Bernard Weisblum

Erythromycin resistance by ribosome modification.

Erythromycin inhibits protein synthesis by its effect on ribosome function (14, 118, 119). The metabolic modifications that enable cells to cope with the inhibitory action of erythromycin fall under major headings that include (i) target site alteration, (ii) antibiotic modification, and (iii) altered antibiotic transport. This minireview concentrates on target site alteration, which for erythromycin is the 50S subunit of the ribosome. The first clinical isolates of macrolide-resistant staphylococci were described in reports from France, England, Japan, and the United States shortly after the introduction of erythromycin into clinical practice in 1953. On the basis of current understanding of the biochemistry of erythromycin’s action, resistance in most of the strains that were described in early reports can be ascribed to a posttranscriptional modification of the 23S rRNA by an adenine-specific N-methyltransferase (methylase) specified by a class of genes bearing the name erm (erythromycin ribosome methylation). The last decade has seen the isolation and characterization of approximately 30 erm genes from diverse sources, ranging from clinical pathogens to actinomycetes that produce antibiotics; for many of these genes, both the respective nucleotide sequences that encode the methylases as well as the flanking sequences that control their expression have been determined. A tabulation of the erm genes that have been described is presented in Table 1. Any discussion of mechanisms of resistance to macrolide antibiotics must include the chemically distinct, but functionally overlapping, lincosamide and streptogramin B families as well. This type of resistance has therefore also been referred to as MLS resistance. Members of the MLS antibiotic superfamily include, among the macrolides, carbomycin, clarithromycin, erythromycin, josamycin, midecamycin, mycinamicin, niddamycin, rosaramicin, roxithromycin, spiramycin, and tylosin; among the lincosamides, celesticetin, clindamycin, and lincomycin; and among the streptogramins, staphylomycin S, streptogramin B, and vernamycin B. The streptogramin family is subdivided into A and B groups or alternatively into M and S groups, respectively. Methylation of A2058 confers resistance to the Band S-group streptogramins but not to the Aand M-group streptogramins. The reason for this grouping was originally based on empirical observations from clinical bacteriology that resistance to one class often involved resistance to the other two classes (11, 16, 35, 39, 41, 135); however, (i) the three classes of antibiotics interact competitively when binding to the 50S subunit, and only one antibiotic molecule can bind per 50S subunit (129, 130); this suggests that the binding sites for these antibiotics overlap or at least functionally interact. (ii) Nucleotide alterations in 23S rRNA, both mutational and posttranscriptional, that confer coresistance to MLS antibiotics appear to cluster in the peptidyltransferase region in 23S rRNA domain V, providing a physical basis and a common location for their sites of action (50, 101–104, 109, 110, 128) (Fig. 1 and Table 2), and (iii) footprinting experiments show that the nucleotides in 23S rRNA domain V are protected by bound MLS antibiotics against modification by agents such as dimethyl sulfate (DMS) and kethoxal that can derivatize purine and pyrimidine bases in single-stranded DNA or RNA (26, 76) (Table 3). The erm family of genes is not alone in conferring clinical resistance to macrolide antibiotics. A notable early exception to the established MLS resistance pattern was the MS pattern reported by Janosy and coworkers (58, 59), who described clinical isolates that were coresistant to erythromycin and streptogramin B but that remained susceptible to lincosamide antibiotics. The molecular basis for resistance in these strains was subsequently shown by Ross et al. (94) to involve the active efflux of erythromycin and streptogramin B but not clindamycin. Additional mechanisms of macrolide resistance, all associated with the acquisition of new genetic information, including structural modification of erythromycin by phosphorylation (82), glycosylation (60), and lactone ring cleavage by erythromycin esterase (2, 83), have been added to the list. Mechanisms involving mutational alteration of genes that normally reside in the host and that encode either ribosomal protein or rRNA have also been described and will be discussed below in detail. Reviews of erythromycin resistance that relate to material covered in the present work have been presented previously (4, 18, 20, 21, 28, 29, 133). Recent developments in the synthetic chemistry of semisynthetic macrolides, including the biological and clinical aspects of their actions, have been reviewed by Kirst (65, 66). A forthcoming review covers the inducible nature of MLS resistance and its implications for the mechanism of action of erythromycin (134).

Antibiotics: Non-haemolytic β-amino-acid oligomers

Pathogenic bacteria are becoming increasingly resistant to common antibiotics, stimulating an intensive search for new ones. Knowing that a class of medium-sized peptides (magainins) are widely used by host organisms as a defence against microbial invasion, we developed a β-amino-acid oligomer (β-peptide) that mimics these natural antibiotics and tested it for antimicrobial activity. We find not only that the activity of our β-peptide is comparable to that of a magainin derivative but also that it is effective against four bacterial species, including two pathogens that are resistant to common antibiotics.

Insights into erythromycin action from studies of its activity as inducer of resistance.

Appreciation of the inducibility of erythromycin resistance began as an observation in the clinical bacteriology laboratory during susceptibility testing of erythromycin-resistant clinical isolates of Staphylococcus aureus. It was noted that inhibition zones surrounding spiramycin, lincomycin, and pristinamycin I (streptogramin B family) test disks placed close to an erythromycin test disk deviated from the expected circular shape and assumed a distorted ‘‘D’’ shape instead. Such observations suggested a possible antagonistic interaction between erythromycin, on the one hand, and spiramycin, lincomycin, or pristinamycin I, on the other (5, 7). The interaction turned out to be a functional rather than a physical antagonism, and out of these observations grew the notion of erythromycin-inducible resistance toward erythromycin, initially (41, 55), and then, more generally, toward all the macrolide, lincosamide, and streptogramin type B (MLS) antibiotics (57). Vazquez (50) and Vazquez and Monro (52) showed that antibiotics belonging to each subclass of the MLS antibiotics competed with chloramphenicol for uptake by intact cells. Competition for binding to purified 50S ribosome subunits was shown only for macrolides and lincosamides but not for streptogramin type B antibiotics. Collectively, these observations suggested that an alteration of 50S subunit function was involved in resistant cells. In a study of the time and concentration dependence of induction, Weisblum et al. (58) showed that (i) the optimal erythromycin concentration for induction was between 10 and 100 ng/ml, the threshold of its inhibitory action, (ii) at the optimal inducing concentration of erythromycin cells became phenotypically resistant within 40 min, and (iii) ribosomes from induced cells apparently bound labeled erythromycin and lincomycin with a reduced affinity. By mixing ribosome preparations from susceptible and resistant cells and noting no loss of expected antibiotic binding activity, it was possible to exclude the alteration of the antibiotic by modifying enzymes present as contaminants in the ribosome preparation. Consistent with this picture, Allen (1) showed that cell extracts of resistant S. aureus carried out erythromycin-resistant protein synthesis in vitro, suggesting that a component of the protein-synthesizing machinery had been altered. A posttranscriptional methylation of a single adenine residue in 23S rRNA, comprising the induced biochemical alteration (30, 31), was located at Escherichia coli coordinate 2058 (A-2058) (47), and translational attenuation (16, 24), the mechanism for its regulation, was proposed. As discussed below, this unusual mechanism of gene regulation requires no repressor proteins but, instead, is based on the conformational isomerization of the ermC message to a translationally active form. How might this be achieved?

Fluorometric properties of the bibenzimidazole derivative hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA

A new fluorescent probe of chromosomal DNA structure in situ, the bibenzimidazole derivative Hoechst 33258, shows enhanced fluorescence with both AT- and GC-rich DNA; however, enhancement by AT-rich DNA is greater than enhancement with GC-rich DNA. When this compound is used as a probe, it produces localized fluorescence which can be correlated with AT concentration in specific chromosome regions. By the use of 33258, Hilwig and Gropp (1972) were able to demonstrate the relatively AT-rich DNA present in centric regions of mouse chromosomes; these regions do not fluoresce brightly when treated with quinacrine because of the presence of guanine residues which are spaced with high periodicity and which therefore efficiently quench quinacrine fluorescence. The data obtained in this study with DNA polymers of defined structure or composition, as test model compounds, suggest that 33258 is a useful cytochemical reagent for generally identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine.

Structure−activity Relationships among Random Nylon-3 Copolymers That Mimic Antibacterial Host-Defense Peptides

Host-defense peptides are natural antibiotics produced by multicellular organisms to ward off bacterial infection. Since the discovery of these molecules in the 1980s, a great deal of effort has been devoted to elucidating their mechanisms of action and to developing analogues with improved properties for possible therapeutic use. The vast majority of this effort has focused on materials composed of a single type of molecule, most commonly a peptide with a specific sequence of alpha-amino acid residues. We have recently shown that sequence-random nylon-3 copolymers can mimic favorable properties of host-defense peptides, and here we document structure-activity relationships in this polymer family. Although the polymers are heterogeneous in terms of subunit order and stereochemistry, these materials display structure-activity relationships comparable to those that have been documented among host-defense peptides and analogous synthetic peptides. Previously such relationships have been interpreted in terms of a specific and regular folding pattern (usually an alpha-helix), but our findings show that these correlations between covalent structure and biological activity do not require the adoption of a specific or regular conformation. In some cases our observations suggest alternative interpretations of results obtained with discrete peptides.

Regulation of Iron Transport in Streptococcus pneumoniae by RitR, an Orphan Response Regulator

RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H(2)O(2) resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR(+)) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H(2)O(2) resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.

N-methyl transferase of Streptomyces erythraeus that confers resistance to the macrolidelincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci

The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.

Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus☆

Abstract Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.

Conformational alterations in the ermC transcript in vivo during induction.

ermC is an inducible antibiotic resistance gene from Staphylococcus aureus, one of several whose expression is regulated at the level of mRNA secondary structure. During induction of ermC, the inhibition of a ribosome active in translation of a short leader peptide by low levels of antibiotic belonging to the macrolide‐lincosamide‐streptogramin b family is believed to cause a rearrangement in mRNA secondary structure. The resultant conformational isomerization unmasks the methylase ribosome binding site and initiator Met codon, causing increased translation of the ermC transcript. Expression of ermC can also be demonstrated in Bacillus subtilis carrying plasmid pE194. To probe the ermC transcript in vivo during induction, ermC was transferred to B. subtilis by transformation and the resultant transformants were treated with dimethyl sulfate which reacts with N‐1 of adenine and N‐3 of cytosine residues in a manner that is sensitive to secondary structure. The bases modified in vivo were detected by primer extension with reverse transcriptase using total cellular RNA as template and a complementary ermC‐specific oligonucleotide as primer. Physical evidence was obtained for the secondary structural rearrangements predicted by the ermC regulatory model. Additionally, physical evidence was obtained demonstrating that during induction, the stalled ribosome protects codons 9 and 10 of the leader peptide from modification by dimethyl sulfate, in agreement with genetic data obtained previously that identified the integrity of codons 5‐9 as critical for induction of ermC by erythromycin.

ermC leader peptide. Amino acid sequence critical for induction by translational attenuation.

The ermC mRNA leader segment, which encodes a 19 amino acid leader peptide, MGIFSIFVISTVHYQPNKK, plays a key role in regulating expression of the ErmC methylase. The contribution of specific leader peptide amino acid residues to induction of ermC was studied using a model system in which the ErmC methylase was translationally fused to Escherichia coli beta-galactosidase as indicator gene. Codons of the ermC leader peptide were altered systematically by replacement of leader DNA segments with double-stranded DNA constructed from chemically synthesized oligonucleotides. Missense mutations that resulted in reduced efficiency of induction involved codons for amino acid residues 5 to 9 (-SIFVI-). Nonsense mutations causing termination of the leader peptide at codons 10 (-S-) or 12 (-V-) remained inducible. These findings suggest that the codons for residues 5 to 9 of the leader peptide comprise the critical region in which ribosomes stall in the presence of erythromycin.