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UNDERSTANDING MECHANISMS OF
REGULATION OF ANTIBIOTIC RESISTANCE
CH397 Mini Project Report
Submitted by
Suketu Datta
22B2736
Under the guidance of
Prof. Ruchi Anand
DEPARTMENT OF CHEMISTRY
INDIAN INSTITUTE OF TECHNOLOGY
BOMBAY 2023 - 2024
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ACKNOWLEDGEMENT
I would like to extend my gratitude to Prof. Ruchi Anand for the
support and guidance. I would also like to extend my thanks to all my
other lab mates.
Suketu Datta
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Certificate
I certify that the work presented in this report has been carried out under my supervision.
Signature and Date (25/11/2024)
Name of the Supervisor: Professor Ruchi Anand
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Table of Contents
Introduction..............................................................................................................................
Objectives..................................................................................................................................
Materials and Methods............................................................................................................
Preparation of Competent Cells.............................................................................................
Site-Directed Mutagenesis.....................................................................................................
Transformation of Plasmids...................................................................................................
Extraction of Plasmid.............................................................................................................
Green Fluorescent Protein (GFP) Assay...............................................................................
Results and Discussion.............................................................................................................
Future Goals...........................................................................................................................
References...............................................................................................................................
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Introduction:
Antimicrobials – including antibiotic, antiviral, antifungal, and antiparasitic – are medicines
used to prevent and treat infectious diseases in humans, animals, and plants. Antimicrobial
Resistance occurs when microbes develop strategies to protect themselves from the action of
antimicrobials. This makes it harder to treat infections, leading to more rapid spread of
diseases and even death. Often called the ‘silent pandemic’, it is an active threat and has
been, as per the Centre for Global Development (2023) report, there is an increase of
resistance from 16% to 44% in 2020, which is tripled from 2001, and leading to 1.3 million
death per year. It is a natural manifestation of the evolutionary theory of ‘survival of the
fittest’, occurring over time through genetic changes in pathogens. It is accelerated by the
misuse and overuse of antibiotics. The diminishing efficacy of common antibiotics against
widespread bacteria is of growing concern and a very active field of research that can help
combat this coming pandemic.
a. c.
b. d.
Figure 1. Death rates per 100,000 attributable to AMR, all ages, 1990, 2021, 2050 [1]. a. Death rate attributable to AMR, all
ages, 1990. b. Death rate attributable to AMR, all ages, 2021. c. Death rate attributable to AMR, all ages, 2050. d. Deaths
attributable and associated with antimicrobial resistance, by detailed age group, for 1990 and 2021
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It has been observed that bacteria employ several mechanisms to confer AMR, some of them
being:
(1) Enzymatic degradation of the drug
(2) Modification or Mutation of Drug target
(3) Changes in Membrane Permeability
(4) Efflux mechanisms
Figure 2. Overview of Mechanisms of Antimicrobial Resistance [2]
Modification of the drug target is observed in the case of Erythromycin Resistance.
Erythromycin, a macrolide antibiotic, is used to treat infections caused by certain strains of
Streptococcus and Staphylococcus[3]. Erythromycin binds to the nascent peptide exit tunnel in
the 50S subunit, interfere with the exit of the newly formed peptide chain. This causes the
ribosome to stall and prevents further protein synthesis, resulting in bacterial death.
Erythromycin-resistant cells produce enzymes called Erythromycin Resistance
Methyltransferases (Erms). Of these, Erm(C) methylates a specific adenine base at 2058
position (A2058) of the 23s ribosomal RNA (rRNA), which hampers the binding of
Erythromycin to ribosomes, thus making cells resistant.
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However, this modification left ribosomes with compromised translation fidelity, which for
longer periods is detrimental to bacteria itself. Thus, how the bacteria regulates the
production of Erm(C) so that it only gets active when necessary is still a question on the
queue. According to the available hypothesis, Erm(C) expression is controlled by the region
at the 5’-end of the gene, the structural and functional details of which are yet to be explored.
According to the hypothesised model for Erm expression, the Ribosome binding site of Erm
(C) is inaccessible to the ribosome under normal conditions due to its specific architecture.
Instead, a short leader peptide sequence preceding the erm(C) sequence called erm(C)L is
expressed. The presence of erythromycin in the cell causes the ribosome to stall, which in
turn leads to conformational changes in the switch region of ErmC mRNA (figure. 3), making
the erm(C) encoding region accessible. The exact mechanisms involved in this are still
unknown, whether the conformational changes are brought about just because of the physical
force exerted by ribosomal stalling or any other factors are involved in this regulation.
Figure. 3 | In absence of erythromycin, ribosome binds to the mRNA on GAGG sequence (RBS1) and synthesizes 19
residues long leader peptide region. In presence of erythromycin, ribosome stalls at IFVI sequence of leader peptide
and this stalling trigger conformational change that disintegrate the two stem-loop regions and form a new stem-loop
that exposes the ribosome binding site (RBS2) prior to ErmC gene.
Objective:
Understanding the mechanism by which erm(C) is expressed is essential for a better
understanding of the mechanisms behind the development of resistance against erythromycin,
which also can help in designing new drug molecules.
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Here, we tried to understand how the mRNA switch behaves to regulate its downstream gene
(ErmC)
Materials and Methods:
Preparation of Competent Cells:
Overnight grown DH5α cells were taken (1 ml) and inoculated into 100 ml Luria Bertani
Broth (LB Broth). At OD 0.4 – 0.5, the culture was divided into two 50 ml sterilised falcons
and kept on ice for 20 minutes. Cells were then centrifuged at 4000 rpm for 15 minutes. The
supernatant was discarded, and the pellet was resuspended with 20 ml of MnCl 2 & CaCl2
solution and kept on ice for 45 minutes. Cells were then harvested by centrifugation at 4000
rpm for 15 mins. The pellet was dissolved into a 1:1 ratio of CaCl 2 and glycerol solution and
aliquoted. Aliquotes were flash-frozen and stored at -20°C.
Site-Directed Mutagenesis:
The ErmC gene with its regulatory switch region was available in a lab in the pGEX-4T-2
vector. Following the switch instead of ErmC, the gene for GFP is inserted so that any effect
due to mutations in the switch region can be monitored by the GFP expression level. The
stem-loop regions of ErmC switch have been mutated through PCR. First, the primers were
designed according to the desired mutation. pGex-4T-2 plasmid containing ErmC switch
region followed by GFP was taken as a template for each PCR. PCR products were
transformed into E. coli (DH5α) cell and mutation was confirmed through sequencing.
Transformation of Plasmids
On successful amplification in PCR, the PCR mix were treated with 1U of Dpn1 enzyme for
2 hours, to digest the template, followed by transformation into DH5α cells. Amplified
products were mixed gently with 100 µl of competent DH5α cells and kept in ice for 30 mins.
Cells were then treated with temperature shock (45 seconds at 42°C, followed by 2 mins in
ice). Cells were then kept for 2 hours at 37°C under shaking conditions to grow. After
centrifugation, the pellet was spread on an LB agar plate and grown overnight at 37°C.
Extraction of Plasmid
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Following transformation, single colonies were taken and inoculated in 5 ml LB media.
Overnight grown cultures were used to extract plasmid. Extraction was done by using the
ExprepTM Plasmid SV kit from GeneAll, and protocol was followed as mentioned in the kit
manual. Extracted plasmids were sent for sequencing to confirm mutations.
Green Fluorescent Protein (GFP) Assay
On confirmation of successful mutation, plasmids were transformed in expression cell line
BL21 (DE3) and a GFP assay was carried out. Overnight grown culture was inoculated into
100 ml LB media and kept at 37oC until it reached OD 0.5. Following that, cells were
aliquoted and treated with 0.3 mM of IPTG and 10 µM of freshly prepared erythromycin to
induce GFP expression. After 3 hours of incubation at 37 oC, cells were harvested at 12500
rpm for 2 minutes, washed with 100 µl of PBS buffer, and lysed with 200 µl of Cell Culture
Lysis Reagent (Promega) for 30 minutes. Lysate was centrifuged at 12500 rpm for 5 mins,
and a supernatant was taken in the plate reader (Calrio Star) to measure GFP fluorescence
intensity.
Result and Discussions
Mutant switches and their corresponding GFP expression level
In order to determine, which conformation of the switch is correct, we have started checking
the switch functionality by mutating its stem-loop regions. In this section, we designed
mutations based on increasing and decreasing the length of the first stem-loop region to
understand how the architecture plays roles in regulating downstream gene expression. First,
C.
.
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Figure. 4 | a. Proposed 2D structure of the ErmC switch. b. Switch construct made by inserting GC base pair (orange
coloured) in the first stem-loop, and its respective GFP count. c. Switch construct made by deleting 1 base pair at the base
of loop region (Grey coloured), and its respective GFP expression.
we inserted a GC base pair in the first stem region, where it shows drop in the expression
level of GFP. We also deleted one base pair from the stem, which showed a similar
expression level compared to the wild-type switch.
Future Goals
The results we have till now are the fuel to carry out further studies. This study has a huge
potential in divulging the core of the regulation mechanisms. Further, we will be trying to
understand the switch behaviour in the presence and absence of drug molecules, followed by
deciphering the conformational dynamics of this RNA.
References:
1. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial
resistance in 2019: A systematic analysis. The Lancet 2022, 399 (10325), 629–655.
[Link]
2. Darby, E. M.; Trampari, E.; Siasat, P.; et al. Molecular mechanisms of antibiotic
resistance revisited. Nat. Rev. Microbiol. 2023, 21 (4), 280–295.
[Link]
3. Van Eldere, J. Multidrug-resistant Staphylococcus aureus. Mayo Clin. Proc. 2012, 87
(7), 713–715. [Link]
4. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol.
Biol. 1983, 166 (4), 557–580. [Link]
5. Zheng, L.; Baumann, U.; Reymond, J.-L. An efficient one-step site-directed and site-
saturation mutagenesis protocol. Nucleic Acids Res. 2004, 32 (14), e115.
[Link]
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