Patent Application
20100292137
The invention relates to the field of cyclic peptide antibiotics and to methods for producing and using cyclic peptides.
Each year about two million people die from tuberculosis (TB), and nine million people become newly infected with its etiological agent, Mycobacterium tuberculosis (Maher and Raviglione, 2005). Incomplete and inadequate antibiotic therapy has led to an increased prevalence of multidrug resistant TB (MDR-TB), which is defined as resistance to two or more first-line antibiotics including isoniazid and rifampicin (Dye et al., 2002). The fall-back position is to treat MDR-TB with second-line antibiotics such as capreomycin (Sharma and Mohan, 2004). Unfortunately, the efficacy of capreomycin is being undermined by its increasing use, which is driving up the frequency of M. tuberculosis strains that are resistant to the drug. The molecular mechanism of capreomycin action has received little attention in recent decades, and a better understanding of this compound is clearly desirable.
Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics. Capreomycin and viomycin are particularly active against mycobacteria, and both inhibit growth by blocking protein synthesis on the ribosome (Gale et al., 1981; Vázquez, 1979), although capreomycin is preferred as a therapeutic agent. The drugs interfere with several ribosomal functions including formation of the 30S subunit initiation complex (Liou and Tanaka, 1976) and tRNA translocation from the A to the P site (Modolell and Vázquez, 1977). No crystal structures, which might explain their mode of action, are available for capreomycin or viomycin bound to their ribosomal target. This contrasts with the wealth of structural information on other antibiotics that bind to a single main target situated either on the 30S or on the 50S ribosomal subunit (reviewed by Poehlsgaard and Douthwaite, 2005). Our current knowledge about capreomycin and viomycin binding has been gleaned from other approaches including drug competition (Misumi et al., 1978), ribosome reconstitution (Yamada et al., 1978), cross-resistance (Suzuki et al., 1998; Taniguchi et al., 1997), chemical footprinting (Moazed and Noller, 1987) and translation inhibition studies (Gale et al., 1981; Liou and Tanaka, 1976; Modolell and Vazquez, 1977; Vázquez, 1979).
The recent identification of several M. tuberculosis strains with an unusual type of resistance to capreomycin and viomycin (Maus et al., 2005a) provides a novel angle from which to investigate the mechanism of drug action. Resistance in these strains is conferred by inactivation of the previously uncharacterized tlyA gene. The tlyA phenotype is specific to capreomycin-viomycin with no cross-resistance to other drugs including aminoglycosides. Capreomycin and viomycin susceptibility could be re-established in a resistant tlyA-strain of M. tuberculosis and a non-tuberculous strain of Mycobacterium smegmatis by complementation with an active copy of tlyA (Maus et al., 2005a).
In the present invention, we among others defined the function of TlyA by analyzing the methylation patterns of rRNAs from drug sensitive and resistant strains of M. tuberculosis and M. smegmatis. We show that TlyA is a 2′-O-ribose methyltransferase, and modifies a cytidine on each ribosomal subunit. The 16S and 23S rRNA nucleosides that are methylated by this enzyme were pinpointed precisely using a combination of reverse transcriptase primer extension and Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The methylated riboses come into close proximity upon association of 30S and 50S subunits and possibly provide a hydrophobic surface that aids capreomycin-viomycin binding across the subunit interface. Methylation at these riboses is not common in other bacteria, and this is consistent with capreomycin and viomycin being particularly active against mycobacteria. The locations of the ribose methylations delineate the site of capreomycin-viomycin binding, and indicate how these drugs inhibit ribosome function.
The invention therefore in one aspect provides a method for determining whether a compound comprises antibiotic activity comprising providing a compound of formula I to a first bacterium that comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli, preferably said ribosomes further comprise 30S subunits wherein a 16S rRNA is not methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli, said method further comprising determining whether said compound inhibits growth of said first bacterium.
wherein R1 or R3 is a molecule such as a hydrogen, a hydroxy group, an aromatic group, an acylgroup, an amino acid (for example a lysine) or other amide-bond forming molecule, a polyketide, a glycosyl group, an alkyl group, a carbohydrate, a halogenated side chain, or a fatty acid, and R2 or R4 is a hydrogen, a methyl group, a hydroxy group, an acyl group. an amino acid or other amide-bond forming molecule, a fatty acid, a carbohydrate, or a polyketide.
In a preferred embodiment new compounds are generated by combining genes in the biosynthesis cluster for cyclic peptide antibiotics with non-ribosomal peptide synthetases (NRPS) derived from any microorganism (WO 2005/021586). An overview of such NRPS is presented Rausch et al. (Rausch et al., 2005). Example of a biosynthesis cluster for an NRPS for the synthesis of tuberactinomycin-type antibiotics (cyclic peptide antibiotics) is the vio cluster from Streptomyces vinaceus for viomycin biosynthesis (Thomas et al., 2003), and more preferably vioA, vioF, vioG, viol, vioM, and vioO.
Examples of NRPS are found in all actinomycetes, such as SCO7682 and SCO7683 in Streptomyces coelicolor, BAC71354 in Streptomyces avermitilis, PhsB in Streptomyces viridochromogenes, BAD55613, BAD59914, and BAD59915 in Nocardia farcinica, MAP3742 in Mycobacterium avium., AAQ17094-17097 in Amycolatopsis lactamdurans, CAJ34374 in Micromonospora ML1, and also mixed NRPS/polyketide synthases such as AAN85522 in Streptomyces atroolivaceus.
Other sources of NRPS clusters are bacteria, including Pseudomonas, Bacillus, Listeria, brevibacillus, Mesorhizobium, Nostoc, Ralstonia, Xantomonas, and preferably culturable and inculturable microbes from soil or marine origin, and fungi (e.g. Aspergillus and more preferably Aspergillus fumigatus; Gibberella, Hypocrea, Cochliobolus).
Also, adding precursor molecules to a fermentation process will alter antibiotic production (Gastaldo and Marinelli, 2003), and can as such be used for the production of new molecules that fit in the capreomycin or viomycin binding site at the interbridge between the two ribosomal subunits. In another embodiment, biologically synthesized variants can be chemically modified. In this way, a person skilled in the art can synthesize any desired new compound. It is part of the invention that the modifications should be such that they do not increase the size of the molecule to such extent that the antibiotic does no longer fit in the binding site defined in
Thus in a preferred embodiment the invention provides a method for determining whether a compound comprises antibiotic activity comprising providing a compound, as generated above and/or generated by other combinatorial approaches by manipulating the synthesis of cyclic peptides in bacteria, to a first bacterium that comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli and determining whether said compound inhibits growth of said first bacterium.
Compounds identified as having antibiotic activity using a method of the invention are typically useful as antibiotics for a large variety of different bacteria. Such compounds are particularly suited antibiotics for the treatment of mycobacterial infections that have developed resistance against capreomycin and/or viomycin.
All bacteria have ribosomes that consist of two subunits. In the present invention these subunits are referred to as the 30S and 50S subunit. These terms identify the subunits for the person skilled in art, although the actual sedimentation characteristics of the subunits in particular cases might be different from the numbers presented.
Antibiotic activity is herein defined as activity at a certain concentration of the antibiotic that inhibits the growth of a tested bacterium when compared to growth of said tested bacterium under comparable circumstances in the absence of said antibiotic. It is preferred that at said concentration, said antibiotic is not toxic to mammalian cells and/or does not inhibit the growth of mammalian cells. Preferably, said mammalian cells are human cells.
The means and methods of the invention and in particular antibiotics of the invention are in one embodiment selected using a first lower eukaryote instead of said first bacterium, and/or a second lower eukaryote instead of said second bacterium. It was found that lower eukaryotes comprise mitochondria that have a similar kind of sensitivity, not necessarily similar amount of sensitivity to the antibiotics of the invention as bacteria specified herein. As a result said mitochondria are at least less functional in the presence of an antibiotic of the invention, leading to at least a reduction in the fitness and/or growth of said lower eukaryote. Thus the invention further provides a use of an antibiotic of the invention for at least reducing the fitness an/or growth of said lower eukaryote. In a preferred embodiment said lower eukaryote comprises a fungus, preferably a yeast or a ascomycete. In another preferred embodiment said lower eukaryote comprises a helminth, preferably a trematode, a cestode or a nematode. Preferred trematodes are Clonorchis, Fasciola and Schistosoma. Preferred cestodes are Taenia and Echinococcus. Preferred nematodes are Trichinella, Strongyloides, Necator and Ancyclostoma. In another preferred embodiment said lower eukaryote comprises a protozoa. Preferred protozoa are Sarcomastigophora, Apicomplexa, Microspora or Ciliophora. Preferred Sarcomastigophora are Amoeba, Tripanosoma or Trichomonas. Preferred Apicomplexa are Plasmodium and Toxoplasma.
In the present invention it was found that bacteria that are normally not very sensitive towards capreomycin or viomycin, can be made more sensitive to the mentioned antibiotics by ensuring that the 23S rRNAs in their ribosomes are methylated at a nucleotide position that corresponds to nucleotide C1920 in Escherichia coli and preferably by also ensuring that their 16S rRNAs in their ribosomes are methylated at a nucleotide position that corresponds to nucleotide C1409 of E. coli. Although ribosomal RNAs are typically well conserved among bacteria, there is some sequence variation. The sequence variation is both in the exact position and the exact base that is present at said position. The exact position of the nucleotide is given relative to the position in E. coli. The corresponding position in another bacterium species can be found according to the E. coli rRNA numbering system (Cannone et al, 2002; Noller 1984) and is used throughout. Sequence variation also occurs in the actual base that is present in said position. In the present invention it is preferred that the base at either the position corresponding to position 23S rRNA C1920 in E. coli or at the position corresponding to position 16S rRNA C1409 in E. coli, or at both positions is a C.
As the present invention identified the three-dimensional spaces where capreomycin and viomycin interfere with the function of the ribosome it has rendered 3D modelling of the interaction of such compounds possible. This knowledge and the knowledge of the methylation dependency of the antibiotic activity can be used to design molecules that resemble the structure of capreomycin and viomycin but that interact differently with the ribosomal groups at that position. This 3D modelling can be used to design compounds with an even higher binding affinity in this position. Alternatively compounds can be produced that exhibit a lower minimum inhibitory concentration (MIC) value, when tested for their antibiotic activity. Thus the invention further provides a method for determining whether a compound inhibits growth of a bacterium, comprising creating a three-dimensional model of a compound and fitting said compound model in a space between the 50S and the 30S subunits where the helix 44 of the 16S rRNA in the 30S subunit meets helix 69 of the 23S rRNA in the 50S subunit in a three-dimensional model of a 30S and a 50S subunit of a ribosome, wherein said fitting comprises establishing interaction between residues of said compound and said subunits in said space, said method further comprises producing said compound or equivalent thereof and contacting said compound or equivalent in vitro with said bacterium, and determining whether growth of said bacterium is at least in part inhibited. Said space in said ribosome for fitting said compound is preferably defined by the nucleotides that surround it.
In a preferred embodiment said space is defined by nucleotides that are at least partially within or touch a sphere of 10 Ångstrom that surrounds the nucleotide that correspond to the position corresponding to position of C1920 in 23S rRNA in E. coli or the position corresponding to position C1409 16S rRNA in E. coli (as shown in
In a preferred embodiment said compound is a cyclic peptide, preferably a tuberactinomycin, a capreomycin, a viomycin or a derivative and/or combination thereof. Cyclic peptides are typically produced by bacteria. As not all bacteria produce all variants of cyclic peptides, it is also possible to produce an equivalent having similar interactions with the modeled ribosome.
A cyclic peptide compound of the invention to be tested in a method of the invention is preferably a compound that was not known for its antibiotic activity prior to the invention. A cyclic peptide compound of the invention to be tested in a method of the invention is preferably a compound that has previously not been tested for antibiotic activity towards mycobacteria.
The site of interaction of capreomycin and viomycin in the ribosome is in a space between the 50S and the 30S subunits where the helix 44 of the 16S rRNA in the 30S subunit meets helix 69 of the 23S rRNA in the 50S subunit (
A method of the invention can be used to find compounds that are more effective for bacteria that lack methylation at one or more of the indicated positions in the 16S and 23S rRNA. Thus in a preferred embodiment of the invention said bacterium comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Preferably said bacterium comprises ribosomes wherein a 16S rRNA in said 30S subunit is not methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli. More preferably said bacterium comprises no methylation at both of said positions.
Alternatively, a method of the invention is used to find compounds that are more effective for bacteria that have methylation at one or more of indicated positions in the 16S and 23S rRNA. Thus in another preferred embodiment of the invention said bacterium comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Preferably said bacterium comprises ribosomes wherein a 16S rRNA in said 30S subunit is methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli. More preferably said bacterium comprises methylation at both of said positions.
In the present invention it was surprisingly found that antibiotic activity of capreomycin and viomycin was dependent on methylation of two positions in rRNA whereas the binding stoichiometry of the antibiotic to a ribosome was close to or exactly 1:1. Thus one capreomycin or viomycin molecule had at least two interaction points with the ribosome when bound thereto. When one of the positions was methylated whereas the other position was not, antibiotic activity was decreased, albeit at a different level than when both positions were not methylated. Thus in a preferred embodiment said compound in the 3D-modelling method is selected on the basis of the fact that best antibiotic activity is obtained when said compound interacts with the ribosome at both of said positions. Either when the nucleotides at these positions are methylated or when they are not methylated. The fitting of said compound thus preferably further comprises selecting a compound that binding interaction is altered and preferably increased at, at least one but preferably at both of said positions.
With the invention it is now also possible to design in vitro selection systems for selecting compounds that bind more efficiently to either methylated or not methylated nucleotides or both at the indicated positions. Thus in another aspect the invention provides a method for determining whether a compound is capable of inhibiting growth of a bacterium, comprising selecting from a collection of compounds a compound that binds to helix 69 of 23S rRNA and providing said compound to a bacterium to determine whether said compound inhibits the growth of said bacterium. In a preferred embodiment said method comprises selecting a compound from said collection of compounds that interacts with said helix 69, and determining whether said compound inhibits translation in a translation assay. Interaction can for instance be inferred by means of chemical footprinting. Preferably, said 23S rRNA is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Said method can be extended to further comprise generating a collection of compounds that bind in vitro to said helix 69 and selecting from said generated collection a compound that binds in vitro to helix 44 of 16S rRNA. This can again be done by selecting a compound from said collection of compounds that interacts with said helix 44, and confirming the interaction by means of, for instance, chemical footprinting. Said compounds are preferably tested on a bacterium comprising the same methylation (be it absent or present) as in the helix used for screening. Said in vitro method is preferably performed using entire ribosomes. Thus in a preferred embodiment said bacterium comprises ribosomal 23S rRNA that is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli.
Selection of compounds according to the invention is preferably combined with a bacterial assay for antibiotic activity of the selected compound. When the compound is selected on the basis of a methylated nucleotide at the herein above indicated position it is preferred that the bacterium used in the assay comprises the same methylation and vice versa, when the compound is selected on the basis of a non-methylated nucleotide at the herein above indicated position it is preferred that the bacterium used in the assay is not methylated at that position. As capreomycin and viomycin have a much higher antibiotic activity on bacteria having ribosomes comprising rRNAs that are methylated nucleotides at said positions, it is preferred to verify whether a compound selected by a method of the invention comprises the same pattern of antibiotic activity, or an altered one. Thus in a preferred embodiment the invention provides a method of the invention further comprising determining whether said compound inhibits growth of a second bacterium comprising the opposite type of methylation pattern when compared to said first bacterium. In other words, when said first bacterium comprises ribosomes with a 23S rRNA that is methylated at the indicated position, said second bacterium preferably comprises the opposite thereof, i.e. 23S rRNA that is not methylated at the indicated position. The same holds true for methylation on 16S rRNA. And the other way around, when said first bacterium comprises ribosomes with a 23S rRNA that is not methylated at the indicated position, said second bacterium preferably comprises the opposite thereof, i.e. 23S rRNA that is methylated at the indicated position. Again, the same holds true for methylation on 16S rRNA. In a preferred embodiment said second bacterium comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Preferably, said second bacterium comprises ribosomes wherein a 16S rRNA in said 30S subunit is methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli. A preferred way of ensuring that said first or said second bacterium comprises methylation of said rRNAs at the indicated positions is to use a bacterium that comprises a tlyA gene. Preferably a bacterium is used that not naturally contains a tlyA gene. Thus in a preferred embodiment the invention provides a method of the invention wherein said first bacterium is a bacterium lacking a functional tlyA gene. Preferably because said bacterium is naturally devoid of a functional tlyA gene. In a preferred embodiment said second bacterium is a bacterium comprising a functional tlyA gene. Preferably, said second bacterium is a bacterium that is naturally devoid of a functional tlyA gene, and said bacterium is transformed with a functional recombinant version of the said tlyA gene. In a preferred embodiment said first and/or said second bacterium is E. coli or Mycobacterium smegmatis. In another preferred embodiment said first and/or said second bacterium is of the class of Actinobacteria, preferably of the order Actinomycetales and more preferably of the genus Streptomyces. In a particularly preferred embodiment said bacterium is not a Mycobacterium. Mycobacteria are not easy to grow and to manipulate in vitro. In another embodiment, said first and/or said second bacterium is a Mycobacterium, preferably Mycobacterium tuberculosis and Mycobacterium smegmatis. Sometimes it is preferred to use a Mycobacterium as these are or closely resemble pathogenic variants.
When in the present invention mention is made of a nucleotide C1409 in 16S rRNA or a nucleotide C1920 in 23S rRNA that is not methylated, it is meant that said nucleotide does not comprises a tlyA protein mediated type of methylation. The nucleotide can, of course comprise other modifications, such as but not limited to methylation at other sites on said nucleotide. When in the present invention mention is made of a nucleotide C1409 in 16S rRNA or a nucleotide C1920 in 23S rRNA that is methylated, it is meant that said nucleotide comprises a tlyA protein specific methylation. The nucleotide can, of course further comprise other modifications, such as but not limited to methylation at other sites on said nucleotide. A tlyA protein specific methylation is a 2′ O-methylation.
Cyclic peptide antibiotics are typically produced by bacteria of the class Actinobacteria. With the identification of the genes involved with the production of cyclic peptides it has become possible to use these genes in combinatorial approaches to generate novel cyclic peptides that have, as yet, no counterpart in nature. In the present invention one or more of these combinatorial approaches are used to generate a collection cyclic peptides that can be used in a selection method of the invention. Thus the invention further provides a method for determining whether a compound comprises antibiotic activity comprising generating a collection of related compounds through combinatorial biosynthesis of cyclic peptides, preferably a collection of capreomycins, tuberactinomycins or viomycins or derivatives or a combination thereof, and determining whether a compound from said collection inhibits the growth of a first bacterium that comprises ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Preferably, said ribosomes further comprise 16S rRNA in said 30S subunit that is not methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli. Again, this method can be further extended by determining the antibiotic activity of said compound on a second bacterium having an alternate methylation pattern. Thus in a preferred embodiment said method further comprises determining whether said compound inhibits growth of a second bacterium comprising ribosomes having a 30S and a 50S subunit, wherein a 23S rRNA in said 50S subunit is methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli. Said second bacterium preferably comprises ribosomes wherein a 16S rRNA in said 30S subunit is methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli. In a preferred embodiment said combinatorial biosynthesis of cyclic peptides comprises combinatorial biosynthesis of capreomycins, tuberactinomycins, viomycins or derivatives or combinations thereof. Preferably, said combinatorial biosynthesis of capreomycins, tuberactinomycins, viomycins or derivatives or combinations thereof comprises providing a bacterium that produces said capreomycin, tuberactinomycin, viomycin or derivative or combination thereof, and altering the biosynthesis pathway thereof. In one embodiment said bacterium comprises a capreomycin, tuberactinomycin and/or viomycin biosynthesis pathway as described in Thomas et al (2003), WO2005/021586 and/or U.S. Pat. No. 2,828,245. In a preferred embodiment said bacterium producing a cyclic peptide is of the class of Actinobacteria, preferably of the order Actinomycetales and more preferably of the genus Streptomyces. In a preferred embodiment said first and said second bacterium are E. coli or of the Actinobacteria, preferably of the order Actinomycetales and more preferably of the genus Streptomyces. Preferably, said first and said second bacterium belong to the same genus, more preferably to the same species.
Compounds that are selected by a method of the invention can be provided with additional features to provide additional properties. This can be done prior to or after a method of the invention. A preferred modification is a modification that stimulates membrane penetration of said compound. Thus in a preferred embodiment a method of the invention further comprises providing said compound with a moiety that stimulates membrane penetration, i.e. a membrane penetration moiety. In a preferred embodiment said moiety comprises a cell penetrating peptide.
In yet another aspect the invention provides a compound obtainable by a method of the invention. Further provided is a composition comprising a compound of the invention. Compounds such a capreomycin, viomycin and equivalents thereof have antibiotic activity against bacteria comprising methylated rRNA at the indicated positions. Capreomycin and viomycin are currently used to treat mycobacterial infections. A problem is however, the occurrence of resistance to these antibiotics. The present invention provides a new class of antibiotics that exhibit increased antibiotic activity on bacteria having no methylation of rRNA at the indicated positions. In a preferred embodiment of the invention, these compounds are used to treat infection with bacteria comprising no-methylation at rRNA at the indicated positions. In a particularly preferred embodiment, the invention provides a composition comprising both said compound and capreomycin, viomycin or an equivalent thereof. Such a composition can advantageously be used to treat mycobacterial infections and thereby circumvent the problem of resistance to said compounds on the basis of the presence or absence of methylation of rRNA on the indicated positions. This is because bacteria are confronted with antibiotics that are effective against bacteria comprising methylation at the indicated positions and bacteria that do not comprise said methylation. Thus in a preferred embodiment a composition of the invention further comprises capreomycin, viomycin or a functional equivalent thereof.
Microorganisms can acquire resistance to antibiotics in various ways. Resistance to cyclic peptide antibiotics is acquired, among others, through mutation or modification of ribosomal RNA. Antibiotics that are selected through a method of the invention are no different in this respect than the classical antibiotics. Thus the use of antibiotics of the invention will select for microorganisms that are, or have acquired resistance to the antibiotic. Thus the present invention further provides a microorganism that has acquired resistance to an antibiotic of the invention. The invention further provides a ribosome and a bacterium comprising said ribosome comprising a mutation in helix 44 of the 16S rRNA together with a mutation in helix 69 of the 23S rRNA other than the nucleotide that corresponds to nucleotide C1920 in the 23S rRNA of E. coli. The invention further provides a ribosome and a bacterium comprising said ribosome comprising a mutation in helix 69 of the 23S rRNA together with a mutation in helix 44 of the 16S rRNA other than the nucleotide that corresponds to nucleotide C1409 in E. coli in the 16S rRNA of E. coli.
In yet another embodiment, the invention provides a microorganism comprising said ribosome. The present invention further provides modelling a mutation that confers resistance to an antibiotic of the invention and designing a compound derived from said antibiotic that is not affected by said resistance conferring mutation.
In one embodiment the invention provides the use of a compound or a composition of the invention, for the preparation of a medicament for at least in part inhibiting growth of a bacterium. Preferably said bacterium is a Gram positive bacterium. Preferably said bacterium is an actinomycete. In another preferred embodiment said bacterium is a Mycobacterium. Preferably said bacterium comprises a C as the nucleotide that corresponds to nucleotide C1920 in the 23S rRNA of E. coli. and a C as the nucleotide that corresponds to nucleotide C1409 in E. coli in the 16S rRNA of E. coli.
The invention further provides a pharmaceutical composition comprising a compound or a composition of the invention, and optionally a pharmaceutically acceptable carrier.
The invention further provides the use of a compound or a composition of the invention, for at least in part inhibiting translational activity by a ribosome of a bacterium. Also provided is a use wherein said translational activity is inhibited by interference with tRNA translocation from the A site to the P site.
The invention also provides a method for producing a modified capreomycin or viomycin, comprising providing capreomycin or viomycin with a substituent that enhances interaction of said antibiotics with 23S rRNA in a ribosome that is not methylated at a nucleotide that corresponds to nucleotide C1920 in E. coli and/or with 16S rRNA in said ribosome that is not methylated at a nucleotide that corresponds to nucleotide C1409 in E. coli, to enhance binding thereto of said modified capreomycin or viomycin. Such a substituent is preferably a hydrophilic substituent.
In yet another embodiment, the invention provides a method for conferring antibiotic sensitivity to a bacterium that is resistant to said antibiotic, comprising inserting a tlyA gene in the genome of said bacterium. In a preferred embodiment said antibiotic comprises capreomycin, viomycin or an equivalent thereof. In a preferred embodiment said bacterium is not a Mycobacterium. In a preferred embodiment said bacterium is E. coli or a bacterium of the class of Actinobacteria, preferably of the order Actinomycetales and more preferably of the genus Streptomyces. Preferably said bacterium comprises a C as the nucleotide that corresponds to nucleotide C1920 in the 23S rRNA of E. coli. and a C as the nucleotide that corresponds to nucleotide C1409 in E. coli in the 16S rRNA of E. coli.
The invention further provides a method for producing an antibiotic of the invention comprising culturing a bacterium producing said antibiotic and harvesting culture medium from said culture. In a preferred embodiment said bacterium is of the class of Actinobacteria, preferably of the order Actinomycetales and more preferably of the genus Streptomyces. Said bacterium can be selected using a method of the invention. In a preferred embodiment said bacterium is Streptomyces vinaceus or Streptomyces capreolus (Skinner and Cundliffe, 1980).
In yet another embodiment the invention provides a bacterium provided with a tlyA gene. In a preferred embodiment said bacterium is not a Mycobacterium. In a preferred embodiment said bacterium is E. coli. The invention further provides the use of a bacterium provided with a tlyA gene for selecting a compound with the antibiotic activity. The invention further provides a use of a tlyA gene or a protein encoded thereby for increasing the antibiotic activity of capreomycin, viomycin or equivalent thereof on a bacterium. Preferably said bacterium comprises a C as the nucleotide that corresponds to nucleotide C1920 in the 23S rRNA of E. coli and a C as the nucleotide that corresponds to nucleotide C1409 in E. coli in the 16S rRNA of E. coli.
As used herein, a tlyA gene sequence comprises a sequence encoding a protein as depicted in
The invention further provides an actinomycete wherein the tlyA gene is functionally deleted. In a preferred embodiment said deletion comprises at least 10 amino acids. Preferably, said deletion comprises the entire coding region. Preferably said deletion comprises an in frame deletion. The deletion preferably leaves other genes and other open reading frames intact. Thus in a preferred embodiment said deletion leaves the expression and integrity of the downstream located genes intact. In a preferred embodiment said actinomycete is a Streptomyces. In a particularly preferred embodiment said Streptomyces comprises S. coelicolor.
Inactivation of the tlyA gene in M. tuberculosis confers resistance to the ribosome-targeting drugs capreomycin and viomycin (Maus et al., 2005a). We show here that the active tlyA gene encodes an enzyme specific for 2′-O-methylation of riboses in rRNA, and that lack of these methylations results in drug resistance. Resistance to ribosome-targeting drugs is generally associated with addition of methyl groups to the rRNA rather than their loss (Cundliffe, 1989; Douthwaite et al., 2005). TlyA thus belongs to an exclusive group of methyltransferases that confer antibiotic resistance by losing their function—the only other characterized group member being KsgA (Heiser et al., 1972; Van Buul et al., 1983). KsgA and TylA are S-adenosylmethionine-dependent methyltransferases and both modify two nucleotides within rRNA, although they differ in their substrate recognition and their sites of nucleoside modification. KsgA catalyzes the N6, N6-dimethylations of the two adenosines A1518 and A1519 in the loop of 16S rRNA helix 45 (
The two sites of TlyA modification come into close proximity when the 30S and 50S subunits associate to form 70S ribosome couples (
The B2a and B2b interbridges have been shown by chemical probing studies to be essential not only for subunit association (Maivali and Remme, 2004; Merryman et al., 1999a; Merryman et al., 1999b), but also for the interaction of tRNAs with the ribosome (Moazed and Holler, 1989a, 1990). The B2a interbridge is at the geometric centre of the subunit interface, and marks the border between the ribosomal A and P sites (
According to earlier studies, viomycin (and, thus, presumably capreomycin) inhibit formation of the translational initiation complex (Liou and Tanaka, 1976), a process that involves placement of the initiator tRNA into the P site. Viomycin also inhibits later stages of translation by blocking translocation of the peptidyl-tRNA from the A to the P site (Liou and Tanaka, 1976; Modolell and Vazquez, 1977). Translocation is blocked after peptide bond formation has occurred and without directly preventing elongation factor G from hydrolyzing GTP (Modolell and Vazquez, 1977; Vázquez, 1979). Reinterpreting these observations in terms of the tRNA hybrid-site model (Moazed and Noller, 1989b), it appears that viomycin confines the peptidyl tRNA to the A/P state, where the 3′-end of the tRNA has moved to the P site on the 50S subunit and has accepted the peptide chain, while the anticodon end of the tRNA remains locked in the A site on the 30S subunit. Viomycin could block the translocation process in at least two ways: by acting as a physical barrier to the passage of the tRNA from the A/P site or, alternatively, by enhancing the association of the ribosomal subunits (Yamada and Nierhaus, 1978) and thereby impairing their relative movement required for translocation. Both putative modes of inhibitory action are consistent with viomycin (and capreomycin) binding to the ribosomal interface at the interbridge region that coincides with the positions of TlyA methylation.
Capreomycin and viomycin are particularly effective against mycobacteria, and capreomycin is presently widely used as a second-line agent against tuberculosis. Homologues of tlyA are absent from many genera of bacteria, and this corresponds with a generally low level of susceptibility to capreomycin and viomycin. For instance, E. coli and other enterobacteria lack a tlyA homologue and the ensuing rRNA methylations (Rozenski et al., 1999 and our unpublished data) and, consistent with this, E. coli is relatively resilient towards capreomycin and viomycin even when its cell membrane is compromised (Table 2). Upon expression of recombinant tlyA in E. coli, the rRNA becomes methylated at the same two ribose sites as in mycobacteria (data not shown), and this is accompanied by a distinct and specific increase in the sensitivity towards capreomycin and viomycin (Table 2). TlyA methylation thus clearly enhances the activity of capreomycin and viomycin and is the cause, at least in part, of the efficacy of these drugs against mycobacteria.
The methylation sites reported here fit well with the earlier observation that changes to the rRNA in either subunit confers viomycin resistance (Yamada et al., 1978). The specific importance of C1409 and neighboring 16S rRNA nucleotides for capreomycin and viomycin activity has previously been established by a series of mutations in this region (Maus et al., 2005a, b; Taniguchi et al., 1997), which support the direct involvement of TlyA methylation at C1409 in drug binding. Of particular interest is the rrl ΔA1916 mutant where the C1409 methylation is maintained while the C1920 methylation is lost, together with the knowledge of the present invention this mutant thus firmly establishes the involvement of 23S rRNA helix 69 in capreomycin-viomycin activity. It remains unclear whether the lack of C1920 methylation and/or the deleted 1916 nucleotide is conferring the resistance phenotype in the rrl ΔA1916 strain, although the higher resistance (compared to the tlyA mutants) indicates that the A1916-deletion disturbs drug binding in a more direct manner than merely by blocking TlyA methylation. It should be noted that we found no support for the claim that loss of N-1 methylation at G745 confers viomycin resistance (Gustafsson and Persson, 1998) (Table 2); neither can we reconcile our findings with the viomycin footprints at 23S rRNA nucleotides U913/G914 obtained using a large molar excess of drug (Moazed and Noller, 1987). The G745 and U913/G914 regions are both located quite distant from nucleotide 1920 in the 23S rRNA tertiary structure.
Questions remain as to how the TlyA methylations enhance the activity of capreomycin and viomycin, and whether both methylations contribute equally to drug binding. The drugs possess numerous hydrophobic edges on their composite ring and alkyl chain structures (Gale et al., 1981; Vázquez, 1979), which could be envisaged to interact with hydrophobic patches provided by the TlyA methylations. The 19 Å to 21 Å distance between the two methylations, deduced from the two highly resolved E. coli ribosome structures (Schuwirth et al., 2005), is just within span of the largest variants of the drugs. These distances quite feasibly reflect just two of the physiological viable conformations that arise from the relative movement of the subunits during translation (Valle et al., 2003b). Presumably, one ribosome conformation provides an optimal spacing between nucleotides C1409 and C1920 that is bound and locked by viomycin and capreomycin.
The mycobacterial strains used in this study are listed in Table 1, and comprise wild-type and capreomycin/viomycin resistant isolates of M. tuberculosis as well as laboratory strains of the non-tuberculous species M. smegmatis. The resistant M. tuberculosis isolates arose from two virulent wild-type strains (H37Rv and Beijing D3); resistant M. smegmatis strains were isolated from the wild-type strain LR222. Growth of all the wild-type strains is completely inhibited by capreomycin and viomycin concentrations of less than 10 μg/ml. The tlyA mutants P2U, 315-A, C-202 and C-211 have capreomycin and viomycin MICs of 20 to 40 μg/ml, while remaining susceptible to the aminoglycosides kanamycin and amikacin (Maus et al., 2005a). No noticeable differences in growth rate were observed between the wild-type strains and the tlyA mutant strains when grown in medium without capreomycin.
One of the spontaneous capreomycin-resistant mutants, C-401, is distinctly different from the other isolates. C-401 has wild-type tlyA and rrs (16S rRNA) sequences, but possesses a deletion in the rrl gene corresponding to loss of 23S rRNA nucleotide A1916 (E. coli numbering is used throughout). The rrl ΔA1916 mutant is resistant to capreomycin (MIC>160 μg/ml) and viomycin (MIC>80 μg/ml) while remaining susceptible to kanamycin and amikacin. In medium without drug there is no discernible difference in the growth rate of the rrl ΔA1916 mutant and the wild-type parent strain; furthermore, low concentrations of capreomycin (at 10 μg/ml) do not noticeably slow the growth rate of the rrl ΔA1916 mutant.
Screening of rRNA from Wild Type and Mutant Strains
We selected seven rRNA regions for our initial screening, covering the majority of known modification sites (Rozenski et al., 1999), which tend to occur in clusters (Brimacombe et al., 1993) within conserved and functionally important ribosomal regions (Gutell et al., 1994). Three regions were in the 16S rRNA and included the sequences around A1408 and G1491, where mutations have been shown to be associated with resistance to capreomycin (Maus et al., 2005a; Taniguchi et al., 1997), and the highly modified A1518 region (
Differences in rRNA Methylation
Differences between the wild-type and mutant primer extension patterns were seen at C1409 in 16S and at C1920 in 23S rRNA. No differences in the methylation patterns were observed in the 16S rRNA 1518 region, nor in the 23S rRNA 745, 2058 and 2552 regions (data not shown), and none of these sequences were subjected to further analysis. The primer extension analysis of the 1491 region in 16S rRNA was ambiguous due to primer hybridization problems; however, this region was later ruled out as a TlyA target using mass spectrometry.
Nucleotides C1409 and C1920 were analysed more closely using primers that hybridize immediately adjacent to these sites (
Mass Spectrometry Analysis of the rRNA
The masses of RNA oligonucleotides can be accurately measured (to within 0.2 Da) by MALDI-TOF mass spectrometry. Modifications such as methylations can thus be readily identified in 16S and 23S rRNAs after digestion with specific RNases to yield oligonucleotides of suitable size. However, digestion of these large rRNA molecules results in numerous oligonucleotides with similar or identical masses, and produces a complex mass spectrum. To circumvent this problem, we selected specific rRNA regions of about 50 nucleotides by hybridizing to complementary oligodeoxynucleotides (
Helix 44 in 16S rRNA
The nucleotide sequences from 1377 to 1427 and 1461-1511 in helix 44 of 16S rRNA (
The m/z 1623 and m/z 1637 peaks were selected and fragmented further by collision-induced dissociation using MALDI quadrupole-TOF tandem MS (
Helix 69 in 23S rRNA
The 23S rRNA sequence encompassing helix 69 (nucleotides 1899 to 1951,
The exact position of the methyl group in the wild-type rRNAs was verified by MALDI quadrupole-TOF tandem MS analysis of the m/z 1301. fragment. From the fragmentation pattern (
Within the 50S subunit of the 70S ribosome, the drugs most notably make interactions between 23S rRNA nucleotides 1900 and 1930, which include helix 69 and its flanking regions. This is evident from the hydroxyl radical footprint showing drug protection around nucleotides C1920 and G1930 (
In the 30S component of 70S ribosomes, drug footprints were also evident, and these were missing when purified 30S subunits were tested for drug binding. On 70S ribosomes, the drugs make a clear interaction with 16S rRNA protecting nucleotide A1408 from modification by DMS (
It can be concluded that binding interactions between the cyclic peptide antibiotics capreomycin and viomycin and the ribosome occur only when the ribosomal subunits are associated into 70S couples. These drug interactions do not occur on the individual subunits. In the associated 70S ribosome, helix 69 in 23S rRNA and nucleotide A1408 of 16S rRNA face each other across the ribosomal interface to form the binding site for the cyclic peptide antibiotics capreomycin and viomycin.
Capreomycin-viomycin binding to 70S ribosomes stabilizes the interaction between the two subunits. This is evident in footprint protections that lie outside the immediate vicinity of capreomycin-viomycin contacts. The majority of these effects are at or close to the subunit interface, which suggests that capreomycin-viomycin binding brings the subunits into closer contact and thereby reduce access of the interface to the chemical reagents. These effects are illustrated by hydroxyl radical footprints for regions of 23S rRNA (
TlyA Proteins from Different Species Methylate rRNA In Vivo
Homologues of the M. tuberculosis and M. smegmatis tlyA gene were amplified by PCR from Serpulina hyodysenteriae (formerly Brachyspira hyodysenteriae), Streptomyces coelicolor, Thermus thermophilus and Geobacillus stereothermophilus (formerly Bacillus stereothermophilus), and were cloned behind the lac promoter in an E. coli expression vector (see below). The origins of the tlyA genes are listed in Table 4.
The rRNAs of E. coli are not naturally methylated at the TlyA targets (C1409 in 16S, and C1920 in 23S rRNA). Induction of the recombinant tlyA genes led to methylation of the E. coli rRNAs in vivo (
Expression of tlyA Homologues Increase Susceptibility to Capreomycin-Viomycin
Expression of the different tlyA genes rendered E. coli more susceptible to the cyclic peptide drugs. Minimal inhibitory concentrations (MICS) for the different recombinants are listed in Table 6. Expression of tlyA lowered the MIC of capreomycin required to inhibit cell growth. The Thermus and Serpulina tlyA recombinants demonstrate that methylation at 23S rRNA nucleotide C1920 is enough to increase drug sensitivity. Thus, loss of this single methylation must confer mild resistance. Expand the point that resistance can be conferred by disruption of either the 16S rRNA or the 23S rRNA drug contact site (the first by mutation at A1408/C1409, the second by loss of methylation).
Activity of tlyA in Streptomyces
To obtain insight as to how resistant wild-type Streptomyces species are to capreomycin, the well-known streptomycetes S. coelicolor M145, S. griseus NRRL B2682, S. lividans 1326 and S. avermitilis MA4630 were streaked on SFM agar plates with different concentrations of capreomycin. This showed that S. avermitilis and S. coelicolor had a MIC value between 1-5 μg/ml capreomycin, while the MIC value of S. griseus and S. lividans lay between 5 and 10 μg/ml capreomycin (
Capreomycin Resistance of S. coelicolor Relates to Spontaneous Mutations in tlyA
The data on Mycobacterium and the high degree of conservation of TlyA in many different bacteria strongly suggest that TlyA should directly relate to capreomycin sensitivity/resistance in all bacteria with a tlyA gene. To obtain more insight in the relationship between TlyA activity and capreomycin resistance in actinomycetes we set out to obtain spontaneous capreomycin resistant colonies. For this, around 109 colony forming units (cfu) of S. coelicolor M145 were streaked on SFM agar plates containing 25 μg/ml capreomycin. All colonies obtained from this first selection experiment were re-streaked on the same media, and stably resistant colonies were selected. In this way, 17 colonies were obtained that were significantly more resistant to the antibiotic than the original strain (
Conceivably, the mechanisms that most readily lead to capreomycin resistance through a single mutation should be (1) mutation of tlyA, (2) mutation of one or more 16S or 23S rRNA genes, or (3) mutation of one or more genes for membrane proteins. The tlyA genes of CAP9, CAP15, CAP16 and CAP17, together with those of the control strain M145, were amplified by PCR with oligonucleotides tlyA-F and tlyA-R and the amplified DNA fragments were sequenced with the same oligonucleotides. This showed that the tlyA genes amplified from CAP9, CAP16 and CAP17 all carried a single mutation, which most likely was the cause of the enhanced resistance to capreomycin. The mutations were: insertion of a G residue after nt position 481 in mutant 17, and insertion of a C residue after nt position 585 in mutants CAP9 and CAP15 (see
By methods described herein, primer extension analysis was used to assess if the 16S and 23S rRNA were methylated at positions C1409 and C1920, respectively. Excitingly, RNA obtained from tlyA mutant CAP9 was not methylated, while rRNA obtained from the same mutant strain complemented with pGWS405 was methylated at both C1409 (16S rRNA) and C1920 (23S rRNA) (
Creating an in-Frame Deletion Mutant of S. coelicolor tlyA
To unequivocally prove that the mutations in tlyA were the sole cause of the observed resistance of S. coelicolor, we created an in-frame deletion mutant by removing the entire coding region of tlyA except for the start and stop codons and the final two codons, so as to allow proper transcription and translation of the downstream-located gene (ppnK, SCO1783). For this, we PCR-amplified 1500 bp of the upstream part of tlyA up to and including the ATG start codon and 1500 bp of the downstream region of tlyA, including the last two codons and the stop codon of tlyA, encompassing exactly nt positions −1500/+3 and +808/+2300 relative to the tlyA start codon, respectively. The oligonucleotides were designed such as to add restriction endonuclease sites to the extremities of the amplified DNA fragments, so as to allow the cloning of the upstream part as an EcoRI-BamHI fragment, and the downstream fragment as a BamHI-HindIII fragment, where the BamHI sites were located closest to the gene. The upstream fragment was cloned as an EcoRI-BamHI fragment into pSET151 (Kieser et al. 2000) digested with the same enzymes, followed by insertion of the downstream fragment as a BamHI-HindIII fragment. This yielded construct pGWS410 designed for the in-frame replacement of nt positions 4-807 of the tlyA gene by the sequence GGATCC (for a BamHI site) on the S. coelicolor genome. The construct was introduced in S. coelicolor M145 by transformation, and positive transformants were identified by selecting for thiostrepton resistance. Since pSET151 cannot replicate in streptomycetes (due to lack of a Streptomyces ori), colonies can only become resistant by having the construct stably integrated in the genome. One such colony was selected, replicated onto SFM agar plates without antibiotics and loss of the disruption vector was selected by screening for loss of thiostrepton resistance. 40 such double recombinants were selected and tested for capreomycin resistance. Excitingly, one strain had acquired extremely high capreomycin resistance and grew very well on MM agar plates containing 100 ug/ml capreomycin, which is more than 100-fold higher than the parent M145 (
In conclusion, TlyA is a capreomycin resistance determinant in antibiotic-producing streptomycetes. On the basis of the identical functions of TlyA in both mycobacteria and streptomycetes, and the proven rRNA 2′ O-methylating activity of TlyA homologues from highly different species such as Geobacillus stearothermophilus, Thermus thermophilus, Serpulina hyodysenteriae, Streptomyces coelicolor, Mycobacterium tuberculosis and Mycobacterium, smegmatis, we claim that removal of TlyA alone is sufficient to significantly enhance capreomycin resistance in all bacteria.
M. tuberculosis wild-type strains (H37Rv and Beijing D3) and M. smegmatis wild-type strain LR222 were obtained from the culture collection at the Mycobacteriology Laboratory Branch, Centers for Disease Control and Prevention. M. tuberculosis tlyA mutant strains 315-A, C-202 and C-211 and M. smegmatis tlyA mutant strain P2U were isolated as previously described (Maus et al., 2005a). The M. tuberculosis C-401 mutant strain was isolated by the same method as C-202 and C-211 (Maus et al., 2005a) by challenging the wild-type Beijing D3 strain with capreomycin (Sigma) at 10 μg/ml. M. tuberculosis cultures were grown in complete Middlebrook 7119 broth (Remel and Difco) at 37° C. or on Middlebrook 7H10 agar with 10% (v/v) oleic acid-albumin-dextrose-catalase (Difco). M. smegmatis cultures were grown at 37° C. with shaking in Middelbrook 7H9 broth containing 0.2% (v/v) glycerol or on Middlebrook 7H10 agar containing 0.5% (v/v) glycerol.
Escherichia coli strain DH1 was used for cloning and expressing recombinant tlyA, and was grown by standard techniques (Sambrook et al., 1989). The hyperpermeable E. coli strain AS19 (Sekiguchi and Iida, 1967) was additionally used to express tlyA.
Cloning of tlyA
The M. smegmatis LR222 tlyA gene was amplified by PCR using the upstream primer 5′-ccgcatatggcacggcgagctcg and the downstream primer 5′-gctcaaagatctttgcggcccttcctcg. The gene was brought under control of the lac promoter by insertion into the NdeI and BglII sites in the plasmid pLJ102 (a derivative of the expression vector pQE-60 from Qiagen) to form plasmid pSJ101. Both pLJ102 and pSJ101 were used to transform E. coli DH1 and AS19 strains.
M. tuberculosis wild-type Beijing D3 and rrl mutant C-401 were inoculated in triplicate into 7H9 media without drug and 7H9 media containing 10 μg/ml capreomycin, and were grown at 37° C. for 21 days. Growth of the cultures was monitored daily at OD600. The MICs of antibiotics were determined for each strain as previously described (Maus et al., 2005a, b).
Overnight cultures of E. coli DH1 and AS19 strains were diluted 105-fold and plated onto agar containing viomycin (US Pharmacopeia), capreomycin, kanamycin or rifampicin (Sigma) with concentrations increasing in two-fold steps. The agar plates were incubated at 37° C.; and after 22 h, MICs were scored as the lowest concentration at which no growth was observed.
Isolation of mycobacterial rRNA
M. tuberculosis wild-type and tlyA mutant bacteria were grown to mid-log phase in 50 ml of 7H9 broth with shaking, and were harvested by centrifugation. Cells were re-suspended in 2 ml Trizol (Invitrogen) and incubated at room temperature for 10 min. RNA was extracted as outlined by DesJardin (DesJardin, 1999), with the exception of the Cleanascite and DNase treatment steps which were omitted.
M. smegmatis strains were grown to late log phase in 200 ml 7H9 broth with shaking, and were harvested by centrifugation. Cells were washed with 10 ml buffer A (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM NH4Cl, 100 mM KCl) at 4° C., and were lysed by sonication in the same buffer. Cell debris was removed by centrifugation twice at 30,000 g for 10 min. Ribosomes were pelleted from the supernatant by centrifugation at 30,000 g for 18 h at 4° C., and were resuspended in 200 μl cold buffer A. Ribosomal proteins were removed by phenol/chloroform extraction, the rRNA was recovered by ethanol precipitation and was redissolved in H2O.
5′-32P-end-labelled deoxynucleotide primers were hybridized to complementary regions of rRNA, and were extended with AMV reverse transcriptase (Finnzymes) as described by Stern et al. (Stern et al., 1988). Initially, primers complementary to the following seven regions were used: 1461-1510, 1505-1524, and 1529-1543 in 16S rRNA; and 803-821, 1955-1974, 2115-2131, and 2591-2610 in 23S rRNA (E. coli numbering). Sites of 2′-O-ribose methylation were detected by reducing dNTP concentrations (Maden et al., 1995). After the initial screening, 16S rRNA nucleotide C1409 was analyzed more closely from a 1412-1432 primer using decreasing concentrations of dGTP (40 μM, 0.2 μM, 0.1 μM and 0.04 μM) while maintaining dATP, dCTP and ddTTP at 40 μM. Nucleotide C1920 in 23S rRNA was analyzed in a similar manner from a primer complementary to nucleotides 1925-1942. Extension products were run on denaturing polyacrylamide/urea gels alongside dideoxy sequencing reactions performed on M. smegmatis P2U rRNAs. Gels bands were visualized using a Typhoon Phosphorimager (Amersham Biosciences).
Specific stretches of rRNA approximately 50 nucleotides in length (
Mass spectra were recorded in reflector and positive ion mode on a PerSeptive Voyager-DE STR mass spectrometer (Applied Biosystems); spectra were smoothed using the software “m/z” (Proteometrics Inc). Tandem mass spectra were recorded in positive ion mode on a MicroMass MALDI Q-TOF Ultima mass spectrometer (Kirpekar and Krogh, 2001). The collision energy used for tandem mass spectrometry was varied between 30 and 110 eV. All tandem mass spectra were smoothed using the MassLynx software supplied by the manufacturer.
Isolation and Cloning of tlyA Genes
The tlyA genes were amplified from genomic DNA that had been extracted from the respective bacteria. For each tlyA gene, two sets of PCR primers were used. First, the DNA sequence including the tlyA gene plus about 30 nucleotides upstream and downstream were amplified using primers that are perfectly complementary to these regions of the genomic DNA (e.g. for M. smegmatis DNA using primers SJ10 and SJ11 in Table 3). This amplification product was then subjected to nested PCR using the second set of primers (e.g. for M. smegmatis DNA using primers SJ8 and SJ9 in Table 3). This amplified the sequence corresponding exactly to the coding sequence of the tlyA gene (defined for each species in Table 2), and placed an Nde1 site at the ATG start codon and a BglII site (or in some cases a BamHI site) at the end of the coding sequence. The nested PCR products were cloned into the same sites in the plasmid expression vector pLJ102 (a derivative of the plasmid pQE-60 from Qiagen). Final constructs contain the tlyA sequence immediately after the lac promoter, and express TlyA with six histidines at the C-terminal end. The plasmid has a β-lactamase gene (AmpR).
Ribosomal particles were isolated from Mycobacterium smegmatis. The particles were kept as associated ribosomes (70S), or were dissociated and purified as ribosomal subunits (30S and 50S). The cyclic peptide antibiotics capreomycin and viomycin were added to 30S, 50S and 70S particles at a molar excess of drug to ribosomal particles. The interactions of the drugs were footprinted using the reagents dimethyl sulphate (DMS) and hydroxyl radical (OH*). Oligodeoxynucleotide primers that were used for the reverse transcriptase analyses of the DMS and OH* modification sites are given in Table 1. The differences in modification patterns (footprints) reveal the interaction sites of the drugs plus any structural rearrangements that occur within or between the ribosomal subunits.
Secondary structures of (A) the 16S rRNA and (B) the 3′-half of 23S rRNA. Sequences isolated by oligodeoxynucleotide hybridization for MS analysis are shown for (A) the A1377-C1427 and G1461-G1511 regions of Mycobacterium 16S rRNA, and (B) the A1899-U1951 region of Mycobacterium 23S rRNA. The E. coli numbering system for rRNA nucleotides (Cannone et al., 2002; Noller, 1984) is used throughout. For reference, the 16S rRNA nucleotide C1409 corresponds to C1402 in M. tuberculosis and C1392 in M. smegmatis; the 23S rRNA nucleotide C1920 corresponds to C2158 in M. tuberculosis and C2144 in M. smegmatis.
Gel autoradiographs of primer extension on the rRNAs from wild-type and mutant mycobacterial strains. Decreasing the dNTP concentration (indicated by wedges) intensifies reverse transcription termination at nucleotides with 2′-O-methylation, as is shown here for the wild-type strains (H37Rv and LR222) at (A) C1409 in 16S rRNA, and (B) C1920 in 23S rRNA. No termination occurs here in the tlyA mutant rRNAs (C-202 and C-211); the 23S rRNA A1916-deletion mutant (C-401) shows no C1920 stop, but has retained the C1409 methylation stop. Reverse transcription was terminated completely at nucleotides A1408 and G1910 by inclusion of ddTTP and ddCTP in the respective extensions; the nucleotide deletion in C-401 is apparent in the shift of the G1910 band. M. smegmatis P2U rRNA was used as the template for the dideoxy-sequencing reactions (lanes C, U, A and G).
MALDI-TOF mass spectra of RNase T1 oligonucleotides from the mycobacterial 16S rRNA C1409 region. (A) Wild-type M. smegmatis LR222 rRNA shows a peak at m/z 1637 corresponding to UCACGp (U1406-G1410) with two methyl groups. Identical spectra were obtained for the M. tuberculosis wild-type rRNAs (Beijing D3 and H37Rv) and for the A1916-deletion mutant, C-401 (not shown). (B) The UCACGp oligo from the M. smegmatis tlyA mutant P2U runs at m/z 1623, and thus contains only one methyl group; identical spectra were obtained for the M. tuberculosis tlyA mutants C-202, C-211 and 315A (not shown). (C) The theoretical masses of protonated RNase T1 fragments from the A1377-C1427 sequence match the empirically measured m/z values to within 0.1 Da in all cases.
Tandem mass spectra of the 16S rRNA sequence U1406-G1410.
(A) The M. tuberculosis H37Rv wild-type oligo (theoretical m/z of 1637.26, measured here at m/z 1637.20) was fragmented resulting in a series of a, b, c and d ions from the 5′-end, and w, x, y and z ions from the 3′-end (McLuckey et al., 1992); for clarity, only a few significant ions are shown. One methyl group, on nucleotide G1410, is evident from the mass of the x1 ion (m/z 440.10), and was localized to the guanine base, which is lost in the y2 ion (m/z 532.10) and appears at m/z 166.07. The second methyl group is at C1409 and could be seen in (for example) the y2 ion (m/z 697.15). This methyl group is retained on the y2 ion at m/z 586.11 despite loss of the cytosine nucleobase; the methyl group is thus on the C1409 ribose, which can be seen at m/z 111.04. The same methylation pattern was seen for the wild-type strains, M. tuberculosis Beijing D3 and M. smegmatis LR222, as well as for the A1916-deletion mutant C-401.
(B) The corresponding spectrum from the C-211 tlyA mutant (theoretical oligo m/z 1623.24; measured m/z 1623.27). G1410 is still methylated on the guanine base (with a visible product ion at m/z 166.07), whereas the C1409 ribose methylation has been lost; an identical pattern was seen for the C-202, 315-A and P2U tlyA strains (not shown).
MALDI-TOF mass spectra of RNase A oligonucleotides from the mycobacteria 23S rRNA C1920 region.
(A) The M. tuberculosis H37Rv wild-type rRNA with a fragment at m/z 1301.2, corresponding to methylated AACCp (A1918-C1921); cleavage between C1920 and C1921 has been blocked by the ribose methylation. The 1301.2 Da peak was also observed in rRNA from the other wild-type strains (M. tuberculosis Beijing D3 and the M. smegmatis LR222).
(B) In the M. tuberculosis C-211 mutant rRNA, RNase A cleaves after C1920, and the 1301.2 Da signal is absent. Nucleotide C1920 runs in the unmethylated AACp fragment (A1918-C1920) at m/z 982.2 together with A1912-C1914. The same pattern was seen for the mutant strains M. tuberculosis C-202, C-401 and 315A, and M. smegmatis P2U (data not shown).
(C) The theoretical masses of protonated RNase A fragments from the A1899-U1951 sequence match the empirically measured m/z values to within 0.1 Da in all cases.
Tandem mass spectra of the A1918-C1921 oligo from M. tuberculosis H37Rv wild-type rRNA. Backbone cleavage of methylated AACCp (theoretical m/z 1301.22; measured m/z 1301.31) results in unmethylated w1 and z1 ions, and methylated y2 and z2 ions, and this localizes the methyl group to nucleotide C1920. No methyl group is attached to the cytosine base because the a3 ion (at m/z 652.16) has lost unmodified adenine and cytosine nucleobases, and the y3 ion (m/z 750.14) is missing two unmodified cytosine bases. However, a methylated ribose is detected at m/z 111.05 (insert). The same fragmentation patterns, showing C1920 ribose methylation, were also obtained from the other wild-type strains, M. tuberculosis Beijing D3 and M. smegmatis LR222 (not shown).
Locations of the nucleotides involved in capreomycin resistance within the tertiary structures of the rRNAs. The 16S (top left hand side; dark grey) and 23S (right hand side; light grey) rRNAs are shown within the 70S ribosome crystal structure (Yusupov et al., 2001); the ribosomal proteins have been removed computationally for clarity. The ribosomal A site is indicated by the bound tRNA (dark molecule between the rRNAs); the nucleotides implicated in capreomycin resistance are C1920 (1) in 23S rRNA, and C1409 (2), A1408 (3) and G1491 (4) in 16S rRNA. Enlargement of this region of interest from the same angle, and viewed from above after rotating 90° (upper and lower panels right, respectively). The four nucleotides are located within close proximity to each other at the interface region between the two subunits. The lower right panel shows that 16S rRNA nucleotides A1408, C1409 and G1491 are located within the ribosomal A site, while 23S rRNA C1920 is located towards the P site (Yusupov et al., 2001).
Protein sequences of TlyA from Mycobacterium tuberculosis and Mycobacterium smegmatis.
(A) Model of the ribosome presenting nucleotides C1409 (16S rRNA in 30S subunit) and C1920 (23S rRNA in 50S subunit). 30S subunit shown in dark grey, 50S subunit in light grey. 2′-O methylation sites are highlighted by elliptical rings.
(B) Close-up of the interface region around nucleotides C1409 (16S rRNA) and C1920 (23S rRNA) involved in capreomycin binding. Distance between the two nucleotides (highlighted by a dashed line) varies between 19.8 and 21.4 Ångstrom, depending on the conformation of the ribosome (data based on Schuwirth et al., 2005). 16S rRNA below (dark), 23S rRNA above (light). 2′-O methylation sites are shown at either end of the dashed line. Relevant nucleotide numbering is presented to provide directionality.
(C) Nucleotides that are at least in part within a 10 Ångstrom radius of 1409C-O-2′ (16S rRNA) or the 2′-O position of 1920C (23S rRNA). nt, nucleotide. In all subfigures the numbers indicate the nucleotide number in E. coli and the corresponding base for said position.
The sum of these effects is summarized in
Sequences of the spontaneous mutants CAP9 and CAP15, which carry an insertion of a C residue after nt position 585, and of CAP17, which carries an inserion of a G residue after nt position 481. Inserted nt are in bold face and underlined in the sequences. Derived amino acid sequences up to an including the first stop codon are shown, with changed amino acid sequence underlined.
M. smegmatis and M. tuberculosis strains used in this study.
M. smegmatis P2U mutant have been described previously
M. smegmatis
M. smegmatis P2U
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
E. coli DH1/pLJ102
E. coli DH1/pSJ102
Growth of the permeable E. coli strain AS19 was consistent with the above values and the effects of tlyA expression (deviating only in the case of rifampin to which AS19 is hypersensitive with an MIC of 0.1 μg/ml). Expression of the plasmid-encoded copy of the mycobacterial tlyA gene led to complete methylation of the 16S rRNA C1409 and 23S rRNA C1920 riboses in E. coli (not shown). Inactivation of the rlmAI gene (formerly rrmA) that encodes a 23S rRNA G745 N-1 methyltransferase in the E. coli AS19 strain (Liu and Douthwaite, 2002) did not affect the MIC values.
Mycobacterium
smegmatis MC2 155
Thermus
thermophilus HB8
Streptomyces
coelicolor A3(2)
Serpulina
hyodysenteriae
Geobacillus
stearothermophilus
thermophilus)
thermophilus)
hyodysenteriae)
ATC CAA TAA TAA AAT GAG
hyodysenteriae)
stearothermophilus)
stearothermophilus)
stearothermophilus)
stearothermophilus)
M. smegmatis tlyA
T. thermophilus tlyA
S. coelicolor tlyA
S. hyodysenteriae tlyA
G. stearothermophilus
| Number | Date | Country | Kind |
|---|---|---|---|
| 06075120.3 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2007/050024 | 1/19/2007 | WO | 00 | 12/3/2008 |
