Julian Davies Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver BC V6T 1Z3, Canada
1.1. Introduction 1 1.2. Mode of Action 5 1.3. Resistance and Aminoglycoside Evolution 7 1.4. Toxicity 9 1.5. Conclusions and Comments 10 Acknowledgments 10 References 11
1.1. INTRODUCTION
Although streptomycin was not the first antibiotic (penicillin, a fungal product, had been isolated some years earlier), its discovery was a landmark in antibiotic history. It was the first effective therapeutic for tuberculosis, a disease that had terrorized humans for centuries and a cause of human morbidity and mortality unmatched by wars or any other pestilence. Streptomycin was the first amino-glycoside to be identified and characterized and is noteworthy in being the first useful antibiotic isolated from a bacterial source. At the present time, the use of streptomycin in infectious disease therapy has largely been replaced by less toxic and equally effective compounds, but it still has significant applications as a second-line treatment for TB and occasionally for the treatment of nosocomial multidrug-resistant gram-positive infections. Streptomycin's preeminent place in the history of antibiotics is assured!
Selman Waksman's commitment to the isolation and screening of soil bacteria in the search for bioactive small molecules, especially potential antibiotics, was validated by the discovery of streptomycin. This led to the creation of the modern biopharmaceutical industry and the subsequent isolation of tens of thousands of bioactive small molecules from soil bacteria and other environments. A proportion of these compounds have become highly successful therapeutics, not only for all types of infectious diseases, but also in the treatment of many other human and animal ailments and as anticancer, immuno-modulatory, and cardiovascular agents. Waksman and Fleming could be considered the fathers of chemical biology (Figure 1.1).
Following on the discovery of streptomycin and its streptamine-based relatives (Figure 1.2), a new generation of the aminoglycosides derived from 2-deoxystreptamine (DOS) was not long in coming (Figure 1.3). For a variety of reasons, many of these compounds have not been employed as human therapeutics; for example, neomycin has rarely been used in the clinic because of its extreme toxicity. Surprisingly, paromomycin, a naturally occurring 6'-desaminoderivative of neomycin, is receiving increasing interest in the treatment of a variety of tropical diseases, including leishmaniasis and certain types of fungal infection. This serves to illustrate that the aminoglycosides (and the related aminocyclitols, such as spectinomycin) have a broad range of biological activities and have found use in a wide variety of applications as indicated in Table 1.1. In addition to these compounds, there is a large group of atypical aminoglycosides, compounds that are of diverse microbial origin, structure, and biological activity (Table 1.2).
Many applications of the aminoglycosides have been of historical significance in genetics and microbiology. For example, mutations to streptomycin resistance were employed as counterselective genetic markers in the historic experiments of William Hayes that demonstrated the existence of bacterial conjugation and the requirement of donor (Hfr or F+) and receptor (F-) species. These experiments showed that conjugal gene transfer occurs with directional polarity and led to the subsequent characterization of sex factors that were ultimately shown to be extrachromosomal DNA elements, or plasmids. The finding by Ruth Sager that streptomycin interferes with chlorophyll production in Chlamydomonas and that high-level streptomycin resistance mutants exhibit cytoplasmic rather than Mendelian inheritance (due to alteration of the chloroplast genome) provided evidence in support of the bacterial (endosymbiotic) origin of chloroplasts in Chlamydomonas and plants. In the early 1970s, kanamycin resistance (encoded by a resistance plasmid) was used as a dominant selective genetic marker for heterologous gene transfer in the seminal recombinant DNA studies of Herbert Boyer, Stanley Cohen, and their colleagues. The later observation that kanamycin and certain other aminoglycosides have inhibitory activity against some types of eukaryotic cells led to their application in the genetic manipulation in higher organisms, including plants. In particular, two antibiotics have been widely used for eukaryotic gene cloning: G-418 (Geneticin[TM]) and hygromycin B. G-418, related to gentamicin, has become the preferred selective agent for mammalian cell studies, and large amounts of the compound are currently employed for this purpose. The neomycin phosphotransferase gene was the first bacterial gene approved in tests of human gene therapy in 1982 and remains the genetic marker of choice for all types of eukaryotic cloning.
1.2. MODE OF ACTION
The biochemical mode of action of the aminoglycosides as antibacterials has long been a topic of great interest. Early experiments carried out soon after the introduction of streptomycin suggested a variety of modes of action, but these conclusions were based largely on symptomatic analyses of antibiotic-treated bacterial cultures. One important experiment done in 1948 showed that streptomycin blocks enzyme induction in susceptible bacteria; this was the closest that anyone came to identifying the mechanism of action at the time.
A series of genetic and biochemical studies in the late 1950s and early 1960s led to the definitive identification of protein synthesis as the primary target for the antibacterial action of streptomycin. Initially, Erdos and Ullmann employed the incorporation of radioactive amino acids to show that production of labeled protein by cell-free extracts of Mycobacterium tuberculosis is effectively blocked by streptomycin. The results of these experiments were subsequently confirmed by others, using defined cell-free translation systems from other bacteria and with synthetic polynucleotides as messenger RNAs. A seminal paper by Spotts and Stanier proposed the ribosome as the probable target for streptomycin action and came up with a plausible biochemical mechanism for the phenomenon of streptomycin dependence. Within the next few years (1962-1965) a flurry of research activity in a number of laboratories confirmed this model, and in vitro translation studies employing hybrids of sensitive and resistant ribosome subunits showed that streptomycin acts by binding to the 30S ribosome subunit. This led to the investigation of the effects of streptomycin and other aminoglycosides on coding fidelity during translation, providing evidence for the active role of the 30S sub-unit in protein synthesis and the important finding that streptomycin and other aminoglycosides induce errors in translation. These studies provided the first evidence that the ribosome is not simply an inert support in the process of peptide bond formation but plays an active role in the selection of aminoacylated tRNAs by the ribosome-bound messenger RNA.
Current work on the three-dimensional structure of ribosome complexes has amply confirmed the dynamic role of the ribosome in translation and the mechanism by which this process is perturbed by the binding of aminoglycosides to specific sites on the 30S subunit. There is now strong genetic and phenotypic evidence for translation misreading by aminoglycosides in living cells. While it has been shown that a number of other translation inhibitors also provoke mistranslation, this may be a symptom of protein synthesis inhibition and not a direct effect on codon reading, as with the aminoglycoside antibiotics. Surprisingly, in the presence of some aminoglycosides, DNA can be accurately read as a messenger on the ribosome and to generate polypeptides in vitro; it is not known if this occurs in vivo. Parenthetically, the ability of aminoglycosides to cause mistranslation has itself been applied recently to an "indirect" form of gene therapy; the administration of gentamicin or related compounds to patients with hereditary diseases such as severe hemophilia or cystic fibrosis can result in partial suppression of the disease. Aminoglycoside-induced read-through of nonsense mutations leads to the production of small amounts of the missing protein and prevents nonsense-mediated decay of messenger RNA.
As is the case with most antibiotics, at subinhibitory concentrations the aminoglycosides induce significant changes in the transcription of some 5% of the genes in susceptible bacteria. The mechanism responsible is not known but may be due to some form of coupling between translation and transcription not previously identified. We can assume that transcription modulation is associated with antibiotic activity in therapeutic use and may contribute to some of the side effects. On the other hand, at low concentrations in the environment, the aminoglycosides and other antibiotics may be acting as cell-signaling molecules. The use of streptomycin or spectinomycin resistance as a genetic marker was critical to the cloning and identification of the gene clusters encoding structural elements of the ribosome in bacteria. Once it had been demonstrated that resistance to streptomycin and spectinomycin is associated with amino acid changes in ribosomal proteins, bacteriophage P1 transduction studies showed that the associated genes are linked in clusters on the bacterial chromosome. Masayasu Nomura and others then used disruption and reconstitution of ribosome particles from 16S rRNA and isolated R proteins to demonstrate the roles of the proteins RpsL (str) and RpsE (spc) in the determination of antibiotic resistance; this confirmed the earlier genetic and phenotypic studies and ratified the role of R proteins in ribosome function. There followed a decade of argument as to the relative importance of ribosomal RNA versus ribosomal proteins in the structure and function of the particle, and a paradigm change occurred when it was shown by numerous sequence and functional studies that the two major rRNA molecules are the structural basis of ribosome function in translation. The fact that these RNA molecules are the targets for the binding and interaction of different antibiotics on the ribosome, resulting in interruption of the translation process, provides strong confirmation of their roles in translation. The spectacularly successful rRNA footprinting studies and X-ray structure analyses carried out by the groups of Noller, Ramakrishnan, and others have amply confirmed this dominant role of rRNA and the consequences of antibiotic binding to the ribosome, initially with the aminoglycosides but subsequently with most ribosomal inhibitors. However, although the primordial template for peptide bond formation is likely to have been RNA alone, the involvement of both RNA and protein is essential in the dynamic role of the "modern" ribosome in translation; this is a topic of continuing interest. To date, it is only in the case of streptomycin that three-dimensional structure analysis of the antibiotic/ribosome complex identifies an interaction of the drug with both R proteins and rRNA. There is increasing evidence for the existence of nonribosomal functions of the protein components of the ribosome. Studies using antibiotics such as the aminoglycosides will undoubtedly continue to play important roles in developing this story.
During these years of exciting revelations concerning aminoglycoside activity and the ribosome, one question relative to the therapeutic use of aminoglycosides has remained unsolved. Unlike most antibiotic inhibitors of protein synthesis in bacteria that lead to bacteriostasis, the aminoglycosides are rapidly bactericidal. The ability of the aminoglycosides to kill bacterial pathogens is an important attribute in their therapeutic use. This action is somewhat surprising when we consider that most inhibitors of ribosome function act in a similar fashion to the aminoglycosides, by binding to target sequences within the 16S or 23S rRNAs (as described above). For example, the aminocyclitol spectinomycin is bactericidal in action. The difference between cidal and static action has been the topic of much discussion and many publications; this work has been largely physiological in nature, and a satisfactory biochemical explanation for the lethal action of the aminoglycosides still eludes us. The possibility that aminoglycosides (as distinct from other translation inhibitors) induce a process of programmed cell death (apoptosis) in bacteria could provide an explanation.
1.3. RESISTANCE AND AMINOGLYCOSIDE EVOLUTION
Antibiotic resistance (both endogenous and acquired) is an important determining factor in the historical development of the aminoglycosides as therapeutic agents. After streptomycin was introduced for the treatment of tuberculosis, it was found that bacterial resistance to the drug often developed; this was shown to be due to spontaneous mutants arising during the course of therapy with the antibiotic, although the biochemical mechanism was not known at the time. Kanamycin, the first useful DOS aminoglycoside, was isolated in Japan in 1957 and rapidly became an antibiotic of choice in that country. However, the appearance of strains resistant to both streptomycin and kanamycin increasingly interfered with their therapeutic use; in addition, hospital infections of Pseudomonas aeruginosa, a bacterium that is naturally less susceptible to antibiotics, were on the rise. A major breakthrough came with the discovery of a novel class of 2-DOS compounds, the gentamicins. These are extremely effective antibiotics with good activity against the pseudomonads and other problem pathogens, such as Proteus and Serratia species, that were being increasingly encountered as nosocomial infections. Gentamicin and related compounds lack the 3' OH group, and the absence eliminates the modification by phosphorylation at this site and confers activity against pathogens possessing aminoglycoside 3' OH phosphotransferases; gentamicin, being a mixture, contains one component with a modified 6' amino group and has reasonable potency against strains harboring plasmid-encoded 6' acetytransferases that inactivate kanamycin. By this time it was known that resistance to antibiotics by enzymic modification could be acquired by plasmid transfer. Gentamicin was also effective for the treatment of staphylococcal and enterococcal infections, frequently being used in combination with a ß-lactam antibiotic in these circumstances.
In spite of its nephrotoxicity, gentamicin was the treatment of choice for gram-negative nosocomial infections for many years, and its success led to the introduction of tobramycin, a related compound. However, novel antibiotic resistance mechanisms began to appear on the scene; of particular concern was the adenylylation of the 2' OH of gentamicin and related compounds that appeared on the scene in 1971 and conferred high-level resistance to the newest generation of aminoglycosides. The increasing, worldwide use of different aminoglycosides led to the appearance of many different types of resistant strains; the local use of specific classes of aminoglycoside often led to the selection of distinct local classes of resistance. (Continues...)
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