movement toward preferred environments. Brownian motion and spontaneous tumbling episodes frequently knock the cells off course, so they must constantly assess their direction of travel with respect to the chemical gradient.
Figure 1: Random and biased walks. Left: A random walk in isotropic environments. When the cell's motors rotate CCW, the flagellar filaments form a trailing bundle that pushes the cell forward. When one or more of the flagellar motors reverses to CW rotation, that filament undergoes a shape change (owing to the torque reversal) that disrupts the bundle. Until all motors once again turn in the CCW direction, the filaments act independently to push and pull the cell in a chaotic tumbling motion. Tumbling episodes enable the cell to try new, randomly-determined swimming directions. Right A biased walk In a chemoeffector gradient. Sensory information suppresses tumbling whenever the cell happens to head in a favorable direction. The cells cannot head directly up-gradient because they are frequently knocked off course by Brownian motion..
The chemotaxis signaling pathway of E.coli
E. coli senses chemoeffector gradients in temporal fashion by comparing current concentrations to those encountered over the past few seconds of travel. E. coli has four transmembrane chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), that have periplasmic ligand binding sites and conserved cytoplasmic signaling domains (Fig. 4). MCPs record the cell's recent chemical past in the form of reversible methylation of specific glutamic acid residues in the cytoplasmic signaling domain (open and filled circles in Fig. 4). Whenever current ligand occupancy state fails to coincide with the methylation record, the MCP initiates a motor control response and a feedback circuit that updates the methylation record to achieve sensory adaptation and cessation of the motor response. A fifth MCP-like protein, Aer, mediates aerotactic responses by monitoring redox changes in the electron transport chain. Aer undergoes sensory adaptation through a poorly-understood, methylation-independent mechanism. The five MCP-family receptors in E. coli utilize a common set of cytoplasmic signaling proteins to control flagellar rotation and sensory adaptation (Fig. 4). CheW and CheA generate receptor signals; CheY and CheZ control motor responses; CheR and CheB regulate MCP methylation state.
Figure 2: Signaling components and circuit logic. E. coli receptors employ a common set of cytoplasmic signaling proteins: CheW and CheA interact with receptor molecules to form stable ternary complexes that generate stimulus signals; CheY transmits those signals to the flagellar motors, CheZ controls their lifetime; CheR (methyltransferase) and CheB (methylesterase) regulate MCP methylation state. Abbreviations: OM (outer membrane); PG (peptidoglycan layer of the cell wall); CM (cytoplasmic membrane)
As in many biological signaling systems, the signaling currency in the E. coli chemotaxis pathway is reversible protein phosphorylation (Fig. 5). However, the principal signaling chemistry is a bit different in prokaryotes and eukaryotes. CheA is a kinase that uses ATP to autophosphorylate at a specific histidine residue. Phospho-CheA molecules then serve as donors for autokinase reactions that transfer phosphoryl groups to specific aspartate residues in CheY and CheB. Phospho-CheY enhances CW rotation of the flagellar motors; phospho-CheB has high MCP methylesterase activity. The active forms of these response regulators are short-lived because they quickly lose their phosphoryl group through spontaneous self-hydrolysis. CheZ further enhances the dephosphorylation rate of phospho-CheY to ensure rapid locomotor responses to changes in the supply of signaling phosphoryl groups to CheY.
CheW couples the autophosphorylation activity of CheA molecules to chemoreceptor control. Receptors, CheW, and CheA form stable ternary signaling complexes that modulate the influx of phosphoryl groups to the CheY and CheB proteins in response to chemoeffector stimuli.
Figure 3: Phosphorelay signaling. The flagellar motors of E. coli spin CCW by default; the signaling pathway modulates the level of phospho-CheY, the signal for CW rotation. Reactions and components that augment CW rotation are depicted in green; those that augment CCW rotation are depicted in red.
Chemoreceptor signaling states in E.coli
The signaling activities of chemoreceptors are described by a two-state model (Fig. 6). Receptor complexes in the CW signaling state activate CheA, producing high levels of phospho-CheY. Receptors in the CCW signaling state deactivate CheA, resulting in low levels of phospho-CheY. Thus, the behavior of the flagellar motors reflects the relative proportion of receptor signaling complexes in the kinase-on and kinase-off conformations. Both chemoeffector binding or release and methylation or demethylation can shift receptor signaling complexes from one state to the other. For example, attractant ligands drive receptors toward the kinase-off state; subsequent addition of methyl groups shifts receptors toward the kinase-on state, reestablishing the steady-state (adapted) balance between the two states and restoring random walk movements.TlpB in Helicobacter Pylori:
H. pylori is a Gram-negative bacterium that resides in thestomachs of over half the world’s population. Its gastric habitatcontains a marked pH gradient from the highly acidic lumen,which can reach pH 2, to the more neutral environment adjacentto the epithelial lining, which is typically pH 7. Based on genome sequence analysis, the H. pylori chemotaxismachinery resembles that of the well-studied model, E. coli,with a few notable variations including the absence of themethylation enzymes involved in receptor adaptation (Sweeneyand Guillemin, 2011).
Figure 5: Chematic representation of chemotaxis signaling in E. coli (A) and H. pylori (B). Chemoreceptors are shown in purple and flagellar motors in greenspanning the cell membrane. Cytoplasmic chemotaxis proteins CheA, CheW, CheY, CheZ, CheR, CheB, CheV, and ChePep are labeled. Protein modificationsare shown as pink circles for phosphorylation and purple hexagons for methylation. Activating interactions between signaling pathway components are indicatedby arrows, and speculative interactions are indicated by dotted lines.
When Helicobacter pylori and Escherichia coli bacteria which belong to two separate systems are examined, we recognize the vast similarity between the two. Both systems have chemoreceptors which belong to the MCP family enable the detection of chemical substances which do chemotaxis. Also, both bacteria also share in common the proteins required to send signals to necessary regions.
H. pylori has four chemoreceptors, TlpA,TlpB, TlpC, and TlpD, and several polar flagella that dictatesmooth swimming behavior in the presence of an attractantand increased stopping behavior in the presence of a repellent(Lowenthal et al., 2009; Rader et al., 2011). Only a small numberof chemotactic signals have been identified for H. pylori. Thebest-haracterized chemoreceptor is TlpB, which is requiredfor chemorepulsive responses to acid, as well as the quorumsensingmolecule autoinducer-2 (AI-2) and H+. TlpB is the first bacterial chemoreceptor of known function shown by crystallography to contain an extracellular PAS domain. Strikingly, a molecule of urea is found within the canonical ligand-binding site of the PAS domain, bound in a manner predicted to be sensitive to changes in pH.
Figure 6: TlpB Forms a Dimer that ContainsUrea-Binding PAS Domains(A) Schematic of estimated TlpB domains is illustrated.Transmembrane region (TM, orange);PAS domain (light gray); HAMP domain (green);chemoreceptor trimer of dimers contact region(blue). The periplasmic domain is from aminoacids 32–209.(B) Ribbon diagram of TlpBpp homodimer andgray/blue/red/white urea molecules, with chaincolor gradation ranging from N terminus (blue) to Cterminus (green) for the monomer on the right andN terminus (light green) to C terminus (red) for themonomer on the left is presented. For orientationthe bacterial inner membrane would be below thelower part of the protein model diagram.(C) Diagram of the TlpBpp urea-binding siteincluding hydrogen bonds (dashed lines) betweenurea (gray, white, blue, and red) and the surroundingresidues and water molecule is demonstrated.Oxygen atoms are shown as red spheres,nitrogen as blue, and hydrogen as white..
Emily Goers Sweeney et.al. report that urea is bound with extremely high affinity andspecificity and is essential for the thermodynamic stability ofthe TlpB molecule. The urea-binding site includes an aspartategroup (Asp114), which we propose to be the key titratableresidue responsible for pH sensing. Mutational and biophysicalanalyses of the urea-binding site support a mechanism in whichthe urea cofactor, by binding in a pH-sensitive fashion, stabilizesthe secondary structure of TlpB and signals a pH response.
Figure 7: Model for how TlpB Senses Acid: In low pH conditions (shown on the left), TlpB’s periplasmic domain is ina ‘‘relaxed’’ or expanded state due to decreased hydrogen bonding to ureaand consequent lowered urea-binding affinity. However, in high or moreneutral pH conditions, TlpB’s periplasmic domain is in a ‘‘tense’’ or condensedstate with increased urea-binding affinity. The state of the periplasmic domainis relayed through the transmembrane region, which affects CheA’s phosphorylationstate, ultimately affecting the flagellar motor and dictating stoppingbehavior. TlpB dimer is shown in orange, CheA in red, CheW in green, urea inpurple, protons in blue, and phosphate in black..
By sequence analysis, H. pylori TlpB is organized like a typical member of the MCP superfamily of transmembrane receptors with two transmembrane helices (tm1, tm2) per subunit bracketing an extracellularsensing domain[2,7]. The extracellular-sensing domain is responsible for detecting ligands directly or indirectly via interactions with periplasmic-binding proteins. Continuing from tm2, the C-terminal portion of the MCP is cytoplasmic and mostly helical. It contains a histidine kinase, adenylyl cyclase, methyl-binding protein, phosphatase (HAMP) domain, followed by a helical domain and a segment that binds to the CheA/CheW histidine autoki-nase complex[2,8]. Phosphorylation of CheA in response to an extracellular signal in turn controls downstream compo-nents that modulate activity of the flagellar motor. MCPs dimerize at the membrane, forming a four helix bundle with ligand-binding domains in the periplasm, whereas trimers of dimers assemble with CheA and CheW to form a high-perfor-mance signaling array located in the cytoplasm[2,9].
In low pH conditions , TlpB’s periplasmic domain is in a ‘‘relaxed’’ or expanded state due to decreased hydrogen bonding to urea and consequent lowered urea-binding affinity. However, in high or more neutral pH conditions, TlpB’s periplasmic domain is in a ‘‘tense’’ or condensed state with increased urea-binding affinity. The state of the periplasmic domain is relayed through the transmembrane region, which affects CheA’s phosphorylation state, ultimately affecting the flagellar motor anddictating stopping behavior
