Antibiotics have highly effective bacteriostatic effects and are widely used in the prevention and treatment of human and animal diseases. The antibacterial mechanism of antibiotics mainly includes hindering the synthesis of bacterial cell wall, changing the permeability of cell membrane, inhibiting the activity of bacterial ribosome, RNA or other related enzymes. The clinical use of natural antibiotics is earlier, and the industrial production system is also becoming more and more perfect, which currently occupies a large market share. Natural antibiotics can be divided into tetracyclines, macrolides, β-lactams and aminoglycosides.
1. Tetracycline Antibiotics
Tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, which are mainly used for infectious diseases caused by Gram-positive and negative bacteria, intracellular mycoplasma, chlamydia and rickettsia.
Ribosomes are the sites of protein synthesis in all living cells and are composed of proteins and RNAs. Many studies have shown that tetracycline can bind to the RNA components of bacterial ribosomes to exert antibacterial activity. That is, the antibacterial mechanism of tetracycline antibiotics is that tetracyclines preferentially bind to bacterial ribosomes, and interact with the highly conserved 16S rRNA target in the 30S ribosomal subunit to form a reversible conjugate, which is charged by steric interference during the extension process. The aminoacyl tRNA binds to the mRNA-ribosome complex, preventing new amino acids from entering the new peptide chain to inhibit the synthesis of proteins necessary for bacterial growth and survival, thereby exerting an antibacterial effect.
Resistance Mechanisms of Tetracycline Antibiotics
Long-term use of tetracycline can cause bacteria to develop obvious resistance to these drugs. So far, 59 tetracycline resistance genes have been reported. They can mediate resistance to tetracyclines through two distinct mechanisms. One is the active efflux of tetracycline through the efflux pump. The most common tetracycline-specific efflux pumps are members of the major dissimilator superfamily of transporters. The latest statistics show that there are 30 different tetracycline-specific efflux pumps in bacteria. The efflux pump gene encodes a membrane-associated efflux protein, which can actively pump drugs out of the cell, resulting in a decrease in the intracellular drug concentration, reducing the binding of ribosomes to tetracyclines, resulting in drug resistance. The second is through the role of bacterial ribosome protection proteins. Tetracycline ribosomal protection proteins (RPPs), originally described in Campylobacter jejuni and Streptococcus, are GTPases with significant sequence and structural similarity to elongation factors EF-G and EF-Tu. Binding of RPPs to ribosomes can reverse the distorted ribosome structure, causing changes in ribosome configuration, directly interfering with the mutual stacking of tetracycline D-ring and 16S rRNA base C1054, so that tetracyclines can not bind to it and release from the binding site 30S dissociated on the subunit, thereby protecting the ribosome.
2. Aminoglycoside Antibiotics
Aminoglycosides are natural or semi-synthetic antibiotics extracted from actinomycetes. Since the first aminoglycoside antibiotic, streptomycin, was first discovered in 1943, it has been widely used in the treatment of infections caused by Gram-negative and Gram-positive bacteria due to its good antibacterial activity and broad antibacterial spectrum.
Aminoglycoside antibiotics are antibacterial drugs in the stationary phase, and can be used in combination with antibacterial drugs (β-lactam antibiotics) in the reproductive period for synergistic antibacterial. The main action site of aminoglycoside antibiotics is also the ribosome, which inhibits bacteria by preventing the initial stage, peptide-chain elongation stage and termination stage of bacterial protein synthesis. During the initiation of prokaryotic peptide chain synthesis, aminoglycoside antibiotics can bind to the 30S subunit and interfere with the assembly of the ribosome 30S initiation complex. During the stage of the peptide-chain elongation, aminoglycosides bind to the A-site of the 30S subunit, resulting in reading errors and abnormal protein synthesis. In the termination stage of peptide chain synthesis, aminoglycoside antibiotics can prevent stop codons from binding to ribosomes, further preventing the release of synthesized peptide chains, and inhibiting the dissociation of 70S ribosomes, preventing the synthesis cycle of proteins.
Resistance Mechanisms of Aminoglycoside Antibiotics
Aminoglycoside resistance occurs in many forms, including enzyme modification leading to inactivation of aminoglycoside antibiotics, changes in target sites, and reduced membrane permeability.
(1) Changes in cell membrane permeability may lead to reduced drug uptake and accumulation by bacteria, leading to bacterial drug resistance
The outer membrane of Gram-negative bacteria is a barrier to both hydrophobic and hydrophilic substances. To overcome this permeability barrier, these microbes have evolved pore proteins, which act as nonspecific entry and exit points for antibiotics and other small organic chemicals. When the numbers of these pore roteins are significantly reduced, bacteria develop drug resistance.
(2) Bacteria can produce aminoglycoside modifying enzymes that inactivate aminoglycoside antibiotics, leading to antibiotic resistance
There are three different types of aminoglycoside modifying enzymes, including aminoglycoside nucleoside transferases (ANTs), aminoglycoside acetyl transferases (AACs) and aminoglycoside phosphotransferases (APHs). Enzyme modification inactivation is the most common mechanism of bacterial resistance to aminoglycoside antibiotics. AMEs covalently binds to functional groups such as -OH or -NH2, which can interfere with the binding of antibiotics to site A in 16S rRNA, resulting in decreased affinity between antibiotics and ribonoyl-TrNA and inactivation of antibiotics.
(3) Ribosome is the target of aminoglycoside antibiotics, and rRNA plays a key role in the binding of antibiotics to ribosome
Because of target change, antibiotics can not bind to ribosomes. For example, plasmid-mediated 16S rRNA methyltransferases RMTs alter specific rRNA nucleotide residues to prevent aminoglycosides from binding effectively to their sites, allowing bacteria to develop high resistance to such drugs.
3. Macrolide Antibiotics
The typical structure of macrolide antibiotics is that the parent nucleus contains a macrolide ring. Macrolide antibiotics are mainly effective against gram-positive bacteria, but have limited effect on gram-negative bacteria. They have very low eukaryotic activity because they have a weak affinity for eukaryotic ribosomes. The mechanism was that macrolide antibiotics irreversibly bound to the bacterial ribosomal 50s subunit and took a place in the nascent peptide exit tunnel (NPET) near the peptide transferase center, inhibiting protein synthesis. After the macrolide is combined with NPET, it can dissociate the peptidyl tRNA from the ribosome during the elongation stage of the peptide chain, so that the ribosome bound to the macrolide can not polymerize the specific amino acid sequence in the nascent protein, thereby blocking transpeptidation and mRNA translocation process, thereby hindering the growth of peptide chains, inhibiting protein synthesis, and finally playing a bacteriostatic effect.
Resistance Mechanisms of Macrolide Antibiotics
At present, the resistance mechanism of macrolides antibiotics mainly includes two tpyes. One is ribosomal modification. Methyltransferase encoded by erythromycin resistant methyltransferase gene can catalyze monomethylation or dimethylation of N6 in nucleotide A2058. This nucleotide has a specific interaction with the carbohydrate located at the C5 position of the macrolactone ring, and methylation interferes with the formation of hydrogen bonds, resulting in a significant decrease in the affinity of the macrolide to the 50S subunit of the ribosomal, making generation of resistant strains. In addition to rRNA methylation, mutations in rRNA can also produce resistance. The A2058 mutation changes the ribosomal target and prevents macrolide binding. The second is reduced intracellular concentrations of macrolides. One way that bacteria can evade the effects of macrolides is by using an efflux pump to reduce intracellular concentrations. The Mef and Msr subfamilies of the efflux pump are closely related to macrolides, which encode on plasmids and are members of the MSF and ATP-binding cassette (ABC) families, respectively. Mef pumps act as reverse transporters, exchanging the bound macrolides with protons.
ATP acts as an energy source to pump antibiotics out of the cell, but utilizes secondary active transport, at which time the energy of ATP is not directly used for transmembrane transport of macrolides. This subfamily protein is one of the important determinants of macrolides drug resistance, Mef(A) and Mef(E) are the most common, both of which can lead to bacterial resistance to 14 and 15 macrolides. Msr proteins provide ribosomal protection by binding to macrolides.
4. β-lactam Antibiotics
β-lactam antibiotics are the most widely used antibiotics. All β-lactams contain a quaternary β-lactam, and in most cases there is also a five- or six-membered ring. According to the different ring structures, β-lactam antibiotics are β-lactam antibiotics are the most widely used antibiotics. All β-lactams contain a quaternary β-lactam, and in most cases there is also a five- or six-membered ring. According to the different ring structures, β-lactam antibiotics are further divided into penicillins, cephalosporins, and atypical β-lactams. β-lactam antibiotics with different structures have the same mechanism of action, and the mechanism of action is mainly that the drug inhibits the transpeptidation of transpeptidase, which leads to the blocking of bacterial cell wall synthesis and induces bacterial death.
Resistance Mechanisms of β-lactam Antibiotics
Peptidoglycan is a major component of bacterial cell walls, and this mesh-like polymer completely surrounds the cell and is the only protective mechanism for Gram-positive bacteria against internal osmotic pressure, enabling them to survive in a hypotonic environment. In recent years, researches have shown that bacterial cell membranes contain several special protein that can form relatively stable complexes with penicillin, known as penicillin-binding proteins (PBPs), which are the main target of β-lactam antibiotics. PBPs are essential enzymes for the synthesis of peptidoglycan, a key component of the cell wall. The structures of penicillin and cephalosporin are similar to the terminal structure of peptidoglycan, D-alanyl-D-alanine, which can competitively bind to the active center of the enzyme by covalent bond and inhibit the catalyzed mucopeptidase. The cross-linking reaction severely disrupts the formation of bacterial cell walls, thereby causing bacteriolysis, resulting in bacterial lysis and death. Mammalian cells have no cell wall and are not affected by this process, so the antibacterial effect of such antibiotics has high selectivity.
There are many complex mechanisms in bacteria that make them resistant to antibiotics. As with most antibacterial drugs, bacteria develop resistance to β-lactam antibiotics through three main mechanisms. Reduced expression or mutation of PBPs is an important mechanism of Gram-positive bacteria resistance to β-lactam antibiotics.
(1) Produce β-lactamase
The β-lactamase produced by bacteria can covalently bind to the carbonyl moiety of the antibiotic, disrupting its cyclic structure, causing the β-lactam antibiotic to be degraded before it reaches its target. β-lactamase can also rapidly and firmly bind β-lactam antibiotics non-hydrolytically, so that it can not enter the target.
(2) Changes of PBPs
PBPs are the targets of β-lactam antibiotics. When PBPs are genetically mutated, the affinity between β-lactam antibiotics and their target PBPs is lost, and the antibiotics can not bind to their action sites to exert their efficacy, which in turn leads to bacterial resistance.
(3) Changes in permeability of the outer membrane
The change of membrane permeability or the increase of efflux pump activity will prevent antibiotics from entering the bacteria, leading to a decrease in the binding of antibiotics to the target, thus reducing the active concentration of antibiotics in the bacteria.