[Frontiers in Bioscience 5, d20-29, January 1, 2000]
BACTERIAL RESISTANCE TO AMINOGLYCOSIDES AND BETA-LACTAMS: THE TN1331 Tn1331 TRANSPOSON PARADIGM
Institute of Molecular Biology and Nutrition, Department of Biological Science, California State University Fullerton, Fullerton, CA
TABLE OF CONTENTS
Aminoglycosides (Ags) are a group of antibiotics that exert their bactericidal activity primarily by inhibition of protein synthesis. Aminoglycoside (Ag)g molecules bind to the bacterial 30S ribosomal subunit rendering the ribosomes unavailable for translation, which results in cell death. Although these antibiotics are and have been very useful to treat a variety of bacterial infections, in recent years the number of Ag resistant and multiresistant isolates has seriously increased. Mechanisms of resistance to Ag include enzymatic inactivation by acetyltransferases, nucleotidyltransferases (adenylyltransferases), and phosphotransferases, ribosomal alterations, and reduced permeability. Of all Ags, amikacin (Ak) is the most resistant to the action of Ag-modifying enzymes. However, AAC(6')-I type enzymes (a group of 6'-N-acetyltransferases) can utilize Ak as substrate and confer resistance to this antibiotic in addition to other Ags. The gene aac(6')-Ib was found in various bacterial species and various research groups performed mutagenesis studies on this or related enzymes. In one case, aac(6')-Ib was identified in a transposable element, Tn1331, included in pJHCMW1, a plasmid isolated from a clinical K. pneumoniae strain. Tn1331 includes genes encoding two Ag-modifying enzymes (aac(6')-Ib and ant(3'')-Ia) and two beta-lactamases (blaTEM and blaOXA-9). Characterization of other functions of the pJHCMW1 plasmid showed the presence of an RNA-regulated replication origin and a functional oriT. Stability by multimer resolution is achieved by the Tn1331 resolvase.
2. AMINOGLYCOSIDE ANTIBIOTICS AND RESISTANCE
The aminoglycosides (Ags) are a family of molecules containing a molecular nucleus, an aminocyclitol ring that can be streptidine or 2-deoxystrepamine and two or more aminosugars linked by glycosidic bonds to the nucleus. The fırst Ag, streptomycin, was introduced in 1944 and was isolated from Streptomyces spp. Other Ags were later derived from the same source such as neomycin, kanamycin (Km), tobramycin (Tm), and paromomycin (sufix "-mycin"). Another source for isolation of Ag antibiotics like gentamicin or sisomicin was Micromonospora spp. (sufix "-micin"). Ags of semisynthetic origin such as amikacin (Ak) (1) and netilmicin (2, 3) were also obtained by chemical modification of the natural occurring kanamycin A and sisomicin antibiotics.
The bactericidal activity of Ags is primarily exerted by inhibition of protein synthesis. Ag molecules bind to the bacterial 30S ribosomal subunit rendering the ribosomes unavailable for translation which results in cell death (4, 5, 6, 7). The importance of the interaction of the antibiotic molecule with the ribosome has been shown using mutants or chemical modification of ribosomal components. Those derivatives that had a reduced affinity for the Ag molecule exhibited reduced levels of susceptibility (8). Furthermore, Fourmy et al. recently determined the structure of the highly conserved site A of the E. coli 16S ribosomal RNA when it is complexed to the Ag paromomycin (9). This study revealed how Ags bind the ribosome. Other major metabolic perturbations caused by Ags, which could be secondary effects, include mistranslation, membrane damage (altered membrane composition and permeability), altered cellular ionic concentrations, and disturbances in synthesis of DNA and RNA (5, 10, 11).
Ags are active primarily against aerobic gram negative bacilli as well as gram positive cocci (12). However, it is well known that bacteria develop resistance to antibiotics and in recent years the number of resistant and multiresistant isolates has seriously increased (8, 13, 14, 15, 16). Mechanisms of resistance to aminoglycosides Ags include enzymatic inactivation by acetyltransferases, nucleotidyltransferases (adenylyltransferases), and phosphotransferases, ribosomal alterations, and loss of permeability (14, 17, 18, 19, 20).
A large number of Ag-modifying enzymes has been described (20) (see also the site "Aminoglycoside Modifying Enzymes" http://www.warn.cas.cz/Topics/miller/amg_root.html by the Schering-Plough group and the Antibiotic Resistance Study Groups). Although detailed studies on several Ag-modifying enzymes have been limited, some mechanistic and mutational studies have been carried out (21, 22, 23, 24, 25, 26, 27) and the crystal structures of two acetyltransferases, a nucleotidyltransferase and a phosphotransferase have been reported (28, 29, 30, 31). Genes encoding Ag-modifying enzymes are often located in plasmids which permits cell to cell dissemination of the Ag resistance trait. Furthermore, several of these genes are also included in transposons (32) and integrons (33) which results in their rapid dissemination at the molecular level. In the next section a transposon, Tn1331, which includes genes encoding resistance to aminoglycosides Ags and beta-lactams as well as a structure resembling the variable portion of integrons is described in some detail.
3. THE MULTIRESISTANCE TRANSPOSON TnN1331
We were originally interested in studying the basis of resistance to Ak in Klebsiella pneumoniae clinical isolates. The semisynthetic aminoglycoside Ak is a derivative of kanamycin A obtained through acylation with the L(-)-gamma-amino-alpha-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety (1). This antibiotic is refractory to most Ag-modifying enzymes and has been a powerful weapon against Klebsiella and other Enterobacteriaceae infections (34, 35). However, AAC(6')-I type enzymes can utilize Ak as substrate and confer resistance to this antibiotic (20). Furthermore, resistance to this antibiotic has been on the raise reaching alarming levels. Lately, there have been several reports of outbreaks of multi-resistant Klebsiella that in some cases include resistance to Ak (36, 37, 38, 39, 40). In a study of one hundred and sixty Klebsiella sp. isolated from patients with nosocomial infections in the Children Hospital of Buenos Aires, Argentina during a 1 year period (1981-1982) it was found that all were multiresistant with 7% being resistant to the Ag amikacin (40). Further studies showed that the percentage of Ak resistant Klebsiella sp. kept increasing yearly. Therefore, years ago we initiated a
project to characterize the nature of this resistance as well as its mechanism of dissemination. A clinical K. pneumoniae strain isolated from a diseased neonate was used as a model for these studies. An 11 kilobase pairs (kb) plasmid, pJHCMW1, was found to harbor a gene encoding an Ag-modifying enzyme responsible for resistance to Ak as well as other Ags such as Km, Tm, Nm, and others (40). Characterization of this enzyme indicated that this enzyme was the acetyltransferase AAC(6')-Ib (40, 41). The AAC(6') enzymes acetylate the Ag molecule at the 6' position (figure 1), and bacteria possessing AAC(6')-I enzymes are resistant to several Ags such as Ak, Km, Nm, sisomicin, Tm, and others, but not to gentamicin.
Figure 1. AAC(6')-Ib-mediated acetylation of Ak.
Analysis of the DNA region where aac(6')-Ib is located in pJHCMW1 led to the identification of integron-like sequences as well as a transposable element, Tn1331, related to Tn3 (42). Further studies on this transposon showed that it mediates resistance to the aminoglycosides mentioned before and in addition it mediates resistance to streptomycin, and some beta-lactams. Resistance to streptomycin is mediated by ant(3'')-Ia which encodes an adenylyltransferase (43). The sequence of this gene is related to those of other adenylyltransferase genes described in other systems (44, 45, 46, 47). Resistance to beta-lactams is due to the presence in Tn1331 of two beta-lactamase genes: blaTEM identical to that one present in Tn3 (48) and blaOXA-9, which codes for an oxacillinase-carbenicillinase (43, 49, 50). The OXA-9 enzyme has a pI of 6.9 and the optimal pH for its activity is 7.7 - 8.2 (51). Stability studies showed that enzyme activity decayed to approximately 56% of the original value after 6 hours pre-incubation of total soluble extracts of E.coli harboring blaOXA-9 at 37˚C. Instead, upon pre-incubation at 42˚C, enzyme activity decayed to approximately 50% of the original value after 30 minutes (51). The activity of the OXA-9 enzyme was inhibited in the presence of p-chloromercuribenzoate, cloxacillin, and clavulanic acid, but not by 200 mM sodium chloride. The inhibition by p-chloromercuribenzoate may indicate the presence of a cysteine residue playing a role in the catalytic action of the enzyme (51).
The genetic map of Tn1331 and its closely related tranposon Tn3 are shown in figure 2.
Nucleotide sequence and mutant complementation analyses demonstrated that Tn1331 can be considered as Tn3 (52, 53) with the addition of a DNA region carrying the antibiotic resistance genes and a 520-bp direct repeat (42, 43) (figure 2). The genes aac(6')-Ib, ant(3'')-Ia and blaOXA-9 are flanked by the direct repeats which include a portion of tnpR, the promoter (PT ) as well as the sequence encoding the first 6 amino acids of the protein encoded by blaTEM (see figure 2). As can be seen in figure 2, this duplication resulted in a fusion of the first 6 amino acids of the TEM beta-lactamase to the rest of the polypeptide that forms the AAC(6')-Ib protein (43, 49, 54).
The DNA region encompassing the three resistance genes aac(6')-Ib, ant(3'')-Ia and blaOXA-9 resembles the variable region of the integrons (33, 55). The nucleotide sequence of this DNA region defines two portions with the characteristics described for the gene cassettes found within the variable region of the integrons (56). In the case of aac(6')-Ib the 5' sequence of the gene cassette does not precede the structural gene but rather is part of it (50). The two genes located downstream of aac(6')-Ib, ant(3'')-Ia and blaOXA-9 , are included in a structure that can be considered a single gene cassette (figure 2) because most of the 59-base element that commonly follows the resistance gene is missing following ant(3'')-Ia (50). Although this is a rare phenomenon, other cases like this have now been described. The aac(6')-Ia gene in Tn2424 (57) is followed, in the same gene cassette, by an ORF with no homology to any sequence in the database (58). This ORF has a Shine-Dalgarno sequence and therefore it may be translated. Also, Roy recently found an integron with the arrangement aadB-ant(3'')-Ia-blaOXA-9 in which the ant(3'')-Ia and blaOXA-9 are arranged as in Tn1331 with no 59-base element at the end of ant(3'')-Ia. (P. Roy, personal communication).
Another unusual feature of the gene cassette including ant(3'')-Ia and blaOXA-9 is the presence of the
sequence AAACAAA preceding blaOXA-9. These are the last nucleotides found in the 5' conserved region of the integrons just before the variable region starts with the sequence GTT. This gene cassette could have been generated by an illegitimate recombination event between the end of ant(3'')-Ia in one integron and the 5' conserved segment in another integron carrying blaOXA-9 generating a new integron that includes a gene cassette with the two genes (50).
This putative new integron may have been the target for a Tn3 transposition event. Two different mechanisms for genesis of Tn1331 starting with transposition of Tn3 into the integron have been postulated based on the mechanism for generation of transposons postulated by Grinsted (figure 3) (43, 59). In one case a defective transposition event consisting of a deletion of the appropriate DNA fragments in the cointegrate could have led to the formation of Tn1331 (figure 3a). The other possibility considers a complete transposition event followed by duplication and deletion of DNA regions that led to the generation of Tn1331 (figure 3b) (43).
Figure 3. Two possible mechanisms for the generation of Tn1331 from Tn3 (interpreted from 43). a. A plasmid carrying Tn3 acts as a donor. The first step of the transposition process, formation of the cointegrate, takes place but before the regular resolution occurs there are two deletion events. The DNA fragments deleted are those indicated in the diagram, as a result of the loss of a copy of the res site the structure is locked generating a new transposon, Tn1331. The box with three arrows represents Tn3, and the three arrows represent the direction of transcription of tnpA, tnpR, and blaTEM genes. The res site is represented by a dot. b. This is a slight modification of one of the mechanisms postulated by Grinsted (59). Tn3 transposes from the donor to a location upstream of the resistance genes in the recipient recipient. Following this event the Tn3 portion encompassing nucleotides 3449 and 4957 undergoes a duplication followed by a deletion starting at nucleotide 3969 of Tn3. The product of this deletion event is the new transposon Tn1331.
The genetic organization of the DNA region encompassing the genes aac(6')-Ib, ant(3'')-Ia, and blaOXA-9 is shown in figure 2. Analysis of insertion and deletion mutants used to analyze expression of ant(3'')-Ia. indicated that the ant(3'')-Ia transcript most probably originates at the promoter PT that also directs transcription of aac(6')-Ib. Expression of the blaOXA-9 gene was studied using another set of mutants and subclones. Minimal inhibitory concentrations and primer extension experiments suggested that transcription of the blaOXA-9 gene can originate at the promoter PT as well as at Px, a promoter located immediately upstream blaOXA-9 (50). In general, genes inserted in integrons are expressed from a common promoter region located in the adjacent 5'-conserved segment (60, 61). Therefore, the expression of blaOXA-9 from two different promoters is an unusual fact.
Therefore, the expression of blaOXA-9 from two different promoters is an unusual albeit not unique fact. Parent and Roy recently found that theThe cat gene present in the Tn2424 integron also possesses a promoter immediately upstream the resistance gene (58).
The aac(6')-Ib gene was also found associated with other integron and transposon sequences in different bacteria (62, 63, 64, 65, 66).
4. Biological properties of pjhcmw1 and dissemination of Tnn1331
Tn1331 was first detected in the multiresistance Klebsiella pneumoniae plasmid pJHCMW1. The replication functions of this plasmid and a functional oriT were located in a 2.4 kb EcoRI (67). In addition a site homologous to cer (mwr) was found in a 0.6 kb EcoRI-SacI fragment (68). The plasmid pJHCMW1 replicates using the RNA-regulated general mechanism described for ColE1, p15A, and several other plasmids (67, 69, 70, 71, 72, 73, 74). Although a very high degree of homology was found between the pJHCMW1 and the p15A origins of replication, compatibility experiments demonstrated that they are incompatible indicating that even when both replicons are highly related they are not sufficiently similar
to be incompatible. The pJHCMW1 2.4 kb EcoRI fragment also includes a sequence with homology to the origin of transfer of other plasmids (67). Conjugation experiments using the helper plasmid pRK2073 (75), a recombinant plasmid that carries the RK2 tra genes and the ColE1 mob genes, indicated that in the presence of the appropriate elements, i.e. the tra and mob encoded products, this origin of transfer is functional making pJHCMW1 a mobilizable plasmid (67).
It is noteworthy to mention that pJHCMW1 possesses a locus, mwr, with homology to cer and psi (76, 77) but that is unable to confer stability by multimer resolution in E. coli (68). Analysis of mwr showed that XerC binds very poorly to the mwr XerC-binding site and resolution of dimers carrying two mwr regions are not resolved efficiently. This fact plus a substandard ArgR binding site could be the reasons why mwr fails to stabilize pJHCMW1. Multimer resolution in pJHCMW1 is achieved by the Tn1331 resolvase at the res site. A site-directed mutagenesis experiment generating the substitution D17Stop in tnpR resulted in destabilization of the mutant derivative which is almost completely lost from E. coli JC8679 after 60 generations (68).
Since transposition and conjugation are important mechanisms of gene exchange we analyzed a number of Ak-resistance clinical isolates for their plasmid content and the presence of Tn1331. The results showed that most strains harbored different Ak-resistance encoding plasmids but in all of them Tn1331 (or its closest relative Tn1331.2) was present. This results showed that Tn1331 was spreading very rapidly (78). It was of interest that some strains harbored a relative of Tn1331, called Tn1331.2, that has the DNA portion including the three genes aac(6')-Ib, ant(3'')-Ia, blaOXA-9 duplicated (79). In particular, a conjugative low copy number plasmid, pMET1, was isolated from a clinical Klebsiella pneumoniae strain that caused a devastating outbreak of hospital infection that led to the deaths of several neonates carried Tn1331.2 (79).
5. Characterization of AAC(6')-Ibb
AAC(6')-I enzymes can inactivate several clinically important aminoglycosides including Ak by acetylation of the 6'-amino group of the molecule (20). Several genes encoding AAC(6')-I enzymes have been described to date (20, 49, 54, 57, 63, 66, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97). Based on deduced amino acid sequences, three subfamilies have been defined [Shaw, 1993 #98](20). AAC(6')-Ib which has been detected in the K. pneumoniae transposon Tn1331 (43, 49), Pseudomonas aeruginosa (98), K. oxytoca (65), and Serratia marcescens (54) is included in subfamily 1(20).
Early studies on AAC(6')-I enzymes clarified different aspects of the biochemical reaction mechanism of acetylation (25, 26). Alignment of the amino acid sequences of several AAC(6')-I and AAC(6)-II enzymes permitted to identify conserved regions among the three subfamilies and in particular, 6 motifs were defined within the four members of subfamily 1 (20). Mutational studies carried out by Rather et al showed that when AAC(6')-Ib was subjected to a modification Leu120Ser the new protein had the resistance profile of AAC(6')-II enzymes, i.e. it could not mediate resistance to Ak but it was capable to mediate resistance to gentamicin (99). Later a spontaneous mutation in aac(6')-Ib that led to conversion of the AAC(6')-I profile to that of AAC(6')-II was found to be identical to that one generated in vitro (63). Interestingly, in vivo mutagenesis experiments carried out in our lab showed that after random mutagenesis followed by selection for resistance to gentamicin, only colonies carrying the Leu120Ser could be isolated. These results confirmed the importance of the role played by this amino acid in substrate specificity with respect to Ak and gentamicin (Tolmasky, unpublished results).
In vivo mutagenesis of aac(6')-Ib also permitted the isolation of other mutants of interest (24). A transition in nucleotide 511 changing the codon TTT to CTT which specifies the amino acid Leu generated the mutant AAC(6')-IbDP1 which has the Phe171Leu modification. Minimal inhibitory concentration (MIC) analyses of E. coli carrying either the mutant AAC(6')-IbDP1 or the wild type showed that, as it is the case for the wild tpye, the mutant enzyme confers a high level of resistance to Km and Tm at either 37˚C or 42˚C. However, in the case of Ak or Nm there was a clear difference in the behavior of E. coli strains carrying either of the enzymes. While the wild type enzyme conferred high levels of resistance to these Ags at both temperatures, the MICs for the same antibiotics dropped sharply at 42˚C for E. coli harboring the mutant derivative. In vitro experiments to determine the acetylating activity of the wild type and mutant enzymes were performed using total soluble protein extracts of E. coli harboring either aac(6')-Ib or aac(6')-Ib DP1. Both enzymes had higher specific activity at 42˚C than at 30˚C when Km or Tm were used as substrates. However, AAC(6')-IbDP1 had a drastically lower specific activity for Ak or Nm at 42˚C than at 30˚C (24). Preincubation of the enzymes at different temperatures demonstrated that AAC(6')-IbDP1 irreversibly looses its ability to modify Ak after a short incubation at 37 or 42˚C. The behavior of the mutant derivative suggested that AAC(6')-IbDP1 specifically looses Ak and Nm acetylating efficiency at 42˚C. A common feature of the chemical structure of both antibiotics is the substitution of the C-1 amino group of the deoxystreptamine moiety (1, 3). AAC(6')-IbDP1 may suffer a conformational change at 42˚C that makes it unable to efficiently modify Ags molecules with a substitution at the C-1 amino group of the deoxystreptamine moiety. Further mutagenesis analysis of the Phe171 showed that this amino acid position has low tolerance to substitutions confirming that it may play an important role in the structure and/or function of AAC(6')-Ib (100). Furthermore, sequence comparisons among AAC(6') enzymes (20) and members of the N-acetyltransferase superfamily (101) indicated that Phe171 is included within the conserved motif B (or motif 5 according to an older nomenclature) and it is one of the most conserved amino acids in this region (20, 101). Mutant AAC(6')-IbDP1 has this nonpolar amino acid replaced by Leu, another nonpolar amino acid. The phenotype of the Phe171Leu substitution taken together with the high conservation of Phe171 observed among AAC(6')-I proteins suggests that this amino acid may be critical for the spatial folding of these acetyltransferases (24). It was described that the hydrophobic effect is an important factor in the three dimensional conformation of proteins (102). Therefore, the nonpolar Phe171 residue may contribute to the structure of AAC(6')-Ib as well as other AAC(6')-I enzymes. The Phe171Leu change decreases the residue volume (103) and eliminates an aromatic ring. These anomalies may contribute to the lower stability of the mutant enzyme, which may be unable to keep the proper spatial conformation at 42˚C. It has been also described that substitutions between Phe and Leu perturb hydrogen bonds that contribute to the conformation of the protein (104). These factors can contribute to the reduction in specific activity of AAC(6')-IbDP1 and its stability at higher temperatures (24).
Another mutant, AAC(6')-IbDP6, has the Tyr80Cys (24) substitution. Although E. coli carrying this mutant gene showed a certain level of resistance to some aminoglycosides, the enzyme AAC(6')-IbDP6 had only marginal levels of acetylating activity in vitro. The change Tyr80Cys is a replacement of a polar residue for a nonpolar amino acid that could not be tolerated by the protein, which lost most of its enzymatic activity. Tyr80 is located within the region encompassing amino acids 77 - 81 (Val-Thr-Pro-Tyr-Ile) which is conserved among three members of the subfamily 1, AAC(6')-Ib, AAC(6')-IIa, and AAC(6')-IIb but not in AAC(6')-Ie (20) where a Val residue is present in the position equivalent to Tyr80. Since Val is hydrophobic, this data suggests that the nonpolar nature of the substituting amino acid is not enough to explain the loss of activity.
Recently, Casin et al. characterized variants of AAC(6')-Ib from clinical Enterobacter cloacae and Citrobacter freundii isolates (105). These variants had substrate profiles intermediate between those of the AAC(6')-Ib and AAC(6')-IIa enzymes. Characterization of these enzymes demonstrated that two of them are 177 or 184 amino acids long while the other is composed of 196 amino acids. All three proteins exhibited a Ser residue at position 119 instead of the Leu found in the wild type AAC(6')-Ib. Size variation in AAC(6')-Ib like proteins was also found in a study of 70 clinical Ak-resistance strains by Tran Van Nhieu et al. (106). These authors found that most amikacin-resistant clinical isolates carried the aac(6')-Ib gene. However, western blots demonstrated that the proteins were not identical, they had molecular weights ranging between 24 and 26 kD. In addition, another AAC(6')-Ib variant with an N terminus modification was published (107). Based on these facts Casin et al. proposed that AAC(6')-Ib variants may have flexible structural requirements for the N terminus(105). This variations in the N terminus, together with the possible alternatives for translation or lack of translation (108, 109) (Brassard and Roy, unpublished results) may indicate that this gene is "hunting" for a good expression element. Fusion to the initial portion of the blaTEM seems to be a good strategy for expression of aac(6')-Ib utilizing the blaTEM promoter (43, 49, 54).
Studies on other AAC(6')-I enzymes showed that the Acinetobacter haemolyticus enzyme AAC(6')-Ig looses acetylating activity against Tm when the Met56 is substituted by an Arg residue (90). In addition these authors found two other natural derivatives unable to confer resistance tom Tm. These mutants have either an insertion of 19 thymines residues in the gene which results in frameshift, or an insertion of a copy of IS17 (90). Wright and Ladak recently purified and characterized the AAC(6')-Ii enzyme from Enterococcus faecium. They concluded that this enzyme is not optimally evolved for Ag inactivation indicating that it probably plays another physiological role(s) (110).
6. Concluding remarks
The transposon Tn1331 is a paradigm of the molecular evolution of drug resistance mechanisms. A transposon carrying only one resistance gene like Tn3 which unlike other tranposons (like the Tn21 subfamily) had shown to be very resilient to changes evolved into a new more dangerous element carrying four genes. This event could represent an important pathway of evolution of transposition elements of the Tn3 family besides other known mechanisms. The addition of the aac(6')-Ib gene to the structure of Tn1331 provided a very effective vehicle for dissemination of the gene and with it resistance to very important antibiotics like Ak. In addition the presence of Tn1331 in mobilizable and conjugative plasmids that in some cases can have a wide host range indicates that the gene will continue to spread to a large variety of bacteria. It is therefore necessary to continue the characterization of different aspects of the mechanism of resistance to antibiotics and dissemination of these traits to design rational methods to overcome the problem.
Some of the work reported in this manuscript was supported by Public Health Service Grant AI39738 from the National Institutes of Health.
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