Lykke H. Hansen

A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid‐encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI‐TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr- strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center

ABSTRACT Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.

A click chemistry approach to Pleuromutilin conjugates with nucleosides or acyclic nucleoside derivatives and their binding to the bacterial ribosome

Pleuromutilin and its derivatives are antibacterial drugs that inhibit protein synthesis in bacteria by binding to ribosomes. To promote rational design of pleuromutilin based drugs, 19 pleuromutilin conjugates with different nucleoside fragments as side chain extensions were synthesized by a click chemistry protocol. Binding was assessed by chemical footprinting of nucleotide U2506 in 23S rRNA, and all conjugates bind to varying degree reflecting their binding affinity to the peptidyl transferase center. The side chain extensions also show various protections at position U2585. Docking studies of the conjugates with the highest affinities support the conclusion that despite the various conjugations, the pleuomutilin skeleton binds in the same binding pocket. The conjugated triazole moiety is well accommodated, and the nucleobases are placed in different pockets in the 50S ribosomal subunit. The derivative showing the highest affinity and a significantly better binding than pleuromutilin itself contains an adenine-9-ylpropylene triazole conjugate to pleuromutilin C-22.

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross‐resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

BackgroundDNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.ResultsWe investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1).ConclusionThe data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

The Order Bacillales Hosts Functional Homologs of the Worrisome cfr Antibiotic Resistance Gene

ABSTRACT The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisome cfr gene and functionally similar genes. Three cfr-like genes from the order Bacillales were identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced in Escherichia coli, and MICs for selected antibiotics indicate that the cfr-like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as the cfr gene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows that cfr-like genes exist in the environment and that Bacillales are natural residences of cfr-like genes.

A cfr-Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as the cfr Gene

ABSTRACT The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostridium difficile) strain does function as a Cfr protein. The enzyme is expressed in Escherichia coli and shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA.

A click chemistry approach to pleuromutilin derivatives, part 2: conjugates with acyclic nucleosides and their ribosomal binding and antibacterial activity.

Pleuromutilin is an antibiotic that binds to bacterial ribosomes and thereby inhibit protein synthesis. A new series of semisynthetic pleuromutilin derivatives were synthesized by a click chemistry strategy. Pleuromutilin was conjugated by different linkers to a nucleobase, nucleoside, or phenyl group, as a side-chain extension at the C22 position of pleuromutilin. The linkers were designed on the basis of the best linker from our first series of pleuromutilin derivatives following either conformational restriction or an isosteric methylene to oxygen exchange. The binding of the new compounds to the Escherichia coli ribosome was investigated by molecular modeling and chemical footprinting of nucleotide U2506, and it was found that all the derivatives bind to the specific site and most of them better than pleuromutilin itself. The effect of the side-chain extension was also explored by chemical footprinting of nucleotide U2585, and the results showed that all the compounds interact with this position to varying degrees. Derivatives with a conformational restriction of the linker generally had a higher affinity than derivatives with an isosteric exchange of one of the carbons in the linker with a hydrophilic oxygen. A growth inhibition assay with three different bacterial strains showed significant activity of several of the new compounds.