Department of Pharmaceutical Biosciences, Division of Microbiology, Biomedical Centre, Uppsala University, Box 581, S-751 23 Uppsala, Sweden1
Author for correspondence: Göte Swedberg. Tel: +46 18 471 46 19. Fax: +46 18 502790. e-mail: gote.swedberg{at}farmbio.uu.se
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ABSTRACT |
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Keywords: Neisseria meningitidis, sulphonamide resistance, dihydropteroate synthase, site-directed mutagenesis
Abbreviations: DHPS, dihydropteroate synthase; pAB, p-aminobenzoic acid; pteridine, dihydropteridine pyrophosphate
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INTRODUCTION |
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Sulphonamide resistance in Neisseria meningitidis may serve as an example of how well-adapted resistant strains have emerged from the original susceptible strains. Sulphonamide-resistant strains have been isolated from patients with meningitis and from healthy carriers that have not been treated with sulphonamide drugs. These strains should thus be considered well adapted since they obviously can survive in competition with other bacteria in humans in the absence of selective pressure caused by the use of sulphonamide drugs (Fermér et al., 1995 ; Fermér & Swedberg, 1997
; R
dström et al., 1992
).
Two types of resistant folP genes have so far been found in clinical isolates of N. meningitidis (Fig. 1). Type 1 may be considered as mutationally altered wild-type genes. These genes have a low sequence divergence (<4%) when compared to folP genes from susceptible isolates (Fermér et al., 1995
). Positions where the folP genes of resistant meningococci differ from those of susceptible isolates were identified. The amino acids at these positions were changed to the consensus sequence by site-directed mutagenesis. The importance of the amino acid changes was shown by changes in the kinetic properties of the mutated enzymes (Fermér & Swedberg, 1997
).
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Here we present results from such a strategy. The effect of removing the Ser-Gly insertion from a type 2 DHPS was studied by enzyme kinetic determinations for pAB and dihydropteridine pyrophosphate (pteridine). An additional amino acid change important for resistance development was identified at position 68. We then took a normal susceptible variant of N. meningitidis DHPS and introduced the additional Ser-Gly dipeptide, as well as the alteration at position 68. Both of these changes slightly changed the Ki for sulphathiazole; however, they also resulted in production of a severely defective enzyme. This finding led to the conclusion that development of type 2 resistance from the susceptible N. meningitidis DHPS is unlikely. These results reinforce the argument that the type 2 resistance gene is a foreign gene that probably has been brought into N. meningitidis by transformation.
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METHODS |
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Site-directed mutagenesis.
The hemimethylation protection method of Vandeyar et al. (1988) was used for construction of most mutants. Primer RM5 (5'-GAATCGACGCGGCCGGGTGCGGATTATGTTTC-3') was used to change T to C in codon 68 in strains MO035 and 418. Primer RM7 (5'-GAATCGACGCGGTCGGGTGCGGATTATG-3') was used to change C to T in codon 68 in strain BT054. The 6 bp insertion (5'-TCCGGC-3') was introduced by the PCR-based megaprimer mutagenesis method (Sarkar & Sommer, 1990
) using primers RM6 (5'-CGCTCGATCCGGGTTTCGGCTCCGGCTTCGGCAAAACCCTGCAACAC-3') and NM2 (R
dström et al., 1992
) in the first PCR. The obtained megaprimer was purified by the QIAquick Gel Extraction kit (Qiagen) and used together with primer NM1 (R
dström et al., 1992
) to recover the whole gene. The required clones were identified by sequence determination using the Cy5 Autoread Sequencing method on an ALFexpress DNA sequencer (Pharmacia Biotech). The insertion in BT054 was also verified by size determinations: the mutated region was PCR amplified with primers NM6 (R
dström et al., 1992
) and NM10 (5'-GCAGGCATCGCACCGCAACG-3') and the amplimer was run on a 8% polyacrylamide gel, which could separate the mutated 110 bp fragment from the original 104 bp fragment. Mutated folP genes were cloned in pUC18 and pUC19 vectors (Yannisch-Perron et al., 1985
) and transformed into DH5
by electroporation (Dower et al., 1988
).
MIC determinations.
Cloned folP genes were transformed into C600folP::KmR. Sulphonamide susceptibility was tested as previously described (Fermér et al., 1995
) using sulphathiazole at concentrations ranging from 0·02 to 5 mM. Some MIC values in this work differ from corresponding values presented earlier (Fermér et al., 1995
) because of the use of different E. coli host cells.
Determination of enzyme kinetic parameters.
Enzyme preparations were made as previously described (Fermér & Swedberg, 1997 ), except that disruption of the cells was by sonication. Enzyme activity was measured by incorporation of radiolabelled [14C]pAB in pseudo-first-order reactions, as previously described (Fermér & Swedberg, 1997
). Km and Ki values were determined using the GraphPad Prism program for Macintosh (GraphPad Software). Standard deviations were calculated from at least five independent measurements.
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RESULTS |
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Introduction of mutations determining resistance into a sulphonamide-susceptible DHPS
The results from the mutagenesis studies presented here were used to try to change the DHPS from a susceptible strain into a form that would confer resistance to sulphonamides. The Ser-Gly insertion was introduced into the BT054 polypeptide at the same position as found in the MO035 enzyme. This had a dramatic effect, raising the Km for both substrates (Table 2). However, the insertion affected the enzymic function adversely, because the ability to complement an E. coli strain lacking the chromosomal folP gene was significantly lower for this mutant than for wild-type genes as well as all the mutants mentioned above. The generation time of C600
folP with a plasmid containing the BT054 gene with the insertion was around 1 h. A clone with the original BT054 gene has a generation time of 30 min. The mutated BT054 enzyme did not allow growth in the presence of any concentration of sulphathiazole tested. However, the Ki for sulphathiazole was slightly raised for the mutant compared to the original BT054 enzyme, but the Km for pAB was raised much more.
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Comparison with plasmid-encoded DHPS mediating sulphonamide resistance
Remarkably, only two types of plasmid-borne genes encoding sulphonamide-resistant DHPS enzymes (sul1 and sul2) have been found so far, mainly in Gram-negative bacteria (Huovinen et al., 1995 ). One reason for the ubiquity of these two plasmid-encoded enzymes is that their respective genes are located on very efficient vehicles for dissemination. An additional explanation is that for a DHPS enzyme to be both efficient in its role in folate biosynthesis and to give a high level of resistance to sulphonamides there is a large constraint on the structure of the enzyme to be able to bind both substrates well and to avoid binding the inhibitor. We determined enzyme kinetic parameters for the plasmid-encoded resistant enzymes Sul1 and Sul2, which were found to have lower Km values for pteridine (1·8 µM and 6·7 µM, respectively) than the chromosomally encoded resistant DHPS enzymes in meningococci. Sul1 also had a Km for pAB that was significantly lower, 0·6 µM, than the values found in resistant meningococcal enzymes. The Sul enzymes have very different amino acid sequences compared to the meningococcal enzymes, so no key amino acids that can explain this difference in Km values can be identified.
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DISCUSSION |
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Sulphonamide-resistant strains are still present in clinical isolates, although the infections caused by N. meningitidis are no longer treated with sulphonamides. We conclude therefore that their DHPS enzymes are well adapted. Km values for pAB, like those found in resistant meningococcal strains, do not seem to be an evolutionary disadvantage, since sensitive strains with similar properties have been found in clinical isolates (Fermér & Swedberg, 1997 ).
When the Km for pteridine is considered, the picture becomes more complicated. The Sul1 and Sul2 enzymes were found to have Km values for pteridine similar to those of sensitive meningococcal enzymes. The Km for pteridine is 1·2 µM for the E. coli DHPS (Dallas et al., 1992 ) and 9·3 µM for Staphylococcus aureus DHPS (Hampele et al., 1997
). Km values for pteridine as high as the ones found for the chromosomal resistant meningococcal DHPS enzymes thus seem to be unusual, but this does not seem to be an obstacle to growth for these strains. One explanation could be that the Km for pteridine is less important if the dihydropteridine pyrophosphokinase (PPPK) has physical contact with the DHPS enzyme during synthesis. If this is the case, the phosphorylated pteridine could be transferred directly from the PPPK enzyme to the DHPS enzyme and the low affinity for pteridine would then have only a minor effect on the reaction rate. In some eukaryotic organisms, including Plasmodium falciparum and Pneumocystis carinii, the PPPK and DHPS enzymic activities are performed by a single, multifunctional enzyme (Brooks et al., 1994
; Triglia & Cowman, 1994
; Volpe et al., 1992
). It is therefore possible that these two enzymes also work in co-operation with each other in bacteria.
In N. meningitidis, two changes in the amino acid sequence of the DHPS enzyme from strains MO035 and 418 that are important for sulphonamide resistance have been identified. The insertion of two amino acids at position 195196 is necessary for the resistance, while the residue at position 68 influences the resistance level. The insertion of additional amino acids in the DHPS enzyme, giving rise to sulphonamide resistance, has been reported for Streptococcus pneumoniae (Lopez et al., 1987 ; Maskell et al., 1997
). The importance of the amino acid at position 68 for sulphonamide resistance in laboratory strains of E. coli has also been noted (Vedantam et al., 1998
), where substitution of Pro by Ser resulted in high-level resistance. Some of the mutations involved in sulphadoxine resistance in Pla. falciparum are found in corresponding parts of the DHPS enzyme (Triglia et al., 1998
). Comparisons with the published DHPS structures of E. coli and Sta. aureus revealed that both positions 68 and 195196 are predicted to be involved in substrate and inhibitor binding (Achari et al., 1997
; Hampele et al., 1997
), consistent with our findings.
The results presented here can be used to propose a possible route of development of a type 2 resistance gene. The original, sensitive gene had no Ser-Gly insertion, a Pro at position 68 and other unidentified differences compared to the present isolates. The Km values for both substrates were probably low, as is usual in wild-type, susceptible enzymes. A replication error caused the insertion of two extra amino acids, generating an enzyme with intermediate resistance to sulphonamides, but low affinity for pAB and pteridine. At this stage, the organism had paid a high cost for the resistance phenotype and had a poorly functioning DHPS enzyme. However, the selection pressure due to the intense use of sulphonamides was sufficiently strong that these bacteria survived. Later, the Pro to Ser substitution at position 68, and possibly other compensatory changes, converted this enzyme into a highly resistant and well adapted enzyme. However, since the original evolution of the resistance gene probably took place in another bacterial species, then it is possible that these two changes alone may have been sufficient to confer high-level resistance. That the resistance evolution probably took place before introduction of the resistance gene into meningococcal strains, is also evident from the identity of a part of the MO035 gene found in strain 418. Both strains have DHPS sequences differing by more than 10% from those of susceptible strains, but the lengths of the replaced segments are considerably different. This finding indicates two different transfer events leading to the generation of the two strains. The change at position 68 in strain 418 must have occurred after the introduction of the foreign DNA, since this amino acid is located outside the replaced region (Fig. 1). This further illustrates the importance of residue 68 for a well adapted resistant type 2 enzyme. If any other mutation in the enzyme is of importance it is hard to identify because of the many remaining differences to the wild-type sequence in the part of the enzyme that is common to 418 and MO035.
When the two changes, identified as important for sulphonamide resistance in type 2 enzymes, were introduced into the sensitive enzyme from BT054, no meaningful increase in the resistance phenotype was seen (Table 2). The modified enzyme is also less able to perform its natural enzymic function, shown by raised Km values for its substrates as well as supporting a slower growth rate when cloned in a folP-deficient E. coli strain. These findings indicate not only that additional changes to DHPS are necessary to develop resistance, but also that it may be difficult to create a resistance gene from a sensitive meningococcal gene by simply introducing sequentially the mutations found in the type 2 resistance genes. The wild-type meningococcal enzymes may differ too much from the type 2 resistant enzymes for such in vitro evolution. The results presented here thus give further evidence for the horizontal-transfer theory of sulphonamide-resistance acquirement, where the type 2 genes are thought to originate from a different organism. Earlier attempts to find the origin of the resistance gene suggested that it may not be derived from commensal Neisseria species, since their available folP sequences are more similar to those from susceptible than from resistant N. menigitidis (Fermér et al., 1995
; Fermér & Swedberg, 1997
). This is in contrast to what has been found earlier for penicillin-binding proteins conferring penicillin resistance in N. meningitidis and Str. pneumoniae, where in both cases related commensal species could be identified as the source of the resistance genes (Spratt, 1994
).
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ACKNOWLEDGEMENTS |
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Received 23 August 1999;
revised 6 December 1999;
accepted 17 January 2000.