A Genetic look at Antibiotic Resistant Tuberculosis:
Inhibiting the BlaC gene and the enzyme B-Lactamase
Introduction
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 B-
Lactam 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.
 
Questions
Questions
What is the most effective way to treat
turburculosis
Can knowledge of Tuberculosis genes help
with the eradication of Antibiotic resistant TB
Jeffrey Welch
Santa Clara University
Methods
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.
Add figure caption
.
Add figure caption.
Add figure captionafter an initial attack size of 1,000 in a population
of 10^7.
Results/Analysis
Results/Analysis
A genetic knockout of the blaC-encoded B-
lactamase in antibiotic resistant strains of
Tuberculosis showed increased sensitivity to B-
Lactams. After cloning the M. tuberculosis BlaC
gene the enzymatic activity of the b-lactamase
was discovered to hydrolyze all penicillin’s (B-
Lactams) 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 acyl-
enzyme 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
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 cross-
linking 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 2-
week trial under aerobic conditions.
Conclusions
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.
 
References
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.