A Genetic look at Antibiotic Resistant Tuberculosis: Inhibiting the BlaC gene and the enzyme B-Lactamase

Jeffrey Welch Santa Clara University

Introduction
Tracy Kidders Mountains beyond Mountains presents Tuberculosis a Mycobacterium, which affects over 2 billion people worldwide (WHO). Tuberculosis is well known for it antibiotics resistance. MDA(multi-drug-resistant) tuberculosis has been found to have mutations in at least nine genes;katG, inhA,aphgC and KasA for isoniazid resistance, r rpoB for rifa,a,picin reistance, rpsL and rrs for streptomycin resistance, embB for ethanbutol resistance and pncA for Pyrazinamide reTracy Kidders Mountains beyond Mountains presents Tuberculosis a Mycobacterium, which affects over 2 billion people worldwide (WHO). Tuberculosis is well known for it antibiotics resistance. MDA (multi-drug-resistant) tuberculosis has been found to have mutations in at least nine genes; katG, inhA,aphgC and KasA for isoniazid resistance, r rpoB for rifampim resistance, rpsL and rrs for streptomycin resistance, embB for ethanbutol resistance, pncA for Pyrazinamide resistance and most importantly BlaC gene for B-Lactam resistance. In the book Mountains Beyond Mountains (Farmer) realizes the growing problem with MDA. He formulates a process, which combats MDA using multiple drugs and DOTS (directly observed therapy short course). After reading Mountains Beyond Mountains and doing research I realized Tuberculosis¶ has an incredible ability to adapt and change in order to survive. I began to wonder how long scientists could continue to develop antibiotics in order to control this ever-changing microbe. In order to actually solve this problem rather than combating these microbes with antibiotics I wondered how we might be able to get down to the genetics and realize a genetic solution to this problem. Jean-Emmanuel Hugonnet answers this question by looking into one of the many genes responsible for drug resistant Tuberculosis, BlaC. The BlaC gene has been found responsible for the inefficiency of the BLactam antibiotics, which are derivatives of penicillin. The M. tuberculosis contains a single, highly active, chromosomally encoded class A (Ambler) B-Lactamase enzyme. When the BlaC gene responsible for the B-lactamase was knocked out the efficiency of B-Lactam antibiotics is far more efficient. To better treat tuberculosis patients the reaction between BlaC gene and multiple antibiotics is extremely important. To understand and cure Tuberculosis the structure of the protein, the chemical reaction and the kinetics of these reactions are essentail.

Methods
BlaC was taken from Tuberculosis and transferred to a plasmid, which was then expressed in Escolar, and B-Lactamase enzyme was recovered. Effectiveness of breaking of the B-Lactam ring was measured at 299nm and various concentrations of Merepenem (antibiotic of interest) were used and standardized using the Michaelis-Mentin equation and were graphed as seen in fig.1 a, b. After B-Lactamase and Merepenem have reacted, the researcher vitrified or made the complex solid. This traps the Merepenem at the active site enabling us to see the structure of the reaction. The solid was then treated (do I need to say how?) and then analyzed by x-ray diffraction in order to study the exact mechanism from how Merepenem acetylates and hydrolyzes B-Lactamase.

Results/Analysis
A genetic knockout of the blaC-encoded Blactamase in antibiotic resistant strains of Tuberculosis showed increased sensitivity to BLactams. After cloning the M. tuberculosis BlaC gene the enzymatic activity of the b-lactamase was discovered to hydrolyze all penicillin¶s (BLactams) at nearly the diffusion-limited rate. The enzyme was even able to hydrolyze and disable the b-lactamase inhibitors sulbactam and tazobactam. However Clavulanic acid was found to be the only FDA approved drug to irreversibly inhibit the BlaC enzyme. Similar in its inhibitory functioning Meroponem, our drug of interest, reacts with the enzyme to form acylenzyme intermediate quickly and then slowly reacts to hydrolyze the enzyme. Because of the slow enzymatic turnover rate Meropenem is a good inhibitor of B-lactamase as shown by the equation Km=3.4 t/_ .7 and Kcat=.08 +/ .01. By Add figure caption tracking B-lactam activity using nitrocefin it is found that with the presence of Meropenem the reaction is non-linear suggesting inhibitory functioning with an inhibitory constant value of Kj-16 +/- 2 mM. Using FTICR(Fourier transform ion cyclotron resonance) the interaction between Mereopenem and B-Lactamase was further shown to react(refer to fig. 2. (A) Peaks of the spectrometer were indicated at 29123.65 and 29167.55 correlating respectively to two covalently acytaled BlaC-Meropenem complex. After 7 minutes of incubation the two separated and the results were as follows: (FIGURE THIS OCHEM SHIT OUT). Different combinations of Mereponem and Clavulanic acid were then administered along with B-Lactams for 5 days to the antibiotic resistant M.tuburculosis under aerobic growth conditions. The colonies number dropped drastically and after 12 days sterilization was complete. When Tuberculosis goes into the persistent, state where no replication occurs, it becomes a problem for antibiotics to kill the Mycobacterium because most antibiotics work during cellular division. In the persistent state L.D trans peptidase changes the peptidoglycan crosslinking and targets B-Lactams as described in another experiment and named the Wayne model. (Maybe delete) Mereponem and Clavulanate were then used in combination with other antibiotics to test effectiveness during this state. Using ATP analysis Mereponem and Clavulanate in addition to B-Lactams were most effective in combating M.tuberculosis in a 2week trial under aerobic conditions.

Conclusions
Multiple drug resistant tuberculosis can be treated by using Meropenem and Clavulanate. Mereponem is the slowest b-lactam antibiotic to be hydrolyzed by b-lactamsae giving it an inhibitory status. (Merepenem basically deals with BlaC gene which makes b-lactamase while other drugs work against the tuberculosis) The actual mechanism is best understood by observing figure 2c. (Describe later but seems pointless). With the combination of these two drugs, the BlaC gene can be combated. Penicillin and it derivatives are the most widely used antibiotic and are very cost effective. Due to the cost efficiency of these drugs, the treatment of tuberculosis can become much cheaper and effective. If clinical trials with Meropenem and Clavulanate are successful this could have large implications for cost effectively treating Tuberculosis. With over 2 million cases of Tuberculosis occurring each year one more drug regiment may save many patients lives who were once thought to be incurable. By taking a closer look at the genetics and transformation of this Mycobacterium we can get right down to the cause of antibiotic resistance and hopefully some day rid our planet of MDA Tuberculosis.

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References
Hugonnet, Jean-Emmanuel. "Science." Science AAAS 323.1215 (2009): 1126-134. Print. Chen, Ling, Xin Gan, Nana Li, Kailun Li, and Hong Zhang. "RpoB Gene Mutation Profile in Rifampicicin-resistant Mycobacterium Tuberculosis Clinical Isolates from Guizhou, One of the Highest Incidence Rate Regions in China." Journal of Antimicrobial Chemotherapy 65.6: 1299-301. Web.

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Questions 
What is the most effective way to treat turburculosis Can knowledge of Tuberculosis genes help with the eradication of Antibiotic resistant TB

Add figure captionafter an initial attack size of 1,000 in a population of 10^7.

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