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A Genetic look at Antibiotic Resistant Tuberculosis:

Inhibiting the BlaC gene and the enzyme B-LactamaseJeffrey Welch

Santa Clara University

Introduction

Introduction

Tracy Kidders Mountains beyond

Mountains presents Tuberculosis aMycobacterium, which affects over 2 billion

people worldwide (WHO). Tuberculosis iswell known for it antibiotics resistance.

MDA(multi-drug-resistant) tuberculosis has

been found to have mutations in at leastnine genes;katG, inhA,aphgC and KasA forisoniazid resistance, r rpoB for rifa,a,picin

reistance, rpsL and rrs for streptomycin

resistance, embB for ethanbutol resistanceand pncA for Pyrazinamide resistance and

most importantly BlaC gene for B-Lactamresistance. In the book Mountains BeyondMountains ( Farmer) realizes the growing

problem with MDA. He formulates a

process which combats MDA using multipledrugs and dots(directly observed therapyshort 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 longscientists will be able to continue and

develop antibiotics in order to resist this

ever changing microbe. In order to actuallysolve this problem rather than combating

these microbes with antibiotics I wonderedhow we might be able to get down to thegenetics and realize a genetic solution to

this problem. Jean-Emmanuel Hugonnet

answers this question by looking into one

of the many genes responsible for drugresistant MDA BlaC. The BlaC gene has

been found responsible for the inefficiency

of the B-Lactam antibiotics. The M.

tuberculosis contains a single, highlyactive, chromosomally encoded classA(Amber) B-Lactamase. When the blaC

gene responsible for the B-lactamase was

knocked out the efficiency of B-Lactamantibiotics(like penicillin) were far more

efficient. With this knowledge it isimportant to understand the structure of theprotein, the exact reaction with the

antibiotics and the kinetics of the r eaction

in order to better treat tuberculosis

patients.

References

References

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

Methods

Methods

Results

Results

BlaC was expressed in E.coli and purified.

Effectiveness of breaking of the B-Lactam ringwere measured at 299nm and various

concentrations of merepenem(antibiotic) wereused and standardized using the michaelus

mentin equation and were graphed as seen in

the conclusion.Soaking of the BlaC with merepenem followed

by vitrification or making the complex solid

traps the merepenem at the active site. The

solid was then treated(do I need to say how?)and then analyzed by x-ray d iffraction in order

to study the exact mechanism from howmerepenem acetylates and hydrolyzes Blac.

Questions

Questions

What is the most effective way to treat

turburculosisCan knowledge of Tuberculosis genes helpwith the eradication of Antibiotic resistant TB

Conclusions

Conclusions

Add figure caption

.

Add figure caption.

Multiple drug resistant tuberculosis can be best

combated while using Meropenem andClavulanate. Mereponem acts as a slow, tight

binding inhibitor of the hydrolysis of the BlaCenzyme. Meroponem reacts with the enzyme to

form acyl-enzyme intermediate quickly and thenslowly reacts to hydrolyze the enzyme. The

reason Meropenem may be a good inhibitor isbecause of this slow enzymatic turnover rate

shown by the equation Km=3.4 t/_ .7 andKcat=.08 +/ .01(just look at the graph).

(Merepenem basically deals with Black while

other drugs work against the tuberculosis) The

actual mechanism is best understood byobserving figure 2c. (Describe later but seemspointless)

In addition to Meropenem the antibioticClavulanate was seen to have similar effects of

slow hydrolysis, as shown by other studies, aswell as enhance the effects for b -lactam

antibiotics. Due to this data differentcombinations of these drugs working together

were administered for 5 days to theM.tuburculosis under aerobic growth conditions.

The colonies number dropped drastically andafter 12 days sterilization was complete.Though the interaction of Mereponem with the

cell walls of tuberculosis is unknown it is clear

that a combination of these two drugs may bevery beneficial in the treatment of tuberculosis.

Hugonnet, Jean-Emmanuel. "Science." Science

AAAS 323.1215 (2009): 1126-134. Print.

Chen, Ling, Xin Gan, Nana Li, Kailun Li, andHong Zhang. "RpoB Gene Mutation Profile in

Rifampicicin-resistant Mycobacterium

Tuberculosis Clinical Isolates from Guizhou,One of the Highest Incidence Rate Regions inChina." Journal of Antimicrobial Chemotherapy

65.6: 1299-301. Web.