Download Metabolism-Antibiotic Sensitivity

Previous Lecture Syllabus Next Lecture
THE BACTERIA
Metabolism-Antibiotic
Sensitivity
MM17-27, 121-131
Table of Contents








Educational Objectives
Microbial Metabolism Overview
Bacterial Cell Wall Biosynthesis
Cytoplasmic Membrane
DNA Replication
Protein Synthesis
Competitive Antagonistic Anitbiotics
Summary
Educational Objectives
General
1. To explore the relationship between bacterial metabolism and susceptibility to
anti-bacterial agents, both physical
and chemical
2.
To define the mode of action of antibiotics
Specific educational objectives (terms and concepts upon which
you will be tested)








Aminoglycoside antibiotics
Antibiotic mode of action
-lactam antibiotics
Cell wall inhibitors
Competitive antagonistic antibiotics
Macrolide antibiotics
Protein synthesis inhibitors
Quinolone antibiotics
Microbial Metabolism as Related to Sensitivity
to Antibiotics-Overview
Many metabolic activities of the bacterial cell differ significantly from those in the
human cell. At least theoretically these differences can be exploited in the
development of chemotherapeutic agents. Ideally, an antimicrobial agent should have
its maximal effect on the bacterial cell and have little or no effect on the human cell. In
reality there is almost always some effect on the human be it induction of
hypersensitivity or liver or kidney toxicity. Despite some adverse reactions in the
human, effective antibiotics have been developed that have one ore more of these
modes of action on the bacterial cell:
A.
Inhibition of cell wall synthesis
B.
Alteration of cell membranes
C.
Inhibition of protein synthesis
D.
Inhibition of nucleic acid synthesis
E.
Antimetabolic activity or competitive antagonism
Bacterial Cell Wall Biosynthesis
Since bacteria have a cell wall made up of repeating units of peptidoglycan and human
cells lack this feature, it would seem that the bacterial cell wall presents an ideal target
for chemotherapy. Indeed, this has been the case; the following antibiotics have been
developed as inhibitors of cell wall synthesis:
A. -lactam antibiotics
1.
Penicillins
Penicillin G
Penicillin V
Oxacillin
Nafcillin
Ampicillin
Ticarcillin
Methicillin
2.
Amoxicillin
Carbenicillin
Cloxaciillin
Dicloxacillin
Piperacillin
Cephalosporins
First Generation
Generation
Second Generation
Fourth Generation
Third
Cefadroxil *
Cefaclor *
Cefdinir
Cefazolin
Cefamandole
Cefoperaxone
Cefelixin *
Cefonicid
Cefotaxime
Cephalothin
Ceforanide
Ceftazidime
Cephaprin
Cefotetan
Ceftibuten
Cephradine *
Cefoxitin
Ceftizoxime
Cefuroxime
Ceftriaxone
Cefepime
* Oral Agent
3.
Monobactams
4.
Thienamycins
5. -lactamase inhibitors (e.g., clavulanic acid)
B.
Cycloserine, Ethionamide, Isoniazid
C.
Fosfomycin (Phosphonomycin)
D.
Vancomycin
E.
Bacitracin
F.
Ristocetin
G.
Fosphomycin (Phosphonomycin)
The biosynthesis of peptidoglycan consists of three stages, each of which occurs at a
different site in the cell.
Stage 1 occurs in the cytoplasm. In this stage the recurring units of the backbone
structure of murein, N-acetylglucosamine and N-acetyl-muramylpentapeptide are
synthesized in the form of their uracil diphosphate (UDP) derivatives. The only
antibiotic that affects this stage of cell wall metabolism is D-cycloserine. D-cycloserine
is a structural analog of D-alanine; it binds to the substrate binding site of two
enzymes, thus being extremely effective in preventing D-alanine from being
incorporated into the N-acetylmuramylpeptide.
Structural relationship between cycloserine (left) and D-ala-nine (right).
Stage 2 of peptidoglycan synthesis occurs on the inner surface of the cytoplasmic
membrane where N-cetylmuramylpeptide is transferred from UDP to a carrier lipid and
is then modified to form a complete nascent peptidoglycan subunit. The nature of the
modification depends upon the organism. This stage terminates with translocation of
the completed subunit to the exterior of the cytoplasmic membrane. The only antibiotic
that affects this stage of cell wall synthesis is bacitracin. Bacitracin is an inhibitor of
the lipid phosphatase.
Bacitracin A. One of a group of polypeptide antibiotics containing a thiazoline ring
structure.
Stage 3 occurs in the periplasmic space (in gram-negative bacteria) and in the growing
peptidoglycan of the cell wall. This is a complex metabolic sequence which offers
multiple targets for chemotherapeutic agents. The earliest acting of these are
vancomycin and ristocetin. They act by binding to the D-alanyl-D-alanine peptide
termini of the nascent peptidoglycan-lipid carrier. This inhibits the enzyme
transglycosylase.
Stage 3 of biosynthesis continues with transpeptidation and the binding of soluble
uncrosslinked, nascent peptidoglycan to the preexisting, crosslinked, insoluble cell
wall peptidoglycan matrix. The -lactam antibiotics are structural analogs of the Dalanyl-D-alanine end of the peptidoglycan strand. In the cell wall there are as many as
seven enzymes (depending on the bacterial species) which bind peptidoglycan units
via their D-alanyl-D-alanine residues. The -lactams fill these substrate binding sites
and thus prevent the binding of D-alanyl-D-alanine residues. Enzymes binding -lactam
antibiotics are known as penicillin-binding proteins.
The Cytoplasmic Membrane as the Site of
Antibiotic Action
The cytoplasmic membrane of bacteria is only affected by two clinically-used
antibiotics. These are polymyxin B and polymyxin E (colistin). They act by
competitively replacing Mg2+ and Ca2+ from negatively charged phosphate groups on
membrane lipids. The result is disruption of the membrane.
DNA Replication as the Site of Antimicrobic Action
The major group of antibacterial agents that act by blocking DNA synthesis/activity is
the quinolone group.
Metronidazole represents as an antibiotic active against DNA in a different way. This
antibiotic, upon being partially reduced, causes the fragmentation of DNA in an, as yet,
undefined way. The antibiotic is only effective against anaerobic bacteria and some
parasites.
The quinolones all act by blocking the A subunit of DNA gyrase and inducing the
formation of a relaxation complex analogue.
DNA gyrase introduces negative superhelical turns into duplex DNA, using the energy
of ATP. This is the crucial enzyme that maintains the negative superhelical tension of
the bacterial chromosome.
The sign-inversion mechanism for DNA gyrase.
The quinolones include:
nalidixic acid - first generation
norfloxacin, ciprofloxacin - second generation
Protein Synthesis as the Site of Antimicrobic
Action
Protein synthesis is the end result of two major processes, transcription and
translation. An antibiotic that inhibits either of these will inhibit protein synthesis.
Transcription
During transcription, the genetic information in DNA is transferred to a complementary
sequence of RNA nucleotides by the DNA-dependent RNA polymerase. This enzyme is
composed of 5 subunits, ß, ß', a, a' and . Antibiotics that either alter the structure of
the template DNA or inhibit the RNA polymerase will interfere with the synthesis of
RNA, and consequently with protein synthesis.
Actinomycin D binds to guanine in DNA, distorting the DNA, and thus blocking
transcription.
Rifampin (Rifampicin or Rifamycin) inhibits protein synthesis by selective inhibiting
the DNA-dependent RNA polymerase. It does this by binding to the ß subunit in a noncovalent fashion.
Translation
In bacterial cells, the translation of mRNA into protein can be divided into three major
phases: initiation, elongation, and termination of the peptide chain. Protein synthesis
starts with the association of mRNA, a 30S ribosomal subunit, and formyl-methionyltransfer RNA (fMet-tRNA) to form a 30S initiation complex. The formation of this
complex also requires guanosine triphosphate (GTP) and the participation of three
protein initiation factors. The codon AUG is the initiation signal in mRNA and is
recognized by the anticodon of fMet-tRNA. A 50S ribosomal subunit is subsequently
added to form a 70S initiation complex, and the bound GTP is hydrolyzed.
In the elongation phase of protein synthesis, amino acids are added one at a time to a
growing polypeptide in a sequence dictated by mRNA. It is this phase that is most
susceptible to inhibition by a number of antibiotics. For many of these the ribosome is
the target site. There are two binding sites on the ribosome, the P (peptidyl or donor
site) and the A (aminoacyl) site. At the end of the initiation stage, the fMet-tRNA
molecule is empty. In the first step of the elongation cycle, an aminoacyl-tRNA is
inserted into the vacant A site on the ribosome. The particular species inserted
depends on the mRNA codon that is positioned in the A site. Protein elongation factors
and GTP are required for polypeptide chain elongation.
In the next step of the elongation phase, the formylmethionyl residue of the fMet-tRNA
located at the peptidyl donor site is released from its linkage to tRNA, and is joined
with a peptide bond to the -amino group of the aminoacyl-tRNA in the acceptor site to
form a dipeptidyl-tRNA. The enzyme catalyzing this peptide formation is peptidyl
transferase, which is part of the 50S ribosomal subunit.
Following the formation of a peptide bond, an uncharged tRNA occupies the P site,
whereas a dipeptidyl tRNA occupies the A site. The final phase of the elongation cycle
is translocation, catalyzed by elongation factor EF-G and requiring GTP. It consists of
three movements:
(1)
the removal of the discharged tRNA from the P site
(2)
the movement of fMet-aminoacyl-tRNA from the acceptor site to
the peptidyl donor site
(3)
the movement or translocation of the ribosome along the mRNA
from the 5' toward the 3' terminus by the length of three
nucleotides.
After translocation, the stage is prepared for the binding of the next aminoacyl residue
to the fMet-aminoacyl-tRNA, each addition requiring aminoacyl-tRNA binding, peptide
bond formation, and translocation. Peptidyl-tRNAa replace the fMet-tRNA in the second
and in all subsequent cycles.
The polypeptide chain grows from the amino terminal toward the carboxyl terminal
amino acid and remains linked to tRNA and bound to the mRNA-ribosome complex
during elongation of the chain. When completed it is released during chain termination.
Termination is triggered when a chain termination signal (UAA, UAG, or UGA) is
encountered at the A site of the ribosome. Protein release factors bind to the
terminator codons triggering hydrolysis by the peptidyl transferase. The polypeptide is
released, and the messenger-ribosome-tRNA complex dissociates.
Several medically important antibiotics owe their selective antimicrobial action to a
specific attack on the 70S ribosome of bacteria, with mammalian 80S ribosomes left
unaffected. Those that act on the 30S ribosome are:






Amikacin
Gentamycin
Kanamycin
Neomycin
Streptomycin
Tobramycin
Macrolides:
Azithromycin
Clarithromycin
Dirithromycin
Erythromycin
Antibiotics that act on the 50S portion of the ribosome include:










Chloramphenicol
Clindamycin
Furadantin
Fusidic acid
Lincomycin
Nitrofuran
Puromycin
Quinopristin/Dalfopristin
Spectinomycin
Tetracycline
Lincomycin
Linezolid
Clindamycin
Puromycin
Competitive Antagonistic Antibiotics
Inhibitors of metabolic pathways via competitive antagonism include:
Isoniazid - Inhibits mycolic acid synthesis
Sulfonamides - Inhibit folic acid biosynthesis
Trimethoprim - Inhibit folic acid biosynthesis
Summary
1. Antibiotics that are active against the cell wall of bacteria include the -lactams,
cycloserine, ethionamide,
isoniazid, phosphomycin, vancomycin, bacitracin and ristocetin.
2. The -lactam antibiotics are related structurally in that they all contain a -lactam
ring. These are the penicillins, cephalosporins, monobactams and thienamycins. They
are all analogs of d-alanyl-d-alanine.
3. Antibiotics that are active against the bacterial cytoplasmic membrane are
polymyxin B and E (colistin).
4. Antibiotics that are active against bacterial DNA are the quinolones (nalidixic acid,
norfloxacin and ciprofloxacin), which inhibit DNA gyrase, and metronidazole, which
fragments DNA.
5.
Antibiotics that block transcription in bacteria are actinomycin D and rifampin.
6. Antibiotics that block translation in bacteria by binding to the 30S ribosome are the
aminoglycosides, nitrofurans, spectinomycin and the tetracyclines.
7. The aminoglycoside antibiotics are related structurally in that they all contain a
unique aminocyclitol ring
structure. These include amikacin, gentamycin, kanamycin, neomycin,
streptomycin and tobramycin.
8. Antibiotics that block translation by binding to the 50S ribosome include
chloramphenicol, erythromycin,
clarithromycin, lincomycin, clindomycin, puromycin, fusidic acid and
quinopristin/dalfopristin.
9. The macrolide antibiotics are related structurally in that they all contain a
macrocyclic lactone ring of 12-22 carbon atoms, to which one or more sugars are
attached. These include erythromycin, clarithromycin, azithrmycin and dirithromycin.
10. Antibiotics that act by inhibiting folic acid biosynthesis include the sulfonamides
and trimethoprim.
Previous Lecture Syllabus Next Lecture