THE BACTERIA Metabolism- Antibiotics

8/7/2019 THE BACTERIA Metabolism- Antibiotics

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THE BACTERIAMetabolism-Antibiotic Sensitivity

Table of Contents

Educational Objectives Microbial Metabolism Overview Bacterial Cell Wall Biosynthesis Cytoplasmic Membrane DNA Replication Protein Synthesis Competitive Antagonistic Anitbiotics Summary

Specific educational objectives (terms and concepts upon which you will be tested) Aminoglycoside antibiotics Antibiotic mode of action

b-lactam antibiotics

Cell wall inhibitors

Competitive antagonistic antibiotics

Macrolide antibiotics

Protein synthesis inhibitors

Quinolone antibiotics

Microbial Metabolism as Related to Sensitivity toAntibiotics-Overview

Many metabolic activities of the bacterial cell differ significantly from those in the humancell. At least theoretically these differences can be exploited in the development ofchemotherapeutic agents. Ideally, an antimicrobial agent should have its maximal effect onthe bacterial cell and have little or no effect on the human cell. In reality there is almostalways some effect on the human be it induction of hypersensitivity or liver or kidneytoxicity. Despite some adverse reactions in the human, effective antibiotics have beendeveloped that have one ore more of these modes of action on the bacterial cell: A. Inhibition of cell wall synthesisB. Alteration of cell membranesC. Inhibition of protein synthesisD. Inhibition of nucleic acid synthesisE. Antimetabolic activity or competitive antagonism
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Bacterial Cell Wall Biosynthesis

Since bacteria have a cell wall made up of repeating unitsof peptidoglycan and human cells lack this feature, itwould seem that the bacterial cell wall presents an idealtarget 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 Oxacillin Ampicillin Amoxicillin Cloxaciillin

Penicillin V Nafcillin Ticarcillin Carbenicillin Dicloxacill

Methicillin Piperacillin

2. Cephalosporins

First Generation Second Generation Third Generation FourthGeneration

Cefadroxil * Cefaclor * Cefdinir Cefepime

Cefazolin Cefamandole Cefoperaxone

Cefelixin * Cefonicid Cefotaxime Cephalothin Ceforanide Ceftazidime Cephaprin Cefotetan Ceftibuten

Cephradine * Cefoxitin Ceftizoxime Cefuroxime Ceftriaxone

* Oral Agent

3. Monobactams4. Thienamycins5. -lactamase inhibitors (e.g., clavulanic acid)

B. Cycloserine, Ethionamide, Isoniazid

C. Fosfomycin (Phosphonomycin)

D. Vancomycin

E. Bacitracin

F. Ristocetin

G. Fosphomycin (Phosphonomycin)


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The biosynthesis of peptidoglycan consists of three stages, each of which occurs at a different site inthe 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 uracildiphosphate (UDP) derivatives.

The only antibiotic that affects this stage of cell wall metabolism is D-cycloserine. D-cycloserine is astructural analog of D-alanine; it binds to the substrate binding site of two enzymes, thus beingextremely 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 completenascent 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 cytoplasmicmembrane. The only antibiotic that affects this stage of cell wall synthesis is bacitracin. Bacitracin is aninhibitor 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 ofthe cell wall. This is a complex metabolic sequence which offers multiple targets for chemotherapeuticagents. 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.


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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 b-lactam antibiotics are structural

analogs of the D-alanyl-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 b-lactams fill these substrate

binding sites and thus prevent the

binding of D-alanyl-D-alanine residues. Enzymes binding b-lactam antibiotics are known as penicillin-

binding proteins.


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The Cytoplasmic Membrane as the Site of Antibiotic Action

The cytoplasmic membrane of bacteria isonly affected by two clinically-usedantibiotics.

These are polymyxin B and polymyxin E(colistin).

They act by competitively replacing Mg2+

and Ca2+ from negatively chargedphosphate groups on membrane lipids. Theresult is disruption of the membrane.


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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 activeagainst DNA in a different way. This antibiotic,upon being partially reduced, causes thefragmentation of DNA in an, as yet, undefinedway. The antibiotic is only effective againstanaerobic bacteria and some parasites.

The quinolones all act by blocking the A subunitof DNA gyrase and inducing the formation of arelaxation complex analogue.

DNA gyrase introduces negative superhelicalturns into duplex DNA, using the energy of ATP.This is the crucial enzyme that maintains thenegative superhelical tension of the bacterialchromosome.

The sign-inversion mechanism for DNA gyrase.

The quinolones include:nalidixic acid - first generationnorfloxacin, ciprofloxacin - second generation


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Protein Synthesis as the Site of Antimicrobic Action

Protein synthesis is the end result of two major processes, transcription and translation. An antibiotic thatinhibits either of these will inhibit protein synthesis.TranscriptionDuring transcription, the genetic information inDNA is transferred to a complementarysequence of RNA nucleotides by the DNA-dependent RNA polymerase. This enzyme is

composed of 5 subunits, , ', a, a' and .

Antibiotics that either alter the structure of thetemplate DNA or inhibit the RNA polymerasewill interfere with the synthesis of RNA, andconsequently with protein synthesis.Actinomycin D binds to guanine in DNA,distorting the DNA, and thus blockingtranscription.

Rifampin (Rifampicin or Rifamycin) inhibitsprotein synthesis by selective inhibiting theDNA-dependent RNApolymerase. It does this by

binding to the subunit in a non-covalent fashion.

Translation

In bacterial cells, the translationof mRNA into protein can bedivided into three major phases:initiation, elongation, andtermination of the peptide chain.

Protein synthesis starts with theassociation of mRNA, a 30Sribosomal subunit, and formyl-methionyl-transfer RNA (fMet-tRNA) to form a 30S initiationcomplex. The formation of thiscomplex also requires guanosinetriphosphate (GTP) and the participation of three protein initiation factors. The codon AUG is the initiationsignal in mRNA and is recognized by the anticodon of fMet-tRNA. A 50S ribosomal subunit issubsequently added to form a 70S initiation complex, and the bound GTP is hydrolyzed.


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In the elongation phase of protein synthesis, amino acids are added one at a time to a growingpolypeptide in a sequence dictated by mRNA. It is this phase that is most susceptible to inhibition by anumber of antibiotics. For many of these the ribosome is the target site. There are two binding sites on theribosome, the P (peptidyl or donor site) and the A (aminoacyl) site. At the end of the initiation stage, thefMet-tRNA molecule is empty. In the first step of the elongation cycle, an aminoacyl-tRNA is inserted intothe vacant A site on the ribosome. The particular species inserted depends on the mRNA codon that ispositioned 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 -aminogroup of the aminoacyl-tRNA in the acceptor site to form a dipeptidyl-tRNA. The enzyme catalyzing thispeptide 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 dipeptidyltRNA occupies the A site. The final phase of the elongation cycle is translocation, catalyzed by elongationfactor 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 threenucleotides.

After translocation, the stage is prepared for the binding of the next aminoacyl residue to the fMet-aminoacyl-tRNA, each additionrequiring aminoacyl-tRNA binding,peptide bond formation, andtranslocation. Peptidyl-tRNAa replacethe fMet-tRNA in the second and in all

subsequent cycles.The polypeptide chain grows from theamino terminal toward the carboxylterminal amino acid and remains linkedto tRNA and bound to the mRNA-ribosome complex during elongation ofthe chain. When completed it isreleased during chain termination.

Termination is triggered when a chaintermination signal (UAA, UAG, or

UGA) is encountered at the A site ofthe ribosome.

Protein release factors bind to theterminator codons triggering hydrolysisby the peptidyl transferase. Thepolypeptide is released, and themessenger-ribosome-tRNA complexdissociates.


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Several medically important antibiotics owe their selective antimicrobial action to a specific attack on the70S ribosome of bacteria, with mammalian 80S ribosomes left unaffected. Those that act on the 30Sribosome are:

Amikacin

Gentamycin

Kanamycin

Neomycin

Streptomycin

Tobramycin


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Macrolides:Azithromycin DirithromycinClarithromycin Erythromycin

A


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Intibiotics that act on the 50S portion of the ribosome include: Chloramphenicol

Clindamycin

Furadantin

Fusidic acid

Lincomycin

Nitrofuran

Puromycin

Quinopristin/Dalfopristin

Spectinomycin

Tetracycline

Lincomycin Clindamycin


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Linezolid

Puromycin


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Competitive Antagonistic Antibiotics

Inhibitors of metabolic pathways via competitive antagonism include:Isoniazid - Inhibits mycolic acid synthesis


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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.


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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 ringstructure. 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.

THE BACTERIA Metabolism- Antibiotics

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