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Mycobacterium tuberculosis (MTB) is a non motile rod-shaped bacterium that is fairly large. The generation time of MTB is slow; 15-20 hours. MTB is surprisingly neither Gram-positive nor Gram-negative because its chemical characteristics are not related to either one. Even though MTB cell walls contain peptidoglycan, when tested with a Gram stain it only stains very weakly as Gram-positive or it does not stain at all, when this occurs these cells are known as "ghosts". This strain of bacteria causes tuberculosis in humans where MTB complexes are known to commonly have an ecological niche within the upper lobes of the lungs. This is because the cells require oxygen for growth (Todar). Â MTB is spread easily in the air when a healthy person is in proximity of an infected person. If the infected person talks, coughs, or breathes they release MTB into the air. Tuberculosis (TB) is able to affect any system in the body (CDC) but with less frequency than infections of the lungs (TBVI). A related strain, Mycobacterium bovis, typically only causes tuberculosis in cows but affects humans rarely. Mycobacterium avium (the cause of a tuberculosis-like disease in AIDS patients) and Mycobacterium leprae (the bacterium that causes leprosy) are also related to M. tuberculosis (Todar).
There are two tests, the TB skin test and the TB blood test, that can be done to see if a person is infected with MTB. These tests cannot verify if a person has a latent TB infection (with no symptoms) or active TB disease. Chest X-rays and sputum sample tests can differentiate between the infection and disease (CDC). Treatment for TB usually is a lengthy process involving two months of a combination of isoniazid, rifampin, pyrazinamide and ethambutol. After this, rifampin and isoniazid are given for four months (Hartkoorn). Prior to the introduction of rifampin treatment could take up to two years; now treatment time has been reduced to a year or less (CDC - treatment). The survival ability of MTB is partly dependent on the fact that it is able to live and replicate within the host's macrophage cells (Read). The prognosis for a patient infected with TB is good; if the patient follows the prescribed medication regimen If the patient strays, the remaining bacteria could potentially become drug-resistant. (Todar). The mortality rate for those infected with TB, who are not HIV positive, was 19 out of 100,000 worldwide for 2009 (WHO).
Due to the overwhelming amount of antibiotic resistance of many TB cases, researchers felt that it was important to find a more efficient way for existing medications to function. Rifampin has become one of the main staples in treating TB infections. Funding of research decreased due to the dwindling number of cases and increased treatment response (CDC). It has been found that rifampin acts by makingÂ RNA polymerase (RNAP) ineffective; inhibiting transcriptionÂ and therefore preventing replication of the bacterium. Research has also shown that rifampin will not inhibit the ability of RNAP in a human cell. (rifampin). Previous studies spent time investigating the mechanism that allows Mycobacterium strains to be resistant to TB medications. Dey et al investigated the effects of a different rifampin type product, rifampicin, andÂ how it interactions with the accessory proteins and RNAP. They also determined that there was a possibility of the rifampicin binding site to overlap with the protein binding site causing transcriptional inhibition. Hartkoorn and associates continued to ask similar questions, which are discussed throughout this paper. They focused on the over expression of sigma factor F (SigF) to see if it caused MTB to be more resistant to rifampin; possibly by binding to the RNAP to change its activity and prevent transcription. Gathering information on thisÂ process may allow the medical field to successfully treat patients with tuberculosis faster and more effectively in the future.
In order to research the functions of rifampin, the researchers established a plan to test the medication in different situations. They were able to purify several different factors that were necessary to complete the experiments that followed. In vitro and in vivo transcription was compared in the presence of rifampin and a complete SigF knockout mutation was also evaluated.
The researchers obtained histadine (his-) tagged RNAP to purify from a similar log-phase strain of mycobacterium, Mycobacterium smegmatis. This bacterium has often been used in the lab setting in place of MTB because it has a fast replicating time and is non-pathogenic (titgemeyer). When purified, RNAP contained both core and holoenzyme (holo-) components. These parts were separated using cation-exchange chromatograph; which is a method that separates molecules based on similar characteristics (Snyder). In order to purify the holo-RNAP contaminants had to be removed by isocratic mobile-phase A and washed with mobile-phase B in a column. A column will collect the wanted material under the correct pH conditions and then can be washed to recover this material. A slow gradient was used to divide the holo- and core RNAP; this separated the fragments by size. Following division, the separated components were pooled. Using a semipermeable membrane, the fractions were separated by dialysis due to unequal diffusion rates (dialysis) and concentrated (Hartkoorn). The purified RNAP was made to be used in real-time and radiometric in vitro transcription (IVT). It was slightly difficult to isolate the two types of RNAP because they have very similar molecular weights. Results of purification are represented in figure 1. They used an SDS-PAGE gel for the RNAP. Proteins were denatured by the sodium didecosulfate (SDS) wash. The his-tagging allowed the RNAP to bind to the Ni2+Â affinity column in the gel. The purified RNAP components were compared to known samples of sigma factor A (sigA) and SigF on the gel. They ran the sample through the gel to prove that native sigma factors were not present in the purified samples.
The process of purifying recombinant sigma factor A (rSigA) and recombinant sigma factor F (rSigF) produced products to be used in the in vitro transcription analysis. The sigA and sigF genes were added into the pHis9GW vector to obtain pHis9SigA and pHis9SigF products. This was done by PCR amplification of genes and again using PCR to recombine linear DNA in another vector (pDONR207). PCR was used to replicate specific DNA material. Single stranded genetic material was used as a template; polymerase can bind to the template and the added deoxynucleotide triphosphates (dNTPs) along with primers that work in each direction. Heat denatured the double stranded DNA and allowed the polymerase, primers, and dNTPs onto the template. As it cooled, replication occurred and this was repeated many times to obtain a high amount of product. After the PCR process the researchers disabled the start codon and added a thrombin site. Â A thrombin was used to recognize amino acid sequences and can cut the bonds between arginine and glycine. Â This was used to get rid of any histadine tags that were upstream (Vanderbilt). Another recombination took place using both a pDONR207 vector and a pHIS9GW vector thus transferring the genes into pHIS9GW. The product that resulted was pHis9SigA and pHis9SigF and was replicated by the use of Escherichia coli.
The next step the researchers needed to take was to produce the necessary proteins. This was done by introducing the plasmids (pHis9SigA and pHis9SigF) and sigma factors into E. coli. Hartkoorn et al found that the different method they were using caused E. coli to grow more slowly when rSigF was introduced to the bacteria. The E. coli was pelleted and resuspended in order to clean the wanted products. Next the bacteria were lysed by sonication. The sonication method was used as it disrupts the cell (sonication) and causes the release of cellular contents into solution. This solution was then pelleted to separate the larger protein fragment from the rest of the solution. Sigma factors with the His-tag then were gathered by the use of affinity chromatography; which is another system to separate the different products based on their desire to bind to other molecules. Then dialysis was used on the sigma containing factors.
Real-time in vitro transcription and radiometric in vitro transcription are two methods the researchers used. They wanted to determine if transcription was inhibited in the presence of rifampin. Also these tests were used to see if inhibition levels changed with the addition of SigF vs SigA. These experiments needed the products described in the two purification methods above.
Real-time transcription is a process that allows researchers to measure RNA synthesis. It is performed with molecular beacons, which are markers that give off light when they hybridize to newly made RNA. They are produced from 2'-O-methylribonucleotides which cannot be copied by RNAP (Marras). The control was a template without any SigF or SigA and the level of transcription was noted. A template for each sigma factor, with a polyA region (recognized by the molecular beacon), promoters, and termination sites was made. The SigA promoter was PlacZ. The SigF had two promoters, PrsbW and PlacZ, each working in different directions. Having the two promoters in the SigF test acted as a control to test if SigA was excluded completely because PlacZ attracts SigA. This ensured that SigF was the only sigma factorÂ affecting the transcription. Transcription happens when the PlacZ promoter gets recognized by the SigA holo-RNAP for the first template. When rSigA was added the rate of transcription increased at the PlacZ promoter and only increased a little bit at the pUC19-PrsbWpolyAT1 location. Similar results were noted when rSigF was added to the other template with a slight change. Transcription from PrsbW increased but not at the PlacZ location (indicating that there was no SigA in the second template). Overall, the addition of either SigF or SigA increased transcription. Transcription was completely halted when a large amount of rifampin was added. Figure 2 in the Hartkoorn research gives a very good overview of this. 2C indicated, by a line graph, that the core enzyme produced a low level photon emission, suggesting a small amount of transcription. When the SigF was added the increase of photon emissions was only slight. Addition of SigA to the core enzyme showed a large increase of photon emission. Figure 2D indicated a similar effect, but with respect to SigF. The SigF-core enzyme had the highest rate of transcription and the SigA-core enzyme was slightly greater than the core enzyme alone. Another line graph, figure 2E, indicates the inhibition of IVT for the two different situations. The transcription at the beginning of the experiment was at 100 percent. As rifampin was added, transcription decreased at about the same rate for each set up. When the concentration of rifampin had reached close to 100nM the transcription stopped.
Radiometric in vitro transcription also showed similar results. PCR was used to make amplified M. bovis BCG hsp60 promoters (specific to SigA) on the linear template and the radiometric label was also added to the PCR. The same procedure was repeated to obtain the M. tuberculosis strain H37Rv rsbW promoter (which is needed for SigF recognition). The transcription happened in the presence of core RNAP, linear DNA templates, recombinant sigma factors, and a radiometric label CTP. This label will attach to the alpha carbon and will give off light when viewed. As transcription increases, the intensity of the light from the label will also increase. A phosphorimage of the products was taken to determine the amount of transcription for each in the presence of rifampin. The SigF recognized promoter produced little transcription with the SigA mediation and the same effect was noted when reversed. When rifampin was added, the amount of transcription was completely terminated at high concentrations.
Figure 3A in the Hartkoorn research showed a picture of the phosphorimage. The two lanes, Phsp60 (SigA) and PrsbW (SigF) promoters, have varying levels of photon emission. The intensity of the photons decreases as the level of rifampin increased. Rifampin amounts ranged from 0nM to 320nM. At the 40nM concentration level, the photon intensity was almost non-existent for both promoters. This indicated that no transcription occurred above 40nM.
Pristinamycin IA purification and Pistinamycin IA-inducible induction of SigF was completed to better understand the way that rifampin effects the viability of a bacterium. Forti et al had previously discovered that inducible gene expression systems were useful in studying the functions of genes and the effectiveness of medications. The over expression of genes in these bacteria allowed researchers to study the genes. For example one of the questions the Hartkoorn study asked was if over expression of sigF could prevent or decrease rifampin's ability to stop transcription in M. tuberculosis. Many different inducible gene systems were previously used but none worked well with mycobacteria genes. Because the previous systems were not useful in vivo the system established by Forti et al was a breakthrough for many, including Hartkoorn et al.
Pristinamycin IA was purified from the medication Pyostacine. It is a protein of Streptomyces coelicolor and a promoter (ptr) of Streptomyces pristinaespiralis (Forti). According to Hartkoorn, Pyostacine was crushed and dissolved in methanol, as it is non-polar. The pristinamycin IA pellet was recovered by centrifuging, then re-dissolved and run through a column. Mass spectrometry was used to verify the recovered material. Mass spectrometry was a way to measure the composition of the molecule and its charge. When the mass spectrometry information was compared to known data, it was possible to determine which substance makes up the sample.
The induction of SigF in M. tuberculosis by the Pristinamycin IA system was completed in order to be able to induce the SigF from M. tuberculosis H37Rv into the ptr promoter (Pptr) found in S. pristinaespiralis in vivo. Pptr was recombined into the sigF gene by PCR and then cloned into pMY769 to get pMYSigF. The control plasmid wasÂ pMY769. Both plasmids were added to M. tuberculosis using electroporation (a way to run a current through the cells forcing them to allow the plasmid through the membrane). Each type of M. tuberculosis (one containing the control plasmid or one with the SigF plasmid) was grown on full media and then the pristinamycin IA was added the cells. Â Time was allowed for SigF induction and the results were observed. The newly produced protein was proven by the use of a Western blot system. A western blot allows the researchers to move proteins from a gel onto a membrane (Snyder) Hartkoorn et al used this technique to transfer protein to a membrane and then stained to confirm the lack of SigF proteins. The negative control for the Western blot was the H37Rv::pMY769 plasmid; no SigF was shown to be present. When the experiment was repeated for the H37Rv::pMYSigF plasmid it was noticed that induction of SigF did occur. H37Rv::pMYSigF was then cultured in the presence of rifampin. Contrary to expectations, SigF did not prevent rifampin from working.
Figure 4 of the Hartkoorn et al work described the results from the induction of sigF and effects of rifampin. First, a general diagram is given of the two pristinamycin plasmids they made (A and B). Figure 4C is a picture of the Western blot results. All of the test situations are represented on the western blot gel. The only indication that the SigF protein was expressed appeared on H37Rv::pMYSigF + pristinamycin IA. Another control was strain H37Rv::pMY769; it showed a growth rate relatively the same with and without pristimycin IA. Figure 4D (the negative control) and 4E are line graph representations of growth curves for the M. tuberculosis strains H37Rv::pMy769 and H37Rv::pMYSigF with and without pristinamycin IA. They hoped to determine if over expression of sigF changed the tolerance of MTB with the assistance of pristinamycin IA. With the presence of pristinamycin IA, the growth curve of H37Rv::pMYSigF was retarded. Without pristinamycin IA, the result appeared similar to the control.
The next part of the research was the most important to test the major hypothesis.. Using a resazurin microtiter assay (REMA) and a CFU determination the researchers tested the rifampin susceptibility of H37Rv::pMY769 and H37RV::pMYSigF (Hartkoorn). REMA is a test commonly used to determine the susceptibility of different MTB strains to antituberculins. This testing is easily accessible because the results are easily visible by looking for a color change from blue to pink and it is an affordable test (Martin). CFU (or colony forming unit) determination measured the number of microorganisms present in a colony (MBL). Samples were grown with and without the presence of pristinamycin IA as well as with and without the presence of rifampin before being plated.
Figure 4F and 4G illustrate the results from the REMA testing. Figure 4F focuses on strain H37Rv::pMY769 while 4G shows H37Rv::pMYsigF. The graphs illustrate cultures with and without pristinamycin I after 3 days of exposure to rifampin. The control group stopped growth at the same rate, regardless of the presence of pristinamycin IA. The strain with the sigF gene expression did not stop growing at the same rate, but it was very similar. When lacking pristinamycin IA, the sample slowed faster. When the control was compared to the sigF gene, the effects were much the same. The REMA test results showed that there was not an increased tolerance to rifampin in the altered sample when compared to the control. Figure 5 showed the results from the CFU determination test. The results show that the induction of SigF did not have an effect on the amount of viable bacteria following the exposure to different amounts of rifampin. This showed that the over expression of SigF in MTB H37Rv gave no increase in either tolerance or susceptibility to rifampin.
Another approach that was taken to see if SigF changed the tolerance to rifampin was performed using a knockout mutation in the M. tuberculosis H37Rv strain. The operon containing the sigF gene was deleted and a hygromycin cassette was made using recombination (Hartkoorn). Â Hygromycin cassettes are generally used to make vectors that have resistance (Zheng). Â Mistranslation occurred between DNA and protein because of translocation interference caused by Hygromycin (Invivogen) which lead to mutations needed for the study. About one thousand base pairs were removed from each side of, and including, rsbW and sigF. Â These genes were amplified using PCR. BamHI and a SpeI (restriction enzymes) (Synder pg 57) were added during this process and joinedÂ to pUC19 producing pUC19-rsbWsigF strain. Next, the amplification of the hygromycin cassette was completed and SpeI sites were introduced alongside the amplicons. This gave the researchers pUC19-rsbW/hygR/sigF. Plasmids were recovered from the clones and the restriction enzyme BamHI was removed. The product from the removed BamHI was used to encourage linear alleles to translocate, causing mutations. The linear allelic product was added to M. tuberculosis with pJV53. The clones that had the hygromycin-resistance and the knockout were collected. The knockout was not able to produce SigF and was used to compare to the wild type strain. The growth patterns of the knockout and the wild-type were the same and indicated that SigF does not change the tolerance of M. tuberculosis to rifampin. Â Figure 6 shows the rifampin susceptibility of wild-type H37Rv and the knockout H37RvÎ”rsbW/sigF. The growth curve is almost exactly the same, which means that the induction of SigF did not result in an increase of susceptibility.
Neutral red staining is the method the researchers employed to detect MTB sulfolipids associated with the cell envelope (Hartkoorn). The neutral red dye is able to be reduced by virulent strains of mycobacteria, which causes them to turn red. Avirulent strains are unable to reduce the dye and therefore do not stain (Gibert). Before staining, the researchers allowed cells to grow and then diluted them. Three days before the staining experiment, pristinamycin IA was added to H37Rv::pMY769 and H37Rv::pMYSigF because this had been an important alteration that had been tested. The bacteria was then fixed, pelleted and finally resuspended with the addition of neutral red and allowed to incubate. The only sample of the Hartkoorn et al experiments that was unable to stain red was their negative control, H37Ra.
Following their research, Hartkoorn and associates found that their original hypothesis was incorrect. They had asked two questions: does over expression of SigF cause MTB to be more resistant to the effects of rifampin? And does SigF change the way that rifampin binds to and modify the activity of RNAP? The answer to both of those questions is no. Their hypothesis was that the deletion of SigF caused an increase in rifampin susceptibility. They ran several tests that showed that induction of sigF did not increase rifampin tolerance or suceptability. The wild-type MTB H37Rv and H37RvÎ”rsbW/sigF had the same values for their minimal inhibitory concentration. The original findings that prompted this research involved MTB strain CDC1551 (Hartkoorn). The researchers used a different strain of MTB for their experiments (H37Rv) and this may have accounted for their hypothesis being false. In their discussion, they noted this discrepancy. CDC1551 did not stain in a neutral red staining test. All of the samples they used from H37Rv stained positive in the same test. They also mentioned that their neutral red staining media may have been different. Ideally, the researchers should repeat their experiments with MTB CDC1551 to see if that strain yields different results.
The research that was done in these experiments is helpful to creating a shorter treatment time for patients with tuberculosis. They hoped to find a way of modifying the bacteria that made it more susceptible to the drug regimen instead of trying to create a new drug. If a method could be found for a shorter treatment, it is a possibility that less people will stray from the prescribed treatment and therefore raise the treatment's success.
While writing the paper, use of Google Documents (Docs) was key to the success and uniformity of the paper. Google Docs provided both authors the ability to be in two different places but still collaborate on a single document; where all work could easily be viewed and edited. Changes could be seen immediately and simultaneously by both parties. This technology also allowed the writers to only meet a few times in person and enabled them to work on the paper it was convenient for them. Other correspondence was made through email and the use of cell phones.
Due to the limited knowledge of the writers, the citations within the original research document were another invaluable tool. There were many things within the research that were difficult to understand without further information. The citations made it possible to grasp the reasons behind the methods. This was very distracting, as the citation trail lead to many other tangents; however interesting, not related to this paper.
Overall, writing this paper was much more time consuming and frustrating than expected. Even so, this project provided a useful strategy that can be used in the future when encountering other research journal articles. An English class offered in place of, or supplementary to, composition two for students in the sciences may be a great resource for students in the future.