subject: Antibiotics and its action on the living cell [print this page] Antibiotics and its action on the living cell
ANTIBIOTICS
Subhas Chandra Roy and Rajesh Kumar Mohapatra
Utkal University, Bhubaneswar
Antibiotics (Greek anti, against, and bios, life)are microbial products or their derivatives that can kill susceptible microorganisms or inhibit their growth. The penicillins were the first antibiotics discovered in 1928 as natural products from the mold Penicillium in response to Staphylococcus aureus by Sir Alexander Fleming, professor of bacteriology at St. Mary's Hospital in London. A very large number of antibiotics have been discovered, but less than 1% has been of practical value in medicine.
Modes of action of antibiotics: Antibiotics are bacteriolytic(they kill and rupture microbial cells), bactericidal(they kill microbes directly) or bacteriostatic(they prevent microbial growth). However, the host's immune systems or phagocytic defenses must still complete the elimination of the invading microbes. Five different modes of action of antibiotics are discussed here:-
Inhibition of cell wall synthesis
Penicillins and Cephalosporins: Gram-positive bacteria possess a thick cell wall composed of peptidoglycan. The polysaccharide portion of the peptidoglycan is made of repeating units of N-acetylglucosamine linked by-1,4 to N-acetylmuramic acid. These molecules are joined by short tetrapeptides consisting of four amino acids. The sugars and tetrapeptides are cross-linked by the enzyme transpeptidase. -lactam antibiotics like penicillins and cephalosporins act at the level of the bacterial cell wall and interfere with the cross-linking of the peptidoglycan chains. The transpeptidases are capable of binding to antibiotics with the -lactam ring. They bind very tightly at the active site of the transpeptidase enzyme by reacting with a serine residue in the transpeptidase and can no longer catalyze the transpeptidase reaction. The cell wall continues to be formed but is no longer cross-linked and becomes progressively weaker as the peptidoglycan backbone is laid down. In addition, the antibiotic-transpeptidase complex stimulates the release of autolysins that digest the existing cell wall. Bacterial cells, especially Gram- positive ones, have a high internal osmotic pressure. Without a normal sturdy cell wall, these cells burst when subjected to the low osmotic pressure of body fluids. Vancomycin and Bacitracin: Vancomycin and bacitracin inhibit cell wall synthesis at an earlier stage than penicillins and cephalosporins. Vancomycin inhibits synthesis and assembly of the peptidoglycan polymers. Its peptide portion blocks the transpeptidation reaction by binding specifically to the D-alanine-D-alanine terminal sequence on the pentapeptide portion of the precursors of the peptidoglycan.Bacitracin inhibits the synthesis of cell walls by interfering with the synthesis of linear strands of peptidoglycans. It inhibits cell wall production by blocking the step in the process (recycling of the membrane lipid carrier Undecaprenol phosphate) which is needed to add on new cell wall subunits.
The inhibition of protein synthesis
Inhibition of peptide bond formation:Antibiotics like chloramphenicol block elongation by inhibiting the formation of peptide bond in the growing polypeptide chain. They react with the 50S subunit of the 70S prokaryotic ribosome subunit and inhibit peptidyl transferase, stopping the binding of new amino acids to the nascent polypeptide chain.They have good coverage of most gram-positive and gram-negative bacteria including anaerobes.
Inhibition of translocation: Antibiotics like erythromycin binds to a specific site on the 23S r RNA of 50S ribosomal subunit and blocks elongation by interfering with the translocation step (movement of ribosome along m RNA).
Inhibition of the binding of aminoacyl t RNA: Antibiotics like tetracycline react with the 30S subunit of the 70S prokaryotic ribosome. The tetracyclines interfere with the attachment of the aminoacyl t RNA carrying the amino acids to the ribosomes, preventing the addition of amino acids to the growing polypeptide chain and thereby blocking continued translation.
Inhibition of the normal pairing between aminoacyl t RNAs and message codons:Antibiotics like streptomycin interfere with the normal pairing between aminoacyl t RNAs and message codons by changing the shape of the 30S subunit of 70S ribosome. This causes the genetic code on the m RNA to be read incorrectly and thereby producing aberrant proteins. Streptomycin causes misreading at relatively low concentration and inhibits initiation at higher concentration.
Inhibition of the proper chain termination:Antibiotics like puromycin causes premature chain termination. A portion of the puromycin molecule resembles the 3' end of the aminoacyl t RNA, so that it can bind to the ribosomal A site and participate in peptide bond formation. Because puromycin resembles only the 3' end of t RNA, it dissociates from the ribosome shortly after it is linked to the c-terminus of the growing polypeptide chain, causing premature chain release.
Disruption of cell membrane function: Antibiotics like polymixin B, valinomycin, gramicidin A etc. bring about changes in the permeability of the plasma membrane.
Gramicidin A is a 15-residue polypeptide, containing alternating L- and D- amino acids. It acts as a cation-specific ion pore, allowing a breakdown in the unequal ratio of K+ and Na+ normally maintained between the inside and outside of living cells. An open pore forms through the plasma membrane when two gramicidin molecules line up to form an end-to-end dimer. K+ ions and, to a lesser extent, Na+ ions can then pass through the channel. Valinomycin is a polypeptide involving three repeats of the sequence (D-valine)-(L-lactate)-(L-valine)-(D-hydroxyisovalerate). Its outer surface (rich in CH3) is hydrophobic, making the molecule soluble in the lipid bilayer, whereas the inside mimics in some ways the hydration shell that the cation would have in aqueous solution. The dimensions of the interior cavity nicely accommodate a K+ ion but do not fit other cations as well. So valinomycin can diffuse to one surface, pick up an ion, and then diffuse to the other surface and release it, this increases the solubility of the ion in the membrane.
Inhibition of nucleic acid synthesis:A number of antibiotics interfere with the process of DNA replication and transcription in microbes. These drugs are not as selectively toxic as other antibiotics because prokaryotes and eukaryotes do not differ greatly with respect to nucleic acid synthesis.
Inhibition of DNA synthesis:Quinolonesact by inhibiting the bacterial DNA gyrase or topoisomerase II. DNA gyrase introduces negative twist in DNA and helps separate its strands. Inhibition of DNA gyrase disrupts DNA replication and repair, bacterial chromosome separation during division, and other cell processes involving DNA.
Inhibition of RNA synthesis:Two related antibiotics rifamycin B and its semi synthetic derivative rifampicin specially inhibit transcription in prokaryotes. Rifampicin inhibits bacterial RNA synthesis by binding to the subunit of RNA polymerase, preventing the promoter clearance step of transcription. Initiation is not completed until several phosphodiester bonds have formed. Rifampicin blocks initiation by preventing the formation of these bonds. The inactivated RNA polymerase remains bound to the promoter thereby block initiation.
Inhibition of both DNA and RNA synthesis:ActinomycinD tightly binds to duplex DNA and strongly inhibits both DNA replication and transcription by interfering with the passage of DNA and RNA polymerases. Its phenoxazone ring system intercalates between the DNA's successive GC base pairs. Actinomycin's chemically identical cyclic depsipeptides extend in opposite directions from the intercalation site along the minor groove of DNA.
Inhibition of synthesis of essential metabolites: Antimetabolites are substances that affect utilization of metabolites and therefore prevent a cell from carrying out necessary metabolic reactions.
Sulfonamidesare the first antimetabolites to be used successfully as chemotherapeutic agents. Sulfanilamide or other sulfa drugs are an analogue of p-aminobenzoic acid or PABA which is used in the synthesis of the cofactor folic acid (folate). When sulfonamides enter a bacterial cell, they compete with PABA for the active site of an enzyme involving in folic acid synthesis, causing a decline in folate concentration. As folic acid is a precursor of purines and pyrimidines which are used in construction of DNA and RNA, the resulting inhibition of purine and pyrimidine synthesis leads to the inhibition of protein synthesis and DNA replication, thus the pathogen dies. Trimethoprim is a synthetic antibiotic that also interferes with the production of folic acid by binding to dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolic acid to tetrahydrofolic acid, competing against the dihydrofolic acid substrate. It can be combined with sulfa drugs to increase efficiency of treatment by blocking two key steps in the folic acid pathway.
Antibiotic resistance: When bacteria are subjected to antibiotics, sometimes they acquire antibiotic resistance and this antibiotic resistance spreads within a bacterial population. Genes for antibiotic resistance may be present on bacterial chromosomes, plasmids, transposons and integrons. Spontaneous mutations in the bacterial chromosome followed by natural selection can make bacteria antibiotic resistant. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion by horizontal gene transfer (HGT). There are three possible mechanisms of HGT; these are transduction, transformation and conjugation.
Types of resistance: Alteration of the antibiotic's target sites: The affinity of ribosomes for erythromycin and chloramphenicol decreases in some bacteria by a change in the 23S r RNA to which they bind. They methylate their ribosomes obscuring the target of antibiotics. Enterococci become resistant to vancomycin (VRE- vancomycin-resistant enterococcus) by changing the terminal D-alanine-D-alanine in their peptidoglycan to a D-alanine-D-lactate.
Degradation of antibiotics:Penicillin G is enzymatically deactivated in some penicillin-resistant bacteria (e.g. Staphylococcus aureus, Streptococcus pneumoniae etc.) through the production of -lactamases (penicillinase). -lactamases cause hydrolysis of the -lactam ring of penicillins and thereby inactivate the antibiotic.
Antibiotic inactivation or modification: Chloramphenicol contains two OH groups that is acetylated and inactivated in some microbes in a reaction catalyzed by chloramphenicol acyltransferase with acetyl CoA as the donor.
Reduced antibiotic accumulation and rapid efflux: Some bacteria have plasma membrane efflux pumps that expel antibiotics. These are relatively non-specific and are called multidrug-resistant pumps. Many are drug/proton antiporters- protons enter the cell and antibiotic leaves.
Alteration of metabolic pathway:Some sulfonamide-resistant bacteria do not require PABA for synthesis of folic acid and nucleic acids. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
Prevention of entry of antibiotics inside cell:Many gram-negative bacteria are unaffected by penicillin G because it cannot penetrate the envelope's outer membrane. Mycobacteria resist many antibiotics because of the high content of mycolic acids in a complex lipid layer outside their peptidoglycan. This layer is impermeable to most water-soluble antibiotics.
Antibiotics have been a much misused products, especially in less developed areas of the world. Antibiotics can almost universally be purchased without prescription in these countries. Extensive antibiotic treatment favors the development and spread of antibiotic-resistant strains because antibiotics destroy susceptible bacteria that would usually compete with drug-resistant strains. Chemotherapeutic antibiotics, particularly broad-spectrum antibiotics, should be used only when definitely necessary. If possible, the pathogen should be identified and the proper narrow-spectrum antibiotics employed. Finally, new chemotherapeutic agents are constantly being produced using several methods for discovering and designed new antibiotics.
