Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1301

Sulphonamide Resistance inNeisseria meningitidis and

Commensal Neisseria Species

BY

YVONNE QVARNSTRÖM

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

Abbreviations

General:

ATP adenosine triphosphate DHF dihydrofolate ET electrophoretic type GTP guanosine triphosphate Ki inhibition constant Km Michaelis constant pAB para-aminobensoate pterin 6-hydroxymethyl-7,8-dihydropterin SuR sulphonamide-resistant SuS sulphonamide-susceptible THF tetrahydrofolate

Gene designations:

aroX putative chorismate mutase gene asmA putative outer membrane biogenesis protein gene dedA putative membrane-associated dedA-family protein gene folK hydroxymethyl-dihydropterin pyrophosphokinase gene folP dihydropteroate synthase gene manB putative phosphomannomutase-phosphoglucomutase gene sul1 plasmid encoded sulphonamide resistance gene 1 sul2 plasmid encoded sulphonamide resistance gene 2

Enzymes:

DHPS dihydropteroate synthase DHFR dihydrofolate reductase GTPCH GTP-cyclohydrolase DHNA dihydroneoaldolase HPPK hydroxymethyl-dihydropterin pyrophosphokinase DHFS dihydrofolate synthase TS thymidylate synthase

Contents

Introduction.....................................................................................................1The use of antibiotics .................................................................................1Development of antibiotic resistance .........................................................1

Resistance mechanisms .........................................................................2Acquired resistance................................................................................3

Mutational adaptation .......................................................................3Horizontal gene transfer....................................................................4

Compensation for fitness costs ..............................................................5The Neisseria family ..................................................................................5

The commensal Neisseria species .........................................................6Neisseria meningitidis ...........................................................................6Genetic variation through horizontal transfer ........................................7

The sulphonamide drugs ............................................................................8The history of sulphonamides................................................................8The action of sulphonamides .................................................................9Resistance to sulphonamides ...............................................................10

Plasmid encoded sulphonamide resistance .....................................10Chromosomal sulphonamide resistance..........................................11

The folate biosynthesis pathway ..............................................................12DHPS...................................................................................................12DHFR...................................................................................................13HPPK...................................................................................................13

Substrate channelling ...............................................................................13

Aims of this thesis.........................................................................................15

Results and Discussion .................................................................................17Sulphonamide resistance in Neisseria ......................................................17

Background:.........................................................................................17Present investigation (Papers I, II and III) ...........................................19

The effect of serine in position 68...................................................19Mutations in the BT054 enzyme.....................................................20The fitness cost of folP mutations...................................................21The degree of horizontal transfer in N. meningitidis.......................23Sulphonamide resistance in commensal Neisseria .........................24Restrictions in transformations between Neisseria species.............25

Interactions of DHPS with HPPK ............................................................26

Background:.........................................................................................26Present investigation (Paper IV):.........................................................27

Characteristics of N. meningitidis HPPK........................................28Substrate channelling ......................................................................28

Concluding remarks ......................................................................................30

Acknowledgements.......................................................................................32

References.....................................................................................................34

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Introduction

The use of antibioticsThe discovery of antibiotics is one of the most important progresses in the

medical history. For the first time, it was possible for physicians to cure many infectious diseases that had troubled humankind for centuries, for example pneumonia, meningitis, sepsis, tuberculosis and syphilis.

The word antibiotic, meaning “against life” in Latin, was first used by the microbiologist Selman Waksman in 1941 to define “natural substances that are produced by microorganisms to prevent growth of other microorganisms” (46). Synthetic substances with growth inhibitory effects on microorganisms do not meet all the criteria of antibiotics, so they are often called chemotherapeutics. However, the distinction between natural and synthetic drugs has become less practical with time, as the proportion of synthetic and semisynthetic drugs has increased. For simplicity, this thesis will therefore use the word antibiotics for all drugs with growth-preventing effect on microorganisms.

The search for antibiotics began at the end of the 19th century, but the first reported substances were either inefficient or toxic for humans (97). The major breakthrough in antibiotic research came in 1928, when Alexander Flemming discovered a mould that contaminated staphylococcal cultivation and seemed to produce a substance capable of killing the bacteria. This substance was indeed found to have a remarkable ability for curing various infectious diseases in humans and Flemming named the substance penicillin. Unfortunately, producing and purifying penicillin on a large scale proved to be very difficult, so the routine use of this miracle drug was delayed until the middle of the 1940s. Penicillin was soon followed by other antibiotics isolated from various moulds and soil-living bacteria, for example tetracycline, erythromycin, chloramphenicol and streptomycin.

Development of antibiotic resistance Bacteria are masters of adaptive evolution. They are present in every

corner of the earth, some have adjusted to life in boiling water, others to high

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salt concentrations or very acid environments. The successful colonisation of bacteria can be partly ascribed to an impressive ability of them to adjust to variability in their environment. The introduction of antibiotics resulted in a rapid environmental change for the bacteria and destroyed the living conditions for the pathogenic species. When faced with this antibiotic threat, the bacteria had the choice of dying or developing resistance to the drugs. Thus, the discovery of each new antibiotic substance was soon followed by the appearance of bacteria resistant to the new drug (18). In the case of penicillin, Flemming made the first observations of resistant bacteria shortly after his discovery of the drug (2).

Resistance mechanisms

Antibiotic resistance in bacteria is due to mechanisms that prevent the active drug from exerting its inhibitory effect. These mechanisms can be divided into five basic groups:

biofilm formation decreasing the drug concentration inside the bacterial cell degradation or modification of the antibiotic drug target bypass drug target alteration or protection

Natural populations of bacteria are commonly organised in so-called biofilms: ordered structures of microcolonies and water channels, surrounded by a polysaccharide-containing matrix (89). Bacteria within biofilms are phenotypically different from free-living bacteria. One of the differences is that biofilm formation renders the bacteria resistant to antibiotic treatment (22).

The second resistance mechanism explains why some Gram-negative bacteria are intrinsically resistant to certain antibiotics: their outer membrane is impermeable to some drugs. Decreased drug concentrations can also be accomplished by so-called efflux proteins that actively transport the drug out of the bacterium and thus prevent the drug from reaching its target. Efflux proteins are, for example, common in erythromycin-resistant bacteria (52).

Destruction of the antibiotic is another method of preventing the antibiotic from reaching the target. Examples include the production of an enzyme ( -lactamase) that degrades penicillin, leading to widespread resistance to this antibiotic, and the production of enzymes that inactivate several other important antibiotics by attaching acetyl-, methyl- or other groups to the drug molecule (60).

Drug target bypass means that the bacteria either circumvent the deleterious effects of target inhibition via an alternative pathway, or, simply lack the drug target. For example, stomach ulcers, caused by Helicobacter

pylori, cannot be treated with trimethoprim since this bacterium lacks the target enzyme dihydrofolate reductase (62).

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The drug target is rendered inaccessible for drug binding by the last mechanism and is accomplished by protection or alteration of the drug target. Resistance due to target protection has so far only been documented for resistance against tetracycline drugs and the exact function of this mechanism is still unknown (11). Drug target alterations, however, are common and result in targets with decreased affinity for the antibiotic. This enables the bacteria to grow even in the presence of the active drug. Alteration of the drug target is the mechanism for resistance against sulphonamide drugs, as will be further presented in this thesis.

Acquired resistance

Although some bacterial species are intrinsically resistant to certain antibiotics due to species-specific characteristics (as illustrated by a few examples above), the alarming clinical problems of antibiotic resistance are caused by acquired resistance. Bacterial species that were originally susceptible to antibiotics have adapted to the antibiotics present in the environment and evolved into resistant variants. Adaptive evolution is consecutively rendering currently available antibiotics ineffective to an escalating number of pathogenic bacterial taxa, making it increasingly difficult to treat infectious diseases (64).

Mutational adaptation

Spontaneous mutations in the chromosome can alter proteins or cell components and result in drug resistance. Hence, mutational adaptation to an antibiotic-containing environment can convert genes already present in a bacterium to resistance genes (18). For instance, chromosomal mutations can alter drug targets, as exemplified with the sulphonamide resistance described in this thesis, and can also affect transport proteins in the outer membrane, as has been reported in multidrug-resistant Escherichia coli (17).

Although chromosomal mutations are crucial for the generation of genetic variation in bacteria, the majority of mutations are detrimental for the individual bacterium. The mutation rate is generally kept at a low level and advantageous mutations affecting drug susceptibility are rare events in normal bacteria. However, an increased mutation rate has been observed in individual bacteria (20). These so-called mutators can increase mutation frequency by 10 000-fold and arise spontaneously in bacterial populations challenged by harsh conditions. Due to their high mutation rate, mutators develop antibiotic resistance more frequently than non-mutators (16), and may therefore contribute to the clinical problem of antibiotic resistance. In particular, pathogenic bacteria tend to benefit from increased genetic variation provided by mutators, presumably because of their need to evade human immune defence. A study concerning the pathogen described in this

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thesis, Neisseria meningitidis, revealed that 57% of the clinical isolates examined had increased mutation rates (76).

Despite the presence of mutators, mutational adaptation is not the preferred method for antibiotic resistance development in most clinically relevant bacteria. There is a much faster alternative: horizontal gene transfer.

Horizontal gene transfer

An alternative approach to mutational adaptation is to receive antibiotic resistance genes from other bacteria. Horizontal genetic transfer allows bacteria to share antibiotic resistance genes with high efficiency and speed. There are three general mechanisms for horizontal transfer of DNA: conjugation, transduction and transformation (Figure 1). In the case of antibiotic resistance transmission, conjugation and transformation are important contributors, but not transduction.

Conjugative plasmids and transposons (mobile DNA elements) carrying antibiotic resistance genes are very common among pathogenic bacteria (60). In some cases, they contain several resistance genes so that one single transfer renders the receiving bacterium resistant to multiple antibiotics. Furthermore, some plasmids have a broad host range and can thereby transfer antibiotic resistance between different bacterial species.

Figure 1: Mechanisms for horizontal genetic transfer

Conjugation is the process where small circular DNA-molecules are transferred from one bacterium to another during close cell-to-cell contact: this requires special transfer proteins that normally are expressed from the conjugative DNA element itself. During transformation, free DNA in the environment is imported and incorporated into the chromosome, only bacterial cells that are competent for DNA uptake can utilise this possibility. Transduction involves so-called bacteriophages (viruses that infect bacteria and thereby inject foreign DNA into the bacterial cell), and each type of bacteriophage infects only one or maybe a few bacterial species. The dotted line symbolises chromosomal DNA and the thick black arrows represent antibiotic resistance genes.

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Transformation involves the uptake of free DNA from the environment. Natural transformation has been discovered in species belonging to the Streptococcus, Bacillus, Haemophilus and Neisseria families, and more recently also in a few other bacteria, such as Campylobacter and Helicobacter species (51). The mechanism of uptake and processing of the imported DNA varies considerably between different bacterial species, but the net result is the same – the replacement of a chromosomal sequence with the imported DNA. If this DNA includes antibiotic resistance genes, the transformed bacterium may become resistant. This mode of resistance transmission is especially important in species where conjugative elements are rare, such as in Neisseria meningitidis.

Compensation for fitness costs

Acquisition of antibiotic resistance has a cost for the bacteria. Antibiotics interfere with the cellular processes required for survival, and changes in these processes are expected to result in unfavourable effects. Mutations or horizontal gene transfers conferring antibiotic resistance are therefore likely to result in a fitness cost for the bacteria (9, 100). In environments free from antibiotics, susceptible bacteria are therefore better equipped than resistant mutants are. Only in the presence of the antibiotic will the resistant mutants have a selective advantage over the susceptible bacteria, making it possible for them to persist in the bacterial population, despite the fitness cost.

If antibiotic resistance were permanently associated with a fitness cost, resistant bacteria would only survive as long as the antibiotic selection pressure was maintained. Without antibiotics, the fitness cost of resistance would force bacteria to revert their resistance or face extinction. However, the evolution of resistant bacteria is more complicated than the above reasoning. Although the acquisition of resistance often entails a fitness cost, this initial disadvantage can be rapidly compensated by additional alterations (54, 63, 98). Such compensatory mutations in the resistance gene or in other locations can restore the fitness while maintaining the resistance phenotype. Once compensation to the original fitness level occurs, there is no evolutionary incentive for the bacteria to sacrifice the resistance phenotype. In other words, well-adapted resistant bacteria with equal fitness as susceptible counterparts have nothing to gain in reverting. This leads to an irreversible resistance development.

The Neisseria family Bacteria belonging to the Neisseria family are Gram-negative diplococci.

The majority of Neisseria species are harmless commensal bacteria that colonise the pharynges in mammals. Neisseria species are competent for

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natural transformation, which promotes a frequent transfer of genetic traits, including antibiotic resistance, between different bacteria.

The commensal Neisseria species

The human body is constantly colonised with various bacteria and other microorganisms. Every surface of the body exposed to the surroundings, such as the skin, the mucus membranes and the intestinal tract, are lined with microorganisms that constitute commensal flora (33, 68). In particular, the intestines are rich in commensal bacteria, which assist in food digestion and provide essential nutrients. Another important function of the commensal flora is protection from pathogenic microorganisms, mainly by occupying available infection sites and thus preventing pathogens from residing in the body (46).

The Neisseria family includes several commensal species that colonise the pharynges in mammals. Although a few species have been associated with rare cases of disease (21, 44), they are mainly classified as harmless bacteria. The Neisseria species included in the human commensal flora are closely related to each other and to the pathogenic Neisseria species (38). Of the commensal species mentioned in this thesis, N. lactamica and N. cinerea

are close relatives to the pathogenic species, whereas N. sicca, N. subflava

and N. mucosa are more distantly related. There are few studies on the carriage of commensal Neisseria: N. lactamica is preferentially isolated from children, whereas colonisation with other species may be more durable (37, 82).

Neisseria meningitidis

Two Neisseria species are human pathogens: N. gonorrhoeae (the gonococcus) that infects the urogenital tract, and N. meningitidis (the meningococcus), named after its ability to infect the meninges. N.

meningitidis colonises the pharynges and can be isolated from around 10% of healthy individuals, with the highest carrier rates for young men aged 15-20 years (15, 71). This carrier state can continue for months without any symptoms and appears to protect the carrier from invasive disease (88). In those individuals that are not so fortunate (especially children), N.

meningitidis can penetrate the mucosa, enter the blood stream and eventually reach the meninges. The growth of bacteria in the blood stream may also result in disseminated meningococcal disease, a very serious condition that can lead to death within a few hours (77).

N. meningitidis strains are divided into serogroups based on the characteristics of their polysaccharide capsule. The five most common serogroups are designated A, B, C, Y and W135 and account for almost all cases of disease (77). Development of vaccines has been difficult: no

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effective vaccine against serogroup B and only short-term protective vaccines against the other four common serogroups are currently available (8).

N. meningitidis causes both sporadic disease and larger outbreaks (epidemics). The lack of preventive vaccines, in combination with the seriousness of the disease, make surveillance programmes important for disease management (103). Isolates responsible for meningococcal disease are collected and characterised at DNA level with a molecular fingerprint method, usually multilocus enzyme electrophoresis. This method assigns each isolate with a unique genetic pattern (80). Isolates with identical fingerprints are designated with the same electrophoretic type (ET) and isolates with similar patterns are clustered in ET-complexes. This makes it possible to determine the role of individual strains or group of strains in epidemics and endemic disease.

Genetic variation through horizontal transfer

Natural transformation is an important mechanism for genetic variation within the Neisseria family (30, 53). Neisseria are naturally competent for transformation, meaning that they can take up free DNA from the environment and incorporate it into their chromosome. The replacement of the original sequence with the imported DNA in the chromosome results in mosaic gene structures, where regions with altered base composition interrupt the original sequence. Horizontal transfer event is therefore implicated when sequence comparisons reveal regions with odd sequence composition: regions with either unusually high sequence divergence, or, identical regions in otherwise dissimilar sequences.

The details of the transformation process are only partly known. Studies on N. gonorrhoeae have determined that uptake of DNA is efficient only if the transforming DNA contains a specific DNA uptake sequence: a 10 base pair sequence repeated in thousands of copies in the gonococcal genome but very uncommon in bacteria belonging to other families (23, 27). Identical DNA uptake sequence has been detected in high numbers in the genome of N. meningitidis (67). The requirement for DNA uptake sequences favours uptake of DNA from related bacteria and probably protects from unfavourable gene replacements.

Horizontal genetic exchange allows transfer of new phenotypes across species barriers. Commensal Neisseria share their growth habitat with N.

meningitidis and may exchange DNA with this pathogen during concurrent colonisation. DNA containing antibiotic resistance can thereby be transferred between commensal and pathogenic Neisseria. This is exactly what happened during the development of penicillin-resistant N. meningitidis:resistance-conferring penA sequences were transferred from the intrinsically

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resistant commensals to the pathogenic Neisseria species by natural transformation (87).

The sulphonamide drugs

The history of sulphonamides

The history of the sulphonamides started in the research department of a dye company in Germany in 1932 (46). During a screening of various dyes for antibiotic activity, the physician Gerhard Domagk discovered that the red dye Prontosil could cure streptococcal infections in mice. Soon, other researchers learned that the antibiotic effect was incurred by sulphanilamide (Figure 2), a sulphonamide released from the dye when the molecule was metabolised in animals (97). This finding led to the development of other sulphonamide drugs with improved pharmacodynamic and pharmacokinetic profiles.

The sulphonamides were the first clinically useful antibiotics available for infection treatment in human medicine and were effective against a wide variety of infections. They were non-toxic, stable, inexpensive and easy to produce in large amounts. The use of sulphonamides quickly led to improvements against many infectious diseases, for example a two-third decrease in mortality from pneumonia (97). Sulphonamides were also highly effective against Neisseria meningitidis (79). In the case of meningococcal disease, sulphonamides were not only used to treat infected patients but also as prophylaxis to prevent epidemics and to eradicate N. meningitidis from asymptomatic carriers.

Unfortunately, treatment with sulphonamides was not always successful. Many patients experienced allergic reactions and other negative effects, and a few suffered from serious complications such as severe anaemia due to bone marrow suppression (83). Hence, other drugs successively replaced the sulphonamides after World War II, as penicillin and other antibiotics with fewer side effects became available. Currently, the use of sulphonamides accounts for only a small portion of antibiotic drugs used (13, 83). Although sulphonamides have limited use against bacterial infections, they are still important against infections caused by yeast and protozoa. For example, sulphonamides are used in the management of Pneumocystis carinii

infections, an increasing problem in AIDS patients and other immunocompromised hosts (57).

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Figure 2.

The molecular structure of a) the red dye Prontosil, b) sulphanilamide, c) the general structure for sulphonamide drugs, where R is a variable structure, and d) para-aminobensoate (pAB).

The action of sulphonamides

The sulphonamide drugs restrict bacterial growth because they inhibit the production of folate. Folate is involved in the biosynthesis of precursors for DNA and proteins and are thus required for growth by all living cells: the reduced form of folate, tetrahydrofolate (THF), participates in several important one-carbon transfers, for example in the biosynthesis of thymidine, glycine and methionine (78). In humans and other higher eukaryotes, folate is a vitamin that has to be provided in the diet and transported into growing cells. Conversely, bacteria and lower eukaryotes lack the proper transport systems for uptake across the cell membrane and therefore depend on folate production within their own cells (83). For this purpose, their genomes code for a series of unique enzymes that together constitute the folate biosynthesis pathway (43). This pathway is depicted in Figure 3.

One of the folate biosynthesis enzymes is dihydropteroate synthase (DHPS). This enzyme uses two substrates, para-aminobensoate (pAB) and 6-hydroxymethyl-7,8-dihydropterin diphosphate (pterin diphosphate), to produce pteroate. Sulphonamides are competitive inhibitors of the DHPS enzyme because they are structurally similar to the normal substrate pAB (Figure 2), and susceptible DHPS enzymes are unable to distinguish between the drug and the normal substrate. In the presence of sulphonamides, DHPS will therefore bind the drug and produce a sulpha-containing product instead of the normal dihydropteroate (10). This halts dihydropteroate production and subsequently causes a depletion of THF, which eventually results in growth inhibition. In addition, a recent study (70) indicate that the sulpha-containing pteroate analogue inhibits another enzyme, dihydrofolate reductase (DHFR), in the folate biosynthesis pathway.

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Figure 3: The folate biosynthesis pathway

The folate biosynthesis pathway converts guanosine triphosphate (GTP: molecule 1) to tetrahydrofolate (THF: molecule 7). The first enzymes in the pathway, GTP cyclohydrolase (GTPCH) and a yet unidentified enzyme, synthesise 7,8-dihydro-neopterin [2]. Dihydroneopterin aldolase (DHNA) converts [2] into 6-hydroxymethyl-7,8-dihydropterin [3]. Hydroxymethyl-dihydropterin-pyrophosphokinase (HPPK) phosphorylates [3] with adenosine triphosphate (ATP) as the phosphate donor, resulting in 6-hydroxymethyl-7,8-dihydropterin diphosphate [4]. Dihydropteroate synthase (DHPS) condenses [4] with para-aminobensoate (pAB), producing dihydropteroate [5]. Dihydrofolate synthase (DHFS) adds one or several glutamate residues to [5], resulting in dihydrofolate (DHF) [6]. In the last step, [6] is reduced to THF [7] by the dihydrofolate reductase (DHFR).

Resistance to sulphonamides

The heavy use of sulphonamides between 1935 and 1945 resulted in extensive development of resistant bacteria (95). Sulphonamide resistance emerges when bacteria gain access to modified DHPS enzymes, which have the capacity to distinguish pAB from sulphonamides. DHPS enzymes conferring resistance to their hosts can be either chromosomal or plasmid encoded.

In the following text, DHPS enzymes mediating sulphonamide resistance will be referred to as sulphonamide-resistant enzymes and DHPS from susceptible strains will be called sulphonamide-susceptible enzymes.

Plasmid encoded sulphonamide resistance

In the majority of Gram-negative pathogenic bacteria, sulphonamide resistance is due to the horizontal transfer of one of two resistance genes,

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designated sul1 and sul2 (74, 91). These sul genes code for DHPS variants with very low affinity for sulphonamides. Even if the bacterial cell continues to express a sulphonamide-susceptible enzyme from its chromosome, the synthesis of folate is secured due to the parallel production of the sul-encoded enzyme.

The origin of the sul genes is unknown. They have a high sequence divergence from each other as well as from all sequenced chromosomal DHPS-coding genes. The sul1 gene is expressed from transposons of the Tn21 class, whereas sul2 is preferentially observed on non-conjugative plasmids (75). A third type of plasmid-borne sulphonamide resistance gene has recently been detected in clinical E. coli isolates from pigs (72) and from humans (28). This new gene is designated sul3.

Chromosomal sulphonamide resistance

Sulphonamide resistance in clinical isolates is not always plasmid encoded. For example, in Neisseria meningitidis and several Gram-positive bacteria, resistance to sulphonamides is caused by alterations in the chromosomal gene coding for the DHPS enzyme (83).

The chromosomal DHPS-coding gene is designated folP. Point mutations or short insertions in the folP gene can decrease the affinity of the DHPS to sulphonamides and confer resistance. However, sulphonamides share their binding site in the protein with pAB, so alterations affecting the drug binding properties are also likely to affect the binding properties of pAB. Lower affinity for pAB can result in decreased fitness, with additional mutations being necessary to improve pAB binding properties and restore fitness. The mutational adaptation of DHPS enzymes is thus a balance between sufficiently low affinity for sulphonamide to confer resistance and sufficiently high affinity for pAB to allow an efficient folate production. An alternative to this successive mutational adaptation is the uptake of folP

genes from already resistant bacteria through natural transformation, instantly replacing the original folP with parts of, or, a complete folP,mediating resistance.

One measurable parameter that reflects substrate affinity of an enzyme is the Km value. This value equals the substrate concentration required to achieve 50% substrate binding to the enzyme. A low Km value means that a low substrate concentration is needed for saturation of the enzyme, leading to a high catalysis rate even at low substrate concentrations. Thus, well-adapted enzymes are expected to display low Km values for their substrates. In the case of DHPS enzymes, this includes low Km for both pAB and pterin diphosphate. Sulphonamide-resistant DHPS should in addition possess a high Km for the sulphonamides (termed as Ki, when referring to an inhibitor). Resistant bacteria, expressing DHPS variants fulfilling these requirements, have equal fitness as susceptible bacteria and have nothing to gain in reverting the resistance. Moreover, reversion of resistance mutations often

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results in lower fitness, since the compensatory mutations are only beneficial when combined with resistance-conferring mutations. This makes chromosomally encoded sulphonamide resistance very stable and explains why high-level resistance is still widespread in bacterial populations, despite low sulphonamide consumption (83).

The folate biosynthesis pathway The folate biosynthesis pathway synthesises folate in bacteria and lower

eukaryotic organisms. This pathway converts guanosine triphosphate (GTP) to tetrahydrofolate (THF) via a series of catalytic steps (Figure 3). Since an adequate supply of folate is a prerequisite for cell division, the normal activities of the folate enzymes are essential for bacterial growth. Drugs inhibiting the folate biosynthesis enzymes, such as sulphonamides, are therefore excellent antibiotics.

DHPS

The biological role of the DHPS enzyme is to condense pterin diphosphate with pAB to produce dihydropteroate, an intermediate in folate biosynthesis. Sulphonamides are substrate analogues that competitively inhibit DHPS.

Being the target for the sulphonamides, the DHPS enzyme has been extensively studied and was first characterised from Streptococcus

pneumoniae (49). Three-dimensional structures have been determined for DHPS from E. coli (3), Staphylococcus aureus (29) and Mycobacterium

tuberculosis (101), and equilibrium binding measurements have been performed on the S. pneumoniae enzyme (6). Taken together, these reports provide a detailed picture of substrate binding and catalytic mechanism, as summarised below:

Active DHPS enzymes are dimers, but only one of the subunits is active in the reaction. Each subunit adopts a so-called TIM-barrel structure with eight parallel -strands surrounded by eight -helices. The loops connecting

-strands and -helices on the C-terminal pole have a flexible conformation and are important for the catalytic and dynamic function of the enzyme. Loops 1, 2 and 6 are highly conserved and contain residues involved in substrate binding. Loop 1 includes an asparagine residue that binds to the phosphate moiety of pterin diphosphate. Loop 2 contains a hydroxyl-containing residue (serine or threonine) and an arginine that are thought to participate in the reaction. Loops 2 and 6 are involved in the formation of the binding site for pAB and sulphonamides. DHPS adopts an ordered binding mechanism, with the pterin diphosphate binding first, deep within the “barrel”. The subsequent binding of pAB results in conformational changes

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in the loops, which seal the protein structure and trap the substrates. During or after the reaction, the loops reopen the structure to facilitate release of the products.

DHFR

Besides DHPS, the only folate enzyme that has been exploited as a target for antibiotics is the dihydrofolate reductase (DHFR). DHFR catalyses the reduction of dihydrofolate to tetrahydrofolate and is especially crucial for the production of thymidine in almost all living cells (12). DHFR is inhibited by trimethoprim and other antifolate drugs. Although both sulphonamides and trimethoprim can be used separately as antibiotics, they are more commonly used in combination (32).

HPPK

The enzyme preceding DHPS in the folate biosynthesis pathway is the hydroxymethyl-dihydropterin-pyrophosphokinase (HPPK). The catalytic role of HPPK in the folate biosynthesis is to transfer two phosphate groups from adenosine triphosphate (ATP) to the pterin molecule. This potential new antibiotic target has recently been the focus for several studies concerning its catalytic properties and three-dimensional structure (for a review, see ref. 36). Potential inhibitors based on the transition-state of the reaction have been synthesised, although the biological relevance of these inhibitors was not addressed (81).

Substrate channelling Enzymatic activities constituting a metabolic pathway can be expressed

either separately as single proteins or together in larger multifunctional enzymes. The folate biosynthesis enzymes are usually expressed as monofunctional proteins, but there are some interesting exceptions. For example, in Lactococcus lactis GTP-cyclohydrolase (GTPCH) and HPPK are expressed as one bifunctional enzyme (92) and Streptococcus

pneumoniae produces a bifunctional enzyme combining the dihydroneoaldolase- (DHNA) and HPPK activities (50). Furthermore, protozoa produce a bifunctional HPPK – DHPS enzyme (69, 96), and fungi express a multifunctional protein with DHNA-, HPPK- and DHPS- activity (102).

One potential benefit with multifunctional enzymes is the increased proximity of the active sites involved in the reactions. Physical connections between active sites facilitate so-called substrate channelling – the direct transfer of an intermediate between two subsequent enzymes in a pathway.

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In a substrate channelling process, the product of the first enzyme is directly transferred to the second enzyme instead of diffusing into the cytoplasm. This leads to increased catalytic efficiency of the reaction, especially if the intermediate is unstable, produced in small quantities or participates in several reactions.

Substrate channelling has been proposed in various metabolic pathways, including the folate-dependent one-carbon metabolism in eukaryotic cells. The most studied example is the bifunctional thymidylate synthase – dihydrofolate reductase (TS-DHFR) enzyme from Leishmania major. The TS domain converts THF to DHF during the synthesis of thymidine (59). The DHF is then channelled along an “electrostatic highway” of positive amino acid residues to the DHFR domain, where it is reduced to the active THF again (39). The pattern of positive charges at the surfaces of TS and DHFR also appear in monofunctional enzymes, suggesting the possibility for electrostatic channelling in organisms with separate enzymes, such as prokaryotes (90). Although substrate channelling has not yet been detected in the folate biosynthesis enzymes, the presence of multifunctional enzymes indicates that physical contact between the enzymes enhances folate production. Protein interactions or channelling events may even be crucial for efficient folate production. Data on such qualities could be exploited in the search for new antibiotics (4).

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Aims of this thesis

This thesis concerns the development and spread of sulphonamide resistance in Neisseria meningitidis and commensal Neisseria species. Sulphonamide resistance in N. meningitidis was first detected in 1963 (65) and the resistance phenotype has remained in the bacterial population since. Although sulphonamides have not been used against N. meningitidis for half a century, recent studies indicate that 60-100% (depending on geographic origin) of meningococcal isolates are resistant to sulphonamides (1, 5, 58). The persistence of sulphonamide resistance, despite a very low selection pressure, indicates that resistant strains are well adapted and have compensated for any fitness cost incurred by the resistance phenotype.

Previous studies have implied a compensatory evolution in some of the resistant strains and horizontal transfer events in other strains (26, 73). One of the aims of this thesis was to confirm these findings and provide further information on the resistance development in N. meningitidis. Even if sulphonamide resistance in N. meningitidis is not a clinical problem, information on development and dispersal could be useful to prevent meningococcal strains from developing resistance against clinically important drugs. In addition, sulphonamides remain an important alternative in the treatment of fungal and protozoal infections, and sulphonamide resistance is a clinically important issue in these organisms (42, 55).

Another aim of this thesis was to evaluate the involvement of the commensal Neisseria species in the resistance development. Antibiotic research has mainly focused on pathogenic bacteria and little is known how the use of antibiotics affects commensal flora. The mode of action of antibiotics is not specific for pathogenic bacteria. Some antibiotic treatments are likely to influence commensal bacteria and may result in the development of resistant commensal populations. In contrast to the pathogen, which is eventually cleared from the body, the resistant commensal bacteria can reside in the body for long periods. Commensal bacteria can therefore act as reservoirs for antibiotic resistance genes and transfer the resistance to new pathogens that enter the body.

Another aspect of antibiotic resistance is that few new drugs are being developed (31). New drugs with alternative modes of action will probably be required to replace current drugs as they become increasingly ineffective due to resistance. One part of this thesis considers a potential new target for

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antibiotic drugs: the folate biosynthesis enzyme HPPK and its physical interactions with other enzymes in the folate biosynthesis pathway.

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Results and Discussion

Sulphonamide resistance in Neisseria

Background:

Neisseria meningitidis strains belonging to the so-called ET-5 complex cause epidemics associated with high frequencies of disseminated meningococcal symptoms (14). The ET-5 strains are also invariantly resistant to sulphonamides, implying that resistance could be used as a virulence marker (40). The resistance determinant is an altered variant of the chromosomal folP gene with high sequence divergence from other meningococcal folP (10% at nucleotide level). The large sequence difference suggests that this resistance gene was acquired by horizontal DNA transfer (73). Sequence comparisons of N. meningitidis strains belonging to other serogroups and complexes have revealed two additional types of resistance genes (Figure 4). The second type of resistance gene is a mosaic gene with the central part identical to the ET-5 folP but the beginning and end identical to folP from susceptible strains (73). The third type of resistance gene is more similar to folP from susceptible strains than the ET-5 folP and is therefore suggested to have evolved by accumulation of point mutations (25). The third type of resistance gene will be referred to as the point-mutated folP gene in the following text. Thus, there are two major routes of resistance evolution: adaptation by point mutations of the chromosomal folP

and horizontal acquisition of folP sequences from another species. If the point-mutated type of resistance gene has evolved by point

mutations, conversion to a susceptibility phenotype should be possible by reverting the mutations. Three amino acids in conserved positions altered in the point-mutated DHPS were examined: a leucine in position 31 (normally a phenylalanine), a serine in position 84 (normally a proline) and a cysteine in position 194 (normally a glycine). All three residues were replaced by the corresponding amino acids found in susceptible strains, both individually and in combinations (26). The analyses identified the leucine in position 31 as the most crucial for the resistance phenotype, whereas cysteine in position 194 has an additional effect on resistance level. By lowering the pAB Km

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value for the resistant enzyme, the serine in position 84 has an apparent compensatory effect on the enzyme. When all three amino acids are changed to the residues found in susceptible DHPS, the resulting DHPS enzyme exhibits sulphonamide susceptibility and kinetic properties comparable with wild type susceptible DHPS. Thus, these mutagenesis experiments supports the conclusion that this type of resistance genes evolved by accumulation of point mutations in the original chromosomal folP gene.

One distinguishing feature of the ET-5 type of resistance genes is an apparent insertion of six base pairs, coding for two extra amino acids (serine and glycine) in positions 195–196 in the DHPS polypeptide. The insertion is flanked by directed repeats of six base pairs with almost identical sequence as the insertion, suggesting that the insertion was generated by a replication error that duplicated the repeat (25). When this insertion is removed by site-directed mutagenesis, the resistance phenotype disappears. The deletion also has a profound effect on substrate binding for the resulting enzyme, raising the Km for pAB more than 10-fold (26).

Figure 4: Schematic representation of folP genes from N. meningitidis.

Comparison between folP from sulphonamide-susceptible (SuS) and resistant (SuR)strains, illustrating differences in sequence. Light shading represents sequence divergence of around 5% from sulphonamide-susceptible strains, and dark shading represents sequence divergence of 10% or more. The filled triangles symbolise the six-base-pairs insertion.

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Present investigation (Papers I, II and III)

The specific aims of this study were: to explore the possibility of converting the ET-5 type of resistant DHPS to a susceptible DHPS with wild type kinetic characteristics to investigate if an insertion of the serine-glycine dipeptide in position 195–196 of a sulphonamide-susceptible DHPS is sufficient to cause a resistance phenotype to evaluate possible fitness costs of the resistance mutations to determine sulphonamide resistant commensal Neisseria in throat specimens, with the aim of identifying the origin of the ET-5 type of resistance to identify possible restrictions in the process of DNA transfer between different Neisseria species

The effect of serine in position 68

The ET-5 folP gene was compared to folP genes from other clinical isolates to identify residues (besides the serine-glycine insertion) with importance for the resistance phenotype. One possible candidate was the invariant proline in position 68, which is a serine in ET-5 strains. By site-directed mutagenesis, this serine was replaced by proline, both in the wild type ET-5 and the mutated ET-5 without the serine-glycine insertion (Paper I). The resulting enzymes had lower Km for pAB compared to before mutagenesis (Table 1). The change in position 68 also influenced the resistance level, because the affinity for sulphonamides was lower for the mutated enzyme with proline in position 68 compared to the original enzyme. These results indicated that serine-68 was necessary for a high level of resistance, but in addition had compensatory effect on substrate binding. However, compensation was modest and the most susceptible enzyme (with proline in position 68 and the insertion removed) still had a 10-fold higher Km value for pAB than enzymes from wild type strains. This indicated that further compensatory alterations were hidden in the divergent sequence.

The residue in position 68 has a crucial role in DHPS. Residue 68 most likely participates in the binding of pAB, since it interacts with bound sulphonamides in three-dimensional structures of DHPS (3). In addition, this residue is located in loop 2 next to threonine-66 and arginine-67, which are thought to participate in the enzyme reaction. Amino acid alterations in this highly conserved region are therefore likely to affect substrate binding and Km values, as well as the resistance level. Mutations in loop 2 have previously been reported from a number of sulphonamide-resistant bacteria, including E. coli (99), Streptococcus pneumoniae (56, 66) and Mycobacterium leprae (35).

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Table 1: Kinetic properties of the ET-5 type of sulphonamide-resistant DHPS before and after site-directed mutagenesis

a results are from ref. 26. SD = standard deviation; MIC = minimal inhibitory concentration; STZ = sulphathiazole; wt = wild type; mut = mutagenised; n.d. = not determined

Mutations in the BT054 enzyme

The serine-glycine dipeptide in the ET-5 type of resistant DHPS is inserted next to the highly conserved glycine at position 194. Removal of this dipeptide had a dramatic influence on the binding properties of both sulphonamides and pAB. The removal of the serine-glycine dipeptide in the ET-5 DHPS abolished the resistance phenotype, which raised the question of whether an insertion of this dipeptide was sufficient to convert a sulphonamide-susceptible enzyme into a resistant one. Therefore, the serine-glycine dipeptide was introduced into position 195-196 of the DHPS from a sulphonamide-susceptible strain, BT054 (Paper I). This led to greatly disturbed kinetic properties of the mutated enzyme, but with no effect on resistance level (Table 2). When compared to three-dimensional structures of DHPS enzymes, this devastating effect on enzyme fitness is understandable _

the amino acids were inserted into loop 6 of the DHPS structure, which contains several residues crucial for the formation of the substrate binding site (6). Thus, the insertion distorted the loop and prevented efficient binding of the substrates.

The proline-68 in the BT054 DHPS was mutated to the serine found in this position in the ET-5 DHPS. As with the insertion, the major influence of this mutation was a raised Km value for pAB (Table 2). Thus, the alterations conferring resistance in the ET-5 strains were not appropriate in the genetic background of sulphonamide-susceptible strains. These results again emphasised the diversity of the ET-5 type from other meningococcal DHPS and supported the hypothesis that it had been horizontally transferred from another species.

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Table 2: Kinetic properties of a sulphonamide-susceptible enzyme before and after site-directed mutagenesis

a results are from (26). SD = standard deviation; MIC = minimal inhibitory concentration; STZ = sulfathiazole; wt = wild type; mut = mutagenised

The fitness cost of folP mutations

The kinetics presented in Paper I were measured in vitro with DHPS enzymes expressed from cloned genes on multi-copy plasmids in E. coli.Suggestions on resistance development were based on the effects of the various mutations on Km and Ki values. It was assumed that mutations in the DHPS, leading to higher Km, also decreased the general fitness of the bacterium. One weakness with this approach was that a correlation between the fitness of a bacterium and substrate affinities of its DHPS enzyme has never been experimentally tested. The effects of various folP mutations on fitness were elucidated by replacing the chromosomal folP of N. meningitidis

strain 952 with wild type and mutated folP variants from other strains (unpublished study). The aim was to obtain a collection of isogenic clones, each one expressing a different folP variant from otherwise identical genetic backgrounds. Any differences between the clones would then have to be a reflection of the expression of the various folP genes.

As N. meningitidis is naturally transformable, the gene replacements were performed by cultivating strain 952 in the presence of DNA containing the desired folP. A transformation vector was constructed by cloning the folP

region from strain MO035 in a pUC18 vector in two steps and concurrently introducing two new restriction enzyme cleavage sites in the meningococcal DNA (Figure 5). The cleavage site for BamHI was used to insert a tetracycline resistance gene [amplified from Tn10, see ref. (93)] between the promoter region of the manB gene and the transcription terminator of folP.This facilitated selection of successful transformants by tetracycline resistance. The introduced SacI cleavage site and the naturally occurring BseRI site were used to replace the original folP with other folP variants in the correct direction in the cloned DNA. The resulting constructs were then separately introduced into strain 952 by natural transformation. Upon uptake,

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the DNA was incorporated into the chromosome by homologous recombination, thus replacing the original folP with the folP from the construct. Successful replacements were confirmed by PCR amplification of a part of the folP gene present in the transformed clone, followed by cleavage with restriction enzymes that discriminated the 952 folP from the replacing variant.

The fitness cost of the mutated folP variants were estimated by comparing the growth rates of clones expressing wild type and mutated DHPS enzymes. Growth rates in early exponential phase were measured in Iso-Sensitest Broth at 37ºC at normal atmosphere. Under these circumstances, the 952-strain had a generation doubling time of 40 minutes and the introduction of the tetracycline resistance gene did not affect this growth rate. In fact, all clones had identical growth rates, although they had different folP genes and thereby produced different DHPS variants. One of the tested clones produced the deletion mutant of the ET-5 DHPS, which had ten-fold higher Km for pAB compared to the wild type ET-5 DHPS. Another clone expressed the point-mutated type of resistance gene, lacking the compensatory mutation in position 84. However, the generation time remained 40 minutes irrespective of which type of folP was expressed. Thus, the conclusion from these experiments was that even a DHPS with high Km values could provide the cell with enough folate to support a normal growth rate. However, the situation may differ when nutrients are limited and the bacteria have to compete for survival. Under such conditions, differences in growth rates, too small to be detectable in this assay, may still be important.

Figure 5: Schematic illustration of the DNA construct used for replacement of the wild type folP in N. meningitidis strain 952.

Open reading frames are symbolised as black arrows pointing in the direction of the reading frame (only the first half of the asmA gene is included in the construct). Horizontal continuous lines mark cleavage sites for restriction enzymes. Dashed lines define regions with different purposes in the construct: the exchangeable folPand the inserted tetB are flanked by sequences that facilitate homologous recombination of the construct into the N. meningitidis chromosome.

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The degree of horizontal transfer in N. meningitidis

Previous work has identified three types of sulphonamide resistance genes in N. meningitidis strains: the ET-5 type and the mosaic type, both resulting from horizontal transfer, and the point-mutated type of resistance gene (25, 73). The two published genomic sequences from N. meningitidis have folP

identical to these types: the MC58 strain has an ET-5 type of folP (94), whereas the folP in strain Z2491 is identical to the point-mutated type of resistance gene (67). Hence, there is no published genomic sequence without alterations in the folP region provoked by sulphonamides. The differentiation of horizontally transferred regions from the original meningococcal sequence in the resistant strains is complicated by this lack of a reference sequence from a sulphonamide-susceptible strain.

Recently, unpublished data from a genome-sequencing project of N.

meningitidis strain 8013 was provided by Christophe Rusniok and Philippe Glaser at the Pasteur Institute. This strain is sulphonamide-susceptible and contains no traces of allelic folP replacements. Comparison of sequence from strain 8013 with sequences from the two published genomes revealed a variable degree of similarity between the strains for the region examined (Paper III). Strain Z2491 was similar or identical to strain 8013 throughout the region examined, except for one region with a very different sequence: the beginning of folP and 14 base pairs preceding the folP start codon (Figure 6). Another distinguishing feature of Z2491 was the absence of the aroX gene upstream of folP. Two additional strains carrying the same type of resistance gene as Z2491 also lacked the upstream gene aroX (Figure 3 in Paper III). A possible explanation is that the ancestor of these strains experienced events that deleted the aroX gene and altered the sequence surrounding the folP start codon.

Strains 8013 and Z2491 were more similar to each other than to the MC58 strain. MC58 deviated from strain 8013 in a mosaic pattern, where similar sequences were interspersed with regions of high divergence. The second half of the dedA gene and most of the folP gene had low sequence similarity compared to strain 8013 and were thus examples of possible horizontal transfers.

Initially, strain 8013 was considered as lacking horizontally transferred sequences in the region examined, but there was a 122 base pairs sequence in the middle of the manB gene, where the sequence from 8013 differed from Z2491 by 20% and from MC58 by 17%. This could be an example of a short horizontal transfer in the 8013 sequence.

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Figure 6: Schematic comparison of genomic sequences from three N.

meningitidis strains.

Boxes represent open reading frames. White regions represent sequences with less than 3% diversity, black regions have a sequence divergence of 10% or more, and the striped boxes mark the aroX gene.

Sequence variation due to horizontal transfer in the folP gene can be easily explained by the selection pressure exerted by the sulphonamides. In contrast, there is no obvious selection for the variation detected in the normal housekeeping genes dedA and manB. Altered variants of dedA and manB

may confer an advantage for the bacteria, but horizontal transfer is possible even without positive selection. Import of genetic material frequently occurs in the N. meningitidis genome and improvements of fitness is not a requirement for a new sequence to remain in the population (104).

Sulphonamide resistance in commensal Neisseria

The origin of the horizontally transferred sulphonamide resistance in N.

meningitidis was assumed a commensal Neisseria species. This is only possible if sulphonamide resistance is present in strains of commensal Neisseria. A collection of throat swabs, sampled from the same individuals at three separate points during one year was used (Paper II). The individuals were not exposed to antibiotics for at least three weeks before the first sample nor throughout the sampling period. Colonies of commensal Neisseria were isolated from the throat swabs and examined with respect to sulphonamide resistance. Highly resistant strains were detected in 2 out of 17 individuals. From repeated throat swabs, both of these individuals had resistant strains that were mixed with susceptible strains. This illustrated that the resistant commensals were not an occasional element due to selection from antibiotic pressure, but rather a well-adapted natural part of the commensal throat flora.

The sulphonamide resistance in the commensal isolates was most likely due to alterations in the chromosomal folP gene, since sequence determination revealed alterations in conserved residues previously reported as crucial for a resistance phenotype. The only identifiable alteration in conserved positions in some resistant isolates was a leucine in position 31. A single alteration in position 31 has previously been reported sufficient for resistance in a N. meningitidis strain and in E. coli (26, 99). Residue 31 is

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thought to interact with the invariant residue in position 68 and thereby influence the crucial loop 2 in the DHPS structure.

Other resistant commensal isolates had a cysteine in position 194 in combination with a methionine in position 66 and one-amino-acid insertion after position 76. In N. meningitidis, a cysteine in position 194 is one of the mutations mediating resistance in the point-mutated type of resistance gene (26). As mentioned previously, position 66 is located in loop 2 and is normally a serine or threonine. The hydroxyl group in these amino acids is thought to participate in the enzyme reaction, so methionine (which lacks a hydroxyl group) in this position is expected to affect the reaction. The insertion after position 75 was also located in loop 2 and it is possible that the combination of methionine-66 and the insertion resulted in an altered but still functional loop structure that conferred resistance without reducing the enzymatic activity. A two-amino-acid insertion in this region in sulphonamide-resistant Streptococcus pyogenes has been reported (34), the position of which corresponds to amino acid 74 in N. meningitidis, and the removal of the insertion in S. pyogenes resulted in decreased resistance and reduced enzymatic efficiency.

The primary objective for detecting sulphonamide-resistant commensal Neisseria was to identify the donor of the ET-5 type of folP. However, the folP in the resistant commensals did not resemble the ET-5 folP, so the origin of the horizontally transferred sulphonamide resistance remains unidentified. Nonetheless, the presence of stable sulphonamide resistance in commensal Neisseria indicates that dissemination of sulphonamide resistance by horizontal transfer from commensal Neisseria is a realistic possibility.

Restrictions in transformations between Neisseria species

Numerous studies have confirmed the frequent exchange of genes within the Neisseria family (48, 53, 84). In contrast, little is known about the mechanisms involved in natural transformation or how the process is regulated. As excessive transformation is likely to disrupt chromosomal stability and function, naturally competent bacteria such as N. meningitidis

must be able to restrict the transformation process to a beneficial level. One restricting factor in pathogenic Neisseria species is the preferential uptake of DNA containing the neisserial DNA uptake sequence (23). This favours genetic material from closely related species, at the expense of DNA from other organisms.

In Paper II, transfer of the sulphonamide resistance from commensal Neisseria isolates to N. meningitidis by transformation was unsuccessful. In order to explain this, the folP gene and surrounding regions from commensal Neisseria strains were examined (Paper III). The species examined could be divided into two groups: one group consisting of N. subflava, N. sicca and N.

mucosa, and the other group of N. lactamica, N. cinerea and the pathogenic

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Neisseria species. The main difference between these groups was an inversion of a 15 kilobases genomic segment, with one breakpoint upstream of the folP gene. Furthermore, the former group had an altered form of the DNA uptake sequence (DUS), where one base pair had been changed compared to the DUS found in pathogenic Neisseria species. This one base pair difference led to almost 50% reduction in transformation efficiency for N. meningitidis.

The differences between commensal species and the pathogenic Neisseria

species found in Paper III are likely to restrict the transformational spread of DNA between distantly related species and could have contributed to the failed transformations in Paper II. However, the ET-5 type of resistance gene could still originate from a distantly related or unrelated species. The introduction of a folP gene mediating resistance to a susceptible bacterium confers a strong selective advantage in a sulphonamide-containing environment, and even very rare events are therefore likely to become fixed in the bacterial population. Laboratory experiments, on the other hand, have a detection limit at 10-8 transformations per recipient.

Another example of very rare events that have been observed in nature, but probably would not be detected in laboratory experiments, is the horizontal transfer from Haemophilus influenzae to N. meningitidis (19, 41). These two species are very distantly related and they require completely different DNA uptake sequences for efficient transformation. The transferred DNA contains genes coding for virulence factors, which are naturally under strong selection pressure in the pathogenic N. meningitidis.

Interactions of DHPS with HPPK

Background:

In Paper I, the affinity of the DHPS enzyme for pAB was complemented with Km measurements for the other substrate, pterin diphosphate. This revealed unexpectedly high values for enzymes from sulphonamide-resistant strains (Table 3). A high Km value means that a high concentration of the substrate is needed to achieve sufficient substrate binding to the enzyme. Wild type enzymes are therefore expected to have low Km values to facilitate efficient reactions even at low substrate concentrations; however, DHPS enzymes with high Km values for pterin were found in apparently well-adapted bacterial isolates. This finding may be explained by a proposal on physical contact between DHPS and the enzyme producing the pterin diphosphate: the HPPK enzyme. A directed transfer of pterin diphosphate from HPPK to DHPS would result in a local very high concentration of pterin diphosphate near the binding site of DHPS, and subsequently a high

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degree of substrate binding despite low affinity. Such substrate channelling would facilitate efficient folate production regardless of the Km value of the DHPS. Another explanation for the high Km values might be that physical interactions between DHPS and HPPK could invoke conformational changes necessary for adopting a correct pterin binding site. In this case, the Km

values obtained for pterin diphosphate in Paper I, measured on isolated DHPS enzymes in the absence of the interacting HPPK partner, would not reflect affinity in the normal in vivo situation.

Evaluation of the channelling of pterin diphosphate from HPPK to DHPS may provide new insights into folate biosynthesis and reveal new drug targets. Two features of the HPPK enzyme support the channelling hypothesis. First, the rate-limiting step in HPPK catalysis is not the chemical reaction but rather the release of products – presumably the pterin diphosphate (47). Physical interaction with DHPS might be required to induce conformational changes that facilitate product release. Second, three-dimensional structures of HPPK revealed that a C-terminal helix becomes highly mobile upon substrate binding, and Keskin et al. (36) suggest that the purpose of this flexibility is to endorse interaction with the DHPS enzyme.

Present investigation (Paper IV):

The specific aims of this study were: to clone the putative folK gene from N. meningitidis to confirm its identity and function to detect possible protein-protein interactions between DHPS and HPPK

Table 3: Pterin diphosphate Km values for wild type DHPS enzymes

SuS = sulphonamide-susceptible; SuR =sulphonamide-resistant; SD = standard deviation

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Characteristics of N. meningitidis HPPK

The gene coding for HPPK is designated folK. A putative folK was annotated in the meningococcal genome sequences based on sequence similarities with folK from other organisms (67). Using PCR primers designed from the genome sequence, the putative folK gene was amplified and sequenced from six clinical isolates of N. meningitidis (Paper IV). The gene product from three of these was also tested for HPPK activity, with positive results for all variants. Thus, the annotation of the folK gene was correct – it coded for a functional HPPK enzyme.

In order to determine if the extensive sequence alterations in the resistant DHPS enzymes required compensatory alterations in the HPPK, the six clinical isolates represented both sulphonamide-resistant and -susceptible strains. However, the folK sequences from the selected isolates had very similar sequences and the few variations present were not linked to sulphonamide resistance. The most variable folK came from the susceptible isolate BT054: this gene had an internal part that differed by 20% from the other folK genes (Paper IV). This mosaic structure was likely to result from horizontal transfer of a part of the folK gene from another species. As noted earlier, interspecies exchanges are common in Neisseria and imported DNA sequences have been detected in several other housekeeping genes in N.

meningitidis (24, 85, 104).

Substrate channelling

Coupling between HPPK and DHPS during folate biosynthesis was previously addressed in a study concerning the bifunctional HPPK-DHPS enzyme from pea leaf mitochondria (61). This work compares kinetic parameters from the HPPK reaction with corresponding parameters from the coupled HPPK-DHPS reaction, but the results could neither support nor exclude channelling of pterin diphosphate within this enzyme. Even if detailed kinetic measurements can provide insights into a possible substrate channelling process, other methods are required to confirm such findings.

Potential protein interactions between HPPK and DHPS from N.

meningitidis were first examined with the yeast two-hybrid system. The formation of a dimer from two DHPS subunits was used as a positive control in this system. However, the two-hybrid assays were unsuccessful in detecting protein interactions between DHPS and HPPK, as well as between two DHPS subunits. This could be caused by an impeding effect of the transcription-factor domains of the hybrid proteins. Another reason for failing to detect HPPK-DHPS interactions could be that these enzymes interact only during active catalysis and not when the enzymes are inactive, as in the two-hybrid assays. Substrate channelling of pterin diphosphate requires physical contact only during the actual transfer, so the interaction between these proteins might be transient. Therefore, a method for studying

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the direct transfer of pterin diphosphate from HPPK to DHPS was developed (Paper IV).

The method was based on the isotope dilution principle (86). The two enzymes involved in an assumed channelling process are incubated with all substances required to complete the two-step reaction. One of the substrates is labelled so that the first enzyme transfers this label to the intermediate and the second enzyme transfers it to the end-product. If a non-labelled intermediate is added and the two enzymes operate separately, the second enzyme can use either the labelled intermediate produced by the first enzyme, or, the additional non-labelled intermediate. Thus, a non-channelled process will result in a mixture of both labelled and non-labelled product: the more intermediate added to the reaction, the less labelled product formed. Conversely, a channelled process will be less affected by the external addition of intermediate, since the second enzyme preferentially uses the intermediate transferred to its binding site directly from the first enzyme. Thus, a channelled process can be distinguished from a non-channelled process by monitoring the decreasing amount of labelled product when an increasing concentration of non-labelled intermediate is added to the reaction.

The method developed in Paper IV used 32P-labelled ATP and a new HPLC assay to separate the compounds after the reaction was completed. Partially purified HPPK and DHPS enzymes from N. meningitidis were used to convert 32P-ATP to pterin 32P-diphosphate (the intermediate) and 32P-pyrophosphate (the end-product). The pyrophosphate was then separated from the other components in a reverse phase HPLC assay based on a complex formation of the phosphate ions with tetra-butyl-ammonium ions. Reaction series with an increasing concentration of non-labelled pterin diphosphate confirmed the identity of the HPLC peaks and that the method functioned as intended. The method will be used in further studies with monofunctional enzymes from N. meningitidis and Streptococcus pyogenes,as well as with the bifunctional enzyme from the malaria parasite Plasmodium falciparum.

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Concluding remarks

Sulphonamide resistance due to alterations in the chromosomal gene for DHPS, folP, has been reported for several species, including bacteria, fungi and protozoa. In Neisseria, mutations resulting in amino acid alterations at positions 31, 66, 68, 84, 194, and insertions after position 75 and 194 contribute to the resistance phenotype. Mutation of residue 31 appears the most efficient alteration. Residue 228 has also been suggested as important for sulphonamide resistance in N. meningitidis. Bennett and Cafferkey (7) observed that all susceptible isolates carry an arginine in this position, whereas resistant isolates invariantly have a serine. Position 228 is located next to an invariant tripeptide that binds to sulphonamides; therefore, mutations in position 228 are likely to affect sulphonamide resistance. Indeed, a mutation in this position confers resistance in clinical isolates of Streptococcus pyogenes (34). However, previous work on N. meningitidis

DHPS does not support this view (26). Mutagenesis experiments were able to convert originally resistant DHPS enzymes to susceptible variants without altering the serine in position 228, suggesting that residue 228 does not contribute to resistance in N. meningitidis. Wild type E. coli also has a serine in this position, without being resistant. Clearly, the contribution of position 228 to sulphonamide resistance varies between different species.

The fact that sulphonamide resistance in N. meningitidis persist, despite decreased selection pressure, suggests that resistance does not incur any fitness cost for the bacteria. This can be explained in three ways. Firstly, resistance evolved by accumulation of point mutations, including compensatory mutations that abolished the fitness costs of the resistance mutations: a view supported by the mutational pattern observed in the point-mutated type of resistance gene in N. meningitidis.

Secondly, mutations conferring sulphonamide resistance lack fitness cost, so mutational adaptation to sulphonamides could occur without concern for the fitness. This is supported by the experiments with isogenic clones expressing mutated folP genes, which illustrated identical growth rates regardless of the properties of the produced DHPS. At least in laboratory experiments, DHPS enzymes with sub-optimal substrate binding properties did not impede growth of N. meningitidis, indicating a minor function for the DHPS activity in the general fitness.

Thirdly, the sulphonamide resistance genes originate from an intrinsically resistant bacterium. There is a natural variability in folP sequences from

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different organisms and it is possible that one of the many folP variants could express a DHPS with naturally low affinity for sulphonamides. Such folP variants could have been transferred to N. meningitidis by natural transformation, instantly providing the recipient with high-level resistance without any fitness costs. The ET-5 type of resistance gene might be an example of such intrinsic resistance.

The widespread occurrence of sulphonamide-resistant N. meningitidis can be explained by a combination of horizontal transfer and an epidemic population structure. Horizontal transfer by natural transformation is a well-documented process in N. meningitidis. Once a folP variant appears in a N.

meningitidis strain, it becomes immediately available to all other strains through natural transformation. In addition, some sulphonamide-resistant N.

meningitidis strains cause large epidemics and the resistance can thereby spread to a large number of humans and result in a high proportion of resistant isolates in some geographic regions.

Transformation is common between different Neisseria species. However, interspecies horizontal transfer is likely to be restricted by sequence diversity, variable genetic organisation and differences in DNA uptake sequence. A comparison of the chromosomal region surrounding the folP

gene from various Neisseria species revealed that distantly related species have a deviating gene succession and altered DNA uptake sequence compared to N. meningitidis. This suggests that horizontal DNA transfer from distantly related commensals to N. meningitidis is restricted by differences in regulatory elements such as the DNA uptake sequence. Almost all examples of DNA import into N. meningitidis involve the closest relatives (see for example 48, 85, 87, 104), whereas transformation events with more distantly related species are much scarcer.

The commensal bacterial species may act as a reservoir for antibiotic resistance genes. Several studies have concluded that antibiotic-resistant commensal bacteria reside in healthy individuals, even if the person has not been treated with antibiotics for a long time. For example, Lester et al. determined a 54–96% carriage rate of E. coli resistant to one or more antibiotics in healthy children, despite at least four months of abstinence from antibiotics (45). The corresponding numbers for sulphonamide resistance was 36–87% of the E. coli isolates. The results presented in this thesis indicate that sulphonamide resistance is also present in the commensal Neisseria flora in the pharynges. Thus, sulphonamide resistance seems to be firmly established in N. meningitidis as well as in the commensal flora.

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Acknowledgements

This work was performed at the Department of Pharmaceutical Biosciences and at the Department of Medical Biochemistry and Microbiology, Uppsala University. I would like to thank all who have contributed to the realisation of this thesis. I am especially grateful to:

My supervisor Göte Swedberg for always taking time to answer my questions and for help and support. Thanks also for allowing me to take a year off when I most needed it!

Ola Sköld for accepting me as a PhD student, for kind interest in my work and for critical reading of my manuscripts. Your comments were really helpful!

Christian Fermér for great supervision during my “exjobb” and for introducing me to the wonders of DHPS activity!

Bjørn-Erik Kristiansen and Peter Rådström for starting the project about sulphonamide resistance in Neisseria meningitidis. Without your work this thesis would never have been written!

Kristina Lundberg, Claës Linder, Lars Sundström and Lars Engstrand for sharing your knowledge and for help in various ways. Additional thanks to Lars S. for nice chats during long evenings at the lab and for allowing me to order my laptop PC.

My former PhD colleagues in the old B9:3 corridor: Katrin Ström, Mats Gullberg, Karin Hansson and Masood Kamali. Thanks for teaching me a lot of useful things during my first year as a PhD student, for friendships and great dissertation parties. Thanks also Masood for correcting my thesis!

My present PhD colleagues Maria Jönsson and Carolina Johansson for friendship, support, help and great pub evenings. A special thanks to Maria for your work on pyrophosphate detection!

The PhD students at the Pharmaceutical Faculty for electing me as chairman for the PhD student association.

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All the students and employees at IMBIM for making it so nice to work here! Thanks to Inger and Kajsa for sequencing assistance and to Kerstin and Olav for excellent computer support, especially when I accidentally deleted my user from my computer. A special thanks also to Aneta for help with the FPLC and silver staining, to Krzysztof for sharing your endless knowledge about chromatography and for lending me your HIC column, to Johan Ledin for help with the HPLC, and to Maarten for your invaluable help with converting my Excel figures.

Ann-Beth Jonsson at KI and her students for allowing me to work with my meningococci in their lab and for company during the Neisseria conferences.

Mira Macvanin at ICM for allowing me to use the Bioscreen reader and for generous help with interpretation of the data.

The undergraduate student Tina Olsson for constructing one DHPS-mutant for me. Thanks also to Adriano Atterman, Laila Bucht, Björn Eklund and Andreas Dahlgren for help with “normalfloreprojectet”.

My friends outside the university for reminding me about life besides my studies. A special thanks to Pelle for encouraging phone calls and pub evenings, to Gabriella for showing how quickly a thesis can be accomplished, and to Lena Klintberg for kind friendship, pep talks and fishes.

My dear family for always being there for me. Thanks to my sisters Camilla, Jessica, Gizela, Kicki and Lotta and their families for friendship and enjoyable dinners and birthday parties. Thanks to my cousin Magnus for inspiring me to start a university education (nu får du snart lyfta på hatten!).

My mother and my father and Eva, for love, support, encouragement and help in various ways. You are the best parents!

Peter, for endless encouragement and for being the best boyfriend imaginable!

This work was financially supported by the Swedish Research Council and the AFA Health Fund. I am grateful to “CD Carlssons stiftelse” and “Apotekarsocietetens resestipendium” for allowing me to participate in international meetings.

34

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