Gives an overview of induction of gens of antimicrobials

1. Submitted By: Samrah Nadeem
Reg. No: 2021-ag-1794
Class: MPhil Biochemistry
(2nd
–M)
Course: Plant Biochemistry
Induction of Gene
of Antimicrobial
Compounds

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Contents
Induction of Gene of Antimicrobial Compounds ........................................................................... 3
Antimicrobial mode of action:........................................................................................................ 3
Mechanisms of resistance ............................................................................................................... 5
(I) Limiting drug uptake: .......................................................................................................... 5
(II) Modification of drug targets............................................................................................. 6
(III) Drug inactivation.............................................................................................................. 7
(IV) Drug efflux....................................................................................................................... 7
Molecular and Cellular Mechanisms of Antimicrobial Action: ..................................................... 8
Genetic Basis of Antimicrobial Resistance................................................................................... 10
1. Mutational Resistance........................................................................................................ 10
2. Horizontal Gene Transfer .................................................................................................. 11

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Induction of Gene of Antimicrobial Compounds
Antimicrobial genes are found in all classes of life. The Bacillus subtilis and Escherichia coli as
target indicator bacteria and transformed them with cDNA libraries. Among thousands of
expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator
bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external
effect from antimicrobial proteins working from outside of the cells) and the high throughput
ability of screening. The B. subtilis is more efficient and reliable than E. coli. Using the B.
subtilis expression system, identified 19 novel, broad-spectrum antimicrobial genes. Proteins
expressed by these genes were extracted and tested, exhibiting strong external antibacterial,
antifungal and nematicidal activities. Furthermore, the newly isolated proteins could control
plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could
inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for
clinical medicine, as they exhibited no hemolytic activity.
Multiple-resistant ‘superbug’ bacteria have the ability to change targets and reduce permeability
against antibiotics by using the phenomena of mutations, whereas antimicrobial peptides often
do not have specific targets and are capable of disrupting the cell membranes, which gives them
durable effects on pathogens and makes them be considered as ideal candidates for clinical
exploitations. Antimicrobial peptides are available not only against human pathogens but also as
promising alternatives for the prevention of plant diseases. Plant resistance can be improved by
spraying antimicrobial peptides on the surface of plants or by creating transgenes. As a defense
response against the invasion of pathogens, plants upregulate a series of genes as part of their
innate immune response. Among them, some genes exhibit a broad range of antimicrobial
activities. In addition to affecting the microbial community directly, some genes can regulate the
innate immune responses of the host and play indirect roles against pathogens. Antimicrobial
peptides encoded by these pathogen infection upregulated host genes are relatively small (<60
amino acids) and have the following advantages: broad antimicrobial spectrum, fast killing, and
potential to be used alone or in combination. These unique features have encouraged researchers
to establish methods for the discovery of new classes of AMPs and their mimics from different
organisms over the last two decades.
Antimicrobial mode of action:
Antimicrobial agents can be divided into groups based on the mechanism of antimicrobial
activity. The main groups are:
a. Agents that inhibit cell wall synthesis
b. Depolarize the cell membrane
c. Inhibit protein synthesis
d. Inhibit nuclei acid synthesis
e. Inhibit metabolic pathways in bacteria.

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Factors that have contributed to the growing
resistance problem include: increased
consumption of antimicrobial drugs, both by
humans and animals; and improper
prescribing of antimicrobial therapy.
Antimicrobial groups based on mechanism of action.
Mechanism of Action Antimicrobial Groups
Inhibit Cell Wall Synthesis β-Lactams
Carbapenems
Cephalosporins
Monobactams
Penicillins
Glycopeptides
Depolarize Cell Membrane Lipopeptides
Inhibit Protein Synthesis Bind to 30S Ribosomal Subunit
Aminoglycosides
Tetracyclines
Bind to 50S Ribosomal Subunit
Chloramphenicol
Lincosamides
Macrolides
Oxazolidinones
Streptogramins
Inhibit Nucleic Acid Synthesis Quinolones
Fluoroquinolones
Inhibit Metabolic Pathways Sulfonamides
Trimethoprim
Disruption and increased permeability of
cytoplasmic membrane
Polymyxins, daptomycin

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Mechanisms of resistance
Antimicrobial resistance mechanisms fall into four main categories:
(1) Limiting uptake of a drug
(2) Modifying a drug target
(3) Inactivating a drug
(4) Active drug efflux.
Intrinsic resistance may make use of
limiting uptake, drug inactivation,
and drug efflux; acquired resistance
mechanisms used may be drug target
modification, drug inactivation, and
drug efflux. Because of differences in
structure, etc., there is variation in the
types of mechanisms used by gram
negative bacteria versus gram
positive bacteria. Gram negative
bacteria make use of all four main
mechanisms, whereas gram positive
bacteria less commonly use limiting
the uptake of a drug (don’t have an LPS outer membrane), and don’t have the capacity for certain
types of drug efflux mechanisms.
(I) Limiting drug uptake:
 There is a natural difference in the ability of bacteria to limit the uptake of
antimicrobial agents. The structure and functions of the LPS layer in gram negative
bacteria provides a barrier to certain types of molecules.
 Certain bacteria modify their cell membrane porin channels thereby preventing the
antimicrobials from entering the cell (Genetics).
 There are two main ways in which porin changes can limit drug uptake:
 A decrease in the number of prorins present.
 Mutations that change the selectivity of porin channel.
Gram positive bacteria like Staphylococcus aureus, developed resistance to vancomycin. Of the
two mechanisms that S. aureus uses against vancomycin, a yet unexplained mechanism allows
the bacteria to produce a thickened cell wall which makes it difficult for the drug to enter the
cell, and provides an intermediate resistance to vancomycin. These strains are designated as
VISA strains.

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(II) Modification of drug targets
There are multiple components in the bacterial cell that may be targets of antimicrobial
agents; and there are just as many targets that may be modified by the bacteria to enable
resistance to those drugs. One mechanism of resistance to the β-lactam drugs used almost
exclusively by gram positive bacteria is via alterations in the structure and/or number of
PBPs (penicillin-binding proteins). PBPs are trans-peptidases involved in the construction of
peptidoglycan in the cell wall.
A change in the number (increase in PBPs that have a decrease in drug binding ability, or
decrease in PBPs with normal drug binding) of PBPs impacts the amount of drug that can bind to
that target. A change in structure (e.g. PBP2a in S. aureus by acquisition of the mecA gene) may
decrease the ability of the drug to bind, or totally inhibit drug binding.
The glycopeptides (e.g. vancomycin) also work by inhibiting cell wall synthesis, and
lipopeptides (e.g. daptomycin) work by depolarizing the cell membrane. Gram negative bacteria
(thick LPS layer) have intrinsic resistance to these drugs.
Resistance to vancomycin has become a major issue in the enterococci (VRE—vancomycin-
resistant enterococci) and in Staphylococcus aureus (MRSA). Resistance is mediated through
acquisition of van genes which results in changes in the structure of peptidoglycan precursors
that cause a decrease in the binding ability of vancomycin.
Resistance to drugs that target the ribosomal subunits may occur via ribosomal mutation
(aminoglycosides, oxazolidinones), ribosomal subunit methylation (aminoglycosides, macrolides
gram positive bacteria, oxazolidinones, streptogramins) most commonly involving erm genes, or
ribosomal protection (tetracyclines). These mechanisms interfere with the ability of the drug to
bind to the ribosome. The level of drug interference varies greatly among these mechanisms.

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(III) Drug inactivation
There are two main ways in which bacteria inactivate drugs;
 By actual degradation of the drug - The β-lactamases are a very large group of
drug hydrolyzing enzymes. Another drug that can be inactivated by hydrolyzation
is tetracycline, via the tetX gene.
 By transfer of a chemical group to the drug - Drug inactivation by transfer of a
chemical group to the drug most commonly uses transfer of acetyl, phosphoryl,
and adenyl groups.
There are a large number of transferases that have been identified. Acetylation is the most
diversely used mechanism, and is known to be used against the aminoglycosides,
chloramphenicol, the streptogramins, and the fluoroquinolones. Phosphorylation and adenylation
are known to be used primarily against the aminoglycosides.
(IV) Drug efflux
Bacteria possess
chromosomally encoded genes for
efflux pumps. Some are expressed
constitutively, and others are
induced or overexpressed (high-
level resistance is usually via a
mutation that modifies the
transport channel) under certain
environmental stimuli or when a
suitable substrate is present. The
efflux pumps function primarily to
rid the bacterial cell of toxic
substances, and many of these
pumps will transport a large
variety of compounds (multi-drug
[MDR] efflux pumps).There are five main families of efflux pumps in bacteria classified based
on structure and energy source:
 The ATP-binding cassette (ABC) family
 The multidrug and toxic compound extrusion (MATE) family
 The small multidrug resistance (SMR) family
 The major facilitator superfamily (MFS)
 The resistance-nodulation-cell division (RND) family.

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Efflux pumps found in gram positive bacteria may confer intrinsic resistance because of being
encoded on the chromosome. These pumps include members of the MATE and MFS families
and efflux fluoroquinolones. There are also gram positive efflux pumps known to be carried on
plasmids. Efflux pumps found in gram negative bacteria are widely distributed and may come
from all five of the families, with the most clinically significant pumps belonging to the RND
family.
Molecular and Cellular Mechanisms of Antimicrobial Action:
The antimicrobial agents in current clinical use may be classified into four major categories, for
they may either
(1) Impede replication of genetic information.
(2) Impair translation of genetic information into protein synthesis.
(3) Alter structure and function of cell wall.
(4) Restrict function of cell membrane.
Classification of Antimicrobial Agents in Current Use According to Molecular
Mechanism of Action

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 Agents That Impede Replication of Genetic Information.
Nalidixic acid and griseofulvin are structurally related to the purine nucleotides. Both
have been shown to block DNA synthesis in sensitive microorganisms.8, 19 Although their
precise locus of action is unknown, their structural similarity to purines suggests that they
may inhibit DNA replication at the level of assembly of the purine nucleotides.
 Agents That Impair Translation of Genetic Information.
These drugs either inhibit protein synthesis (chloramphenicol, the tetracyclines,
erythromycin and lincomycin) or induce formation of defective protein molecules
(kanamycin, neomycin and streptomycin). The former are bacteriostatic, the latter
bactericidal.
Chloramphenicol has been to abolish protein synthesis in bacterial cells. That this effect
is not limited to microorganisms has also been well recognized. The universality of inhibition
of protein synthesis by chloramphenicol readily accounts for its major toxicity in man-
impairment of hemoglobin synthesis. Chloramphenicol has no effect upon cell synthesis of
mucopeptides, respiration or permeability.
The aminoglycoside antibiotics, streptomycin, kanamycin and neomycin, appear to act by
similar mechanisms. These drugs produce specific misreading in the genetic code at the level
of the ribosome. After their attachment to the ribosome, they appear to permit incorporation
of one or more incorrect amino acids into a growing peptide chain, resulting in synthesis of
defective proteins. Since these drugs are bactericidal, it may be reasoned either that these
defective proteins are lethal to the cell or that the deficiency in normal proteins created by
their synthesis leads to failure of one or more vital metabolic functions of the cell.
Convincing evidence in support of either of these hypotheses has not been presented.
 Agents That Alter Structure and Function of the Bacterial Cell
The antimicrobial agents known to affect bacterial cell walls adversely are vancomycin,
ristocetin, bacitracin, cycloserine, penicillins and cephalothin and its analogues. These drugs
are bactericidal for sensitive cells.
Cycloserine is a structural analogue of n-alanine e of the substrates for the penta-peptide
side chain within the cell wall. This drug appears to prevent assembly of the side chain
containing n-alanine by competitively inhibiting one or both of the enzymes alanine
racemase or n-alanyl-n-alanine synthetase. Vancomycin, ristocetin and bacitracin appear to
inhibit the action of the enzyme glycopeptide synthetase, which is responsible for
condensation of the glycopeptide backbone of the cell wall. Penicillin and cephalothin
prevent cross-linking (transpeptidation) of the glycopeptide. Thus each of the antibiotics in
this group is capable of weakening the physical support of the cell. This may lead to rupture

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and dissolution of the cell if the osmotic pressure of the environment does not approximate
that of the cell.
 Agents That Restrict Function of the Cell Membrane
The cell or plasma membrane of bacteria serves two major functions, one bio-energetic
and the other partitioning. Two infrequently used antimicrobial agents, gramicidin and
tyrocidin, uncouple oxidative phosphorylation and decrease respiration, with leakage of
amino acids from the cell. Polymyxin B and colistin (polymyxin E) act as cationic detergents
with affinity for phosphate radicals and thereby alter the osmotic barrier function of the cell
membrane.
Consequently there is a release of amino acids, purines and pyrimidines from the cell, and
dilution of the substrates available for synthetic processes. The polyene antifungal agents,
amphotericin B and nystatin, destroy the osmotic barrier activity of the plasma membrane by
binding to a sterol, present only in sensitive cells. This binding presumably reorients the
lamellar structure of the membrane. The lysis of human erythrocytes observed clinically
during use of amphotericin B appears also to result from binding of this drug to sterol groups
on the surface of the red cell.
Genetic Basis of Antimicrobial Resistance
Bacteria have a remarkable genetic plasticity that allows them to respond to a wide array of
environmental threats, including the presence of antibiotic molecules that may jeopardize
their existence. As mentioned, bacteria sharing the same ecological niche with antimicrobial-
producing organisms have evolved ancient mechanisms to withstand the effect of the harmful
antibiotic molecule and, consequently, their intrinsic resistance permits them to thrive in its
presence. From an evolutionary perspective, bacteria use two major genetic strategies to
adapt to the antibiotic “attack”, i) mutations in gene(s) often associated with the mechanism
of action of the compound, and ii) acquisition of foreign DNA coding for resistance
determinants through horizontal gene transfer (HGT).
1. Mutational Resistance
A subset of bacterial cells from a susceptible population develops genetic mutations
affecting the activity of the drug, resulting in preserved cell survival in the presence of
the antimicrobial molecule. Once a resistant mutant emerges, the antibiotic eliminates the
susceptible population and the resistant bacteria predominate. In general, mutations
resulting in antimicrobial resistance alter the antibiotic action via one of the following
mechanisms,
i. Modifications of the antimicrobial target (decreasing the affinity for the
drug)

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ii. A decrease in the drug uptake,
iii. Activation of efflux mechanisms to extrude the harmful molecule
iv. Global changes in important metabolic pathways via modulation of
regulatory networks.
2. Horizontal Gene Transfer
Acquisition of foreign DNA material through HGT is one of the most important
drivers of bacterial evolution and it is frequently responsible for the development of
antimicrobial resistance. Most antimicrobial agents used in clinical practice are (or derive
from) products naturally found in the environment (mostly soil). Bacteria sharing the
environment with these molecules harbor intrinsic genetic determinants of resistance and
there is robust evidence suggesting that such “environmental resistome” is a prolific
source for the acquisition of antibiotic resistance genes in clinically relevant bacteria.
Classically, bacteria acquire external genetic material through three main strategies,
i) Transformation (incorporation of naked DNA)
ii) Transduction (phage mediated)
iii) Conjugation (bacterial “sex”).
Finally, one of the most efficient mechanisms for accumulating antimicrobial resistance genes is
represented by integrons, which are site-specific recombination systems capable of recruiting
open reading frames in the form of mobile gene cassettes. Integrons provide an efficient and
rather simple mechanism for the addition of new genes into bacterial chromosomes, along with
the necessary machinery to ensure their expression; a robust strategy of genetic interchange and
one of the main drivers of bacterial evolution.