the mechanism of action of fosfomycin (phosphonomycin)

THE MECHANISM OF ACTION OF FOSFOMYCIN (PHOSPHONOMYCIN)

Frederick M. Kahan, Jean S. Kahan, Patrick J. Cassidy, and Helmut Kropp

Merck Institute f o r Therapeutic Research Rahway, New Jersey 07065

INTRODUCTION

The discovery of fosfomycin, a new antibiotic produced by strains of Streptomyces, was announced under its former name phosphonomycin by Hendlin and colleagues in 1969.l The chemical structure shown in FIGURE 1 combines two unusual features: an epoxide ring, rare among antibiotics, and a carbon-phosphorus bond which is seen to occur here for the first time among the natural products of the bacteria.

Most of the present account concerns the determination of the enzymatic step in cell wall biosynthesis that is ultimately blocked by fosfomycin. We compare in detail the action of that enzyme’s catalytic center upon the antibiotic and its normal substrate. We also describe the role of two stereospecific nutrient transport systems that by mediating the entry and accumulation of fosfomycin comprise the determining factors in the sensitivity of various bacteria to this polar antibiotic.

Although the main conclusions of these mechanism of action studies were briefly stated by us in the initial announcement,’, the present publication is the first in which there appears any portion of the original experimental data that support those conclusions. Several reports have appeared from other laboratories 4, and from our own IJ in which certain basic findings and meth- odology were reproduced in the course of pursuing the independent goals of their studies. Their additional contributions to the understanding of fosfomycin action are cited at appropriate points in the succeeding text.

MATERIALS AND METHODS

Experiments performed with Salrnonella typhirnuriuni (MB 1997) employed Nutrient Broth (Difco) as the growth medium. With Staphylococcus aureus (Duncan), we use the media and “crude extract” preparation described by lto and Strominger.’ Escherichia coli strains L-1 and L-217 were provided by Dr. E. C. C. Lin and were grown in Nutrient Broth.

The sources and syntheses of radioactive substrates and other reagents have been previously described.O Pyruvyl transferase assays employed a buffer that contained 0.125 M sodium maleate (pH 6.8), 10 mM potassium fluoride, and 10 mM 2-mercaptoethanol. Substrate concentrations, unless otherwise specified in legends, are 3 mM UDP-GlcNAc and 0.5 mM pho~pho[~‘C]enolpyruvate. Measurements of the radioactive product were obtained as described.fi Inactiva- tion and labeling studies were performed throughout in 20 mM KPO, (pH 7.2) that contained 5 mM 2-mercaptoethanol (KPM) .

3 64

Kahan et al.: Fosfomycin Action Mechanism 3 65

The “purified pyruvyl transferase” from Micrococcus lysodeikticus (MB 1784) corresponds to the “phosphocellulose enzyme” of Cassidy and Kahar~ .~

Identification of the amino acid residue in pyruvyl transferase that is labeled by fosfomycin started from 620 mg of “phosphocellulose enzyme” that had been reacted with 70 pM [1,2-3H]fosfomycin (11 pCi/pmole) and 6 mM UDP- GlcNAc in 30 ml of 20 mM KPO, (pH 7.4) that contained 10 mM 2-mercaptoethanol for 30 min at 37” C. Fosfomycin rendered acid insoluble in this reaction amounted to 35 nmoles (210,000 cpm). After extensive dialysis against 0.2 M KPO, (pH 7.4), the reacted enzyme solution was heated to 100” C for 5 min. Pronase was then added to 3 mg/ml, and incubation was continued at 45” C for 18 hr under a layer of toluene. After removal of insoluble proteins by filtration, the soluble peptides (which contained 95 % of initial protein-bound radioactivity) were concentrated under reduced pressure and chromatographed on BioGel P-2 (200-400 mesh). The radioactivity eluted in two peaks between the internal markers, cyanocobalamin and P,. A subsequent elimination of most of the unlabeled peptides that remained in both peaks was effected by passage of the BioGel fractions, concentrated and adjusted to pH 4.5, through Dowex@-50-X2 (H+) . Radioactivity was retarded well behind the salt peak and emerged between 2 and 4 column volumes on continued elution with water. Both of the BioGel fractions, after Dowex-50 chromatography and additional treatment with Aminopeptidase Mg [30 U/ml, 0.2 M Tris (pH 8.2) for 3 hr at 37” C], yielded a radiochemically homogeneous and identical product, as

H C--c/ FIGURE 1. Structure of fosfornycin (~-cis-1,2- H,

epoxypropylphosphonic acid, MW 138). CH3‘ \d ‘P03H2

judged by paper electrophoresis in 8% formic acid (pH 1.9). This product was purified further by preparative paper electrophoresis in the same buffer, and this final fraction comprises the “isolated adduct” referred to in the text. The authentic 2-S-~-cysteinyl-l-hydroxypropylphosphonic acid preparations were purified from the crude reaction mixtures (see text) by analogous means, which employed gel chromatography for initial desalting of the chemical preparations and retardation on Dowex-50 (Hf) . Cocrystallization of the “isolated adduct” after thioglycolic acid reduction and preparative electrophoresis described in the text was effected as the benzylammonium salt from initial solu- tions of 7 mM in 80% isopropanol at 50” C. After prolonged crystallization at 0” C, 30% of the ninhydrin-assayable material remains in solution.

RESULTS AND DISCUSSION

Selective Inhibition by Fosfornycin o f Mucopeptide Biosynthesis in Intact Bacteria

The first indication that fosfomycin inhibited cell wall biosynthesis was the observation that bacteria exposed to inhibitory concentrations of fosfomycin in media of high osmolarity formed spheroplasts.’ Direct evidence of such inhibition is presented in FIGURE 2, which represents the rate of wall synthesis as the incorporation of diamino[l.’C]pimelic acid into acid-precipitable material.

366

E

H i.0 0. 8

2 0.6

0. 4

- ._

al

- m +i 0. 2 0”

T 4 s x

g 3 C

g 2 K .- 2 1

ml

Annals New York Academy of Sciences

0 control Q 5pM 0 50vM

turbidity

/ mucopeptide synthesis

P’

m

F:

viability x

2. 0 - .A I W

L E 0

1. 0 ; c

0.6 g 0. 2

0

7 4 0 -

E x

3 :

2 s

c ._ - a 0 c ._

Time - minutes

FIGURE 2. Selective inhibition of cell wall synthesis in S. typhirnuriurn. Inoculum was grown in broth at 37” C supplemented with 10 mM L-lysine and harvested when the A m reached 0.5. Cells were resuspended to Aem=0.5 in replicate flasks that contained broth supplemented with 20 mM L-lysine, 2 mM L-leucine, 70 pM DL-diamino- pimelic acid (DAPA) , and the indicated level of fosfomycin. For “mucopeptide synthesis,” sufficient [I-”CIDAPA was added to achieve final specific activity in the broth of 0.8 pCi/pmole; for “protein synthesis,” ~-[l-“C]leucine was added to 0.1 pCi/ pmole. After the indicated times of incubation at 37” C, 10-ml samples of labeled cells were brought to 0.3 M in trichloroacetic acid, heated to 90” C for 5 min, and washed by multiple cycles of centrifugation and resuspension in cold acid. “Muco- peptide synthesis” data were corrected by subtracting 30 cpm for each minute of labeling. This corresponds to the amount of label incorporated into protein-bound lysine (determined by chromatography of 6 N HCl hydrolysates of late time samples).

This rate declines rapidly to zero, at which time the viability of the culture begins to drop, followed minutes later by frank lysis. Because fosfomycin, even at the higher concentrations employed, failed to inhibit incorporation of leucine into protein (prior to the onset of bacterial lysis), we concluded that it interferes with cell wall synthesis in a selective fashion and is without effect on the general metabolic pathways that serve protein synthesis.

We tried to determine the stage at which cell wall biosynthesis was blocked, by analyzing the N-acetylamino sugar ester pool of fosfomycin-treated cells. All known inhibitors of cell wall synthesis induce the accumulation of these compounds in certain strains of Staphylococcus; the composition of the attached peptide chain is diagnostic of the site of antibiotic action. In TABLE 1 we show, however, that fosfomycin fails to induce the accumulation of such sugar esters in a strain that, in control experiments, responded as expected to the addition of either penicillin G or cycloserine. That fosfomycin does, nevertheless, affect

Kahan et al. : Fosfomycin Action Mechanism 3 67

the flow of N-acetylamino sugar esters into the cell wall at an early stage is shown by the diminished effectiveness of penicillin treatment in inducing the accumulation of these precursors when superimposed on cells first exposed to fosfomycin for 30 min.

A survey of the sensitivity to fosfomycin of enzymes concerned with the incorporation of alanine into cell wall precursors proved essentially negative. By employing extracts prepared from Staphylococcus and assayed by the methods of Ito and Strominger,T* we found the alanine racemase, the D-alanyl-D alanine ligase, and the D-alanyl-D-alanine “adding enzyme” to be inhibited less than 20% by 7 mM fosfomycin. The L-alanine “adding enzyme” was inhibited as much as 92% by 25 mM and 50% by 2.5 mM antibiotic. This effect could not, however, account for the ability of 0.02 mM fosfomycin to inhibit growth of that strain of Staphylococcus, and it lost all significance when compared with the profound effect of fosfomycin, which will now be described, on an even earlier step in the biosynthetic sequence.

Fosfomycin Znactivation of “Pyruvyl Transferase,” the First Step in Cetl Wall Biosynthesis

The branch-point between general nucieotide-sugar metabolism and N- acetylmuramyl peptide synthesis is the reaction of UDP-N-acetylglucosamine (UDP-GlcNAc) and phosphoenolpyruvate to form UDP-GlcNAc-3-enolpyrvyl ether.’*. lR The enolpyruvate product is subsequently reduced to UDP-N- acetylmuramic acid, from whose free carboxyl group a pentapeptide sequence is then initiated. The enzyme that catalyzes the transfer of the enolpyruvate group to UDP-GlcNAc, phosphoenolpyruvate:UDP-GlcNAc-3-Oenolpyruvyl

TABLE 1 FOSFOMYCIN SUPPRESSION OF ACCUMULATION OF CELL WALL PRECURSORS

INDUCIBLE BY PENICILLIN *

N-Acetylamino Sugar Esters Concentration Exposure (pmoles/ml packed cells t )

Antibiotic (mM) (min) Total Accumulated

1.04 (0) None - - Fosfomycin 0.3 Z 45 1.29 0.25

3 .O 45 1 .so 0.46 Penicillin G 0.05 45 26.0 25.0 Cycloserine 0.5 45 15.0 14.0

,,} § 3.4 2.4

0.05 15 7.25 6.0

Fosfomycin 0.3

Penicillin G 0.05 final 15 Penicillin G

+ > * Staphy~ococcus aureus (Duncan). ’r Extracted and estimated by the methods of Strominger.” Z Lysis of culture begins 60 min after addition. § Penicillin G added to culture 30 rnin after fosfomycin and simultaneously to an

untreated control; incubation continued for 15 min.

368 Annals New York Academy of Sciences

transferase, will be referred to as pyruvyl transferme. We find (TABLE 2) that pyruvyl transferase activity can be totally inhibited by fosfomycin and that it exhibits a significant response to this antibiotic at levels only threefold greater than those that kill the organism from which the extract is prepared. The apparent percentage inhibition increases with time of incubation, which suggests that the enzyme becomes progressively inactivated. Increasing the concentration of the substrate UDP-GlcNAc has little effect upon the degree of inhibition observed. The relative degree of inhibition decreases markedly, however, as the concentration of phosphoenolpyruvate in the reaction mixture is increased. These data imply that fosfomycin inactivates the transferase by serving as a phosphoenolpyruvate analog.

It was therefore anticipated that the pyruvyl transferase would show maximum sensitivity to fosfomycin when preincubated in the absence of competitive phosphoenolpyruvate. In fact, under these conditions, complete inactivation was effected (TABLE 3) by concentrations of fosfomycin comparable to growth- inhibiting levels. Enzyme activity could not be restored, even by prolonged dialysis. An unexpected finding, shown in TABLE 3, is that the second non- competitive substrate, UDP-GlcNAc, was an obligatory cofactor for inactivation during the prior incubation phase. Subsequent experiments revealed that the rate of inactivation at a fixed level of fosfomycin displayed a concentration dependency for UDP-GlcNAc that coincided with the Michaelian saturation curve for UDP-GlcNAc as substrate. Furthermore, UDP-GlcNAc could not be replaced in its cofactor role by unesterified hexosamines, their N-acetyl

TABLE 2 FOSFOMYCIN INACTIVATION OF THE PYRUVYL TRANSFERASE OF Staphylococcus

BY AcnoN AS AN ANALOG OF PHOSPHOENOLPYRUVATE"

[2-"C] Phosphoenol- Reaction

UDP-GIcNAc pyruvate T Fosfornycin Time Inhibition (mM) (mM) (mM) (min) (% 1

2.5 99 0.25 81

0.13 70 0.06 37

2.0 1 .o 45

15 54 2.0 1 .O 0.13 3 0 74

45 77

6.0 1 .O 0.13 45 78

3 . 0 54 2.0 1 .o 0.13 45 70

0.3 87

__

* Reaction mixtures of 0.1 ml contained 0.2 mg protein with an activity of 0.067

.t Specific activity, 0.6 mCi/mmole. nmoles/rnin measured at highest indicated substrate concentration.


TABLE 3 INACTIVATION OF PYRUVYL TRANSFERASE * IN THE ABSENCE

OF PHOSPHOENOLPYRUVATE

Product Formed in Prior Incubation Complete Reaction

without Phosphoenol- Mixture 'i pyruvate (nmoles)

Concentration GlcNAc Time Inhibition Fosfomycin UDP-

(PM) (mM) (min) +Drug -Drug (% 1 125 - 0 0.5 1 0.683 26

6 20 0.025 0.505 95 0 20 0.28 0.35 21 - 0 0.65 0.683 5

6 20 0.122 0.505 76 0 20 0.338 0.35 4

6 - 0 0.67 0.683 2 6 20 0.346 0.505 3 1

1 1

25

1 :> Crude extract of S. aurerts, 0.2 mg protein/reaction. i Complete reaction mixtures contained phospho['"C]enolpyruvate ( 3 mM) and

UDP-GlcNAc (2 mM). Incubation was conducted for 15 min at 37" C.

derivatives, or by other UDP-sugars (e.g., -glucose, -mannose, and -xylose) . This unusual role of UDP-GlcNAc as "gate-keeper" of the pyruvyl transferase greatly simplified subsequent studies by providing an internal control that would immediately authenticate the specificity of binding of radioactive fosfomycin and phosphoenolpyruvate to crude or only partially purified (5-100- fold) enzyme preparations.

Inactivation of Pyruvyl Transferase in Vitro Results in Covalent Attachment of Fosfomycin to Protein

The finding of irreversible inhibition effected by an epoxide-bearing structure led naturally to the expectation that fosfomycin is covalently linked in that process to enzyme. This was confirmed (TABLE 4) in experiments that employed [ 1 ,2-3H]fosfomycin. Covalent binding of fosfomycin to extract protein was observed only when UDP-GlcNAc was simultaneously present during the incubation. In such reactions, similar amounts of radioactivity were found either directly precipitable by acid or associated with the protein fraction that emerged in the excluded volume from gel permeation columns. All of the latter radioactivity was also acid precipitable. Despite the obligatory participation of UDP-GlcNAc in the reaction of fosfomycin with enzyme, radioactivity from UDP-[14C]GlcNAc did not become acid precipitable in the course of the inactivation. Coincubation with fosfomycin also did not increase the small amount of nonspecific association of UDP-GlcNAc with the native protein separated from reaction mixtures by gel permeation chromatography.


We have found that [32P]fosfomycin (obtained by fermentation) is bound to the same extent as the synthetic 1,Ztritiated form, which demonstrates that the entire propylphosphonate backbone is incorporated into the enzyme. The stability of the linkage was affirmed by the retention of acid precipitability of the bound fosfomycin after exposure to 0.3 M NaOH for 10 min at 37" C or 0.3 M trichloroacetic acid at 100" C for 10 min.

The cofactor role of UDP-GlcNAc in both the inactivation of pyruvyl transferase activity and the incorporation of fosfomycin into protein argues strongly that these are due to the same chemical event. Further support for this conclusion derives from the parallel kinetics of inactivation and labeling (TABLE 5 ) . The ratio of inactivated pyruvyl transferase to radioactivity acquired in precipitated protein remained constant over the time course of treatment and provides a measure of the catalytic turnover number (Kcat) of the enzyme. Note the similar magnitude of Kcat values for partially purified enzyme from M. Zysodeikticus (TABLE 4) and for the present example, crude extracts of S . typhimurium. This finding is consistent with the assumption that fosfomycin has reacted uniquely and with the same type of protein in the two different cases.

TABLE 4 REACTION OF PARTIALLY PURIFIED PYRUVYL TRANSFERME

WITH RADIOACTIVE FOSFOMYCIN AND UDP-Glc-NAc *

Analysis of Proteins Separated from Substrates on Gel Direct Analysis of

Reacted Protein Permeation Columns

Precipitable Radioactivity Enzyme Radioac- Enzyme (pmoles/mg ) Activity tivity Activity (nmoles/ (pmoles/ (nmoles/ Precipi-

Reagents min/mg) mg) min/mg) Total table

[gHIFosfomycin 9.3 0.4 9.7 5 < 0.3

[W]Fosfomycin 0.6 40.0 0.6 5 1 46.0

UDP-GlcNAc (3 mM)

UDP-["C]GkNAc + I - 2.0 9.6 31 1.8 ( 3 mM)

+ Fosfomycin

(70 PM)

* Reaction mixtures contained in 0.75 ml KPM, 15 mg enzyme protein plus the indicated reagents. Incubation was conducted for 30 min at 37" C. Each reaction was applied to a 1s-ml column of BioGel P-6 (100-200 mesh), equilibrated with KPM, and was eluted at 0" C with KPM. Fractions were assayed for precipitable radioactivity and pyruvyl transferase activity by described methods." The enzyme's catalytic turnover rate, K,.t, is 211-232 min-' on the basis of the above data.

(70 PM)

UDP-["C]GlcNAc 1.5 0.3 32 2.0 I -

Kahan et al. : Fosfomycin Action Mechanism 371

TABLE 5 KINETICS OF INACTIVATION AND LABELING OF CRUDE PYRUVYL TRANSFERASE

BY [8H]FOSFOMYCIN *

Acid-Precipitable Transferase Activity Time Radioactivity (nmoles/min/rng) K,,t (min) (pmoles/mg) Remaining Inactivated (rnin-I)

0 1 5

15

0.21 1.86 “0” - 4.45 1.22 0.64 144 8.8 0.17 1.69 192

10.7 0.06 1.80 168

0.15 UDP-GlcNAc l5 omitted 2.04 - -

* Crude extract of S. typhinzrrrircnz (MB-1997) was prepared by exposure of a 20% cell suspension in KPM to sonic disintegration for 1 min at 0” C. After centrifugation at 10,OOOg for 10 min, the supernatant was precipitated with 3 volumes of saturated ammonium sulfate. The precipitate was redissolved to 10 mg/ml and dialyzed against KPM. Inactivation was initiated by adding 70 pM [*H]fosfomycin (1 1 pCi/pmole) to extracts supplemented, except in the last row, with 3 mM UDP- GlcNAc. Aliquots of 1 rnl were removed for analysis by acid precipitation of radioactivity bound, and at that time, 50 pl were added to 50 pl of the assay mixture to measure residual pyruvyl transferase activity. The turnover number, K,., , is the ratio of pyruvyl transferase inactivated/fosfomycin bound.

Fosfomycin Inactivation and Labeling of Pyruvyl Transferme in the Intact Bacterium

To demonstrate the relevance of the above enzymological findings to the mechanism of inhibition of bacterial growth, we have analyzed extracts of a strain of S. typhimuriurn, which was selected for its high sensitivity to fosfomycin, after 30 min of growth at levels of tritiated fosfomycin that ranged from threefold below to threefold above the minimum inhibitory concentration (1.1 pM) in that broth (TABLE 6). A self-consistent relationship should hold between the response of this strain to fosfomycin in vivo (FIGURE 2 ) , the kinetics of inactivation of its pyruvyl transferase in vitro, and the attachment of labeled drug to protein.

We first found that enzymatic activity in extracts of treated cells was markedly reduced, even at subinhibitory levels of antibiotic in the growth medium (TABLE 6, sample 2). However, only in cells exposed to the inhibitory range (samples 3 and 4) did the residual activity drop below 10% of the initial value. At the 10% level (-0.2 nmole/min/mg protein) under optimum conditions, just enough UDP-GlcNAc enolpyruvate would be provided for the synthesis of 10 mg of completed murein per gram of soluble protein in each 30-min doubling time. This corresponds to the usual estimates (1% dry weight) of murein content in the gram-negative organisms. Thus, the inhibition of the pyruvyl transferase observed is sufficient to account for the bactericidal action of the antibiotic.

Next, we determined that the incorporation of radioactive fosfomycin into acid-precipitable material paralleled the degree of inactivation of pyruvyl transferase activity, as reflected in the constancy of the ratio Kcat (TABLE 5).

3 72 Annals New York Academy of Sciences

Furthermore, upon reexposure of extracts to tritiated fosfomycin and UDP- GlcNAc, the additional labeling where residual activity was present did not raise the maximum specific radioactivities above those achievable by in vivo exposure. In the “cold-drug’’ control (sample 5 ) , as expected, there was no acquisition of radioactivity during the reexposure to tritiated fosfomycin. These data prove that the population of proteins labeled in vivo are identical with those studied at length in vitro.

Importantly, the radioactivity found attached to extracted proteins was no greater than that which was acid precipitable in aliquots of unbroken cells.

TABLE 6 INACTIVATION OF PYRUVYL TRANSFERASE AND INCORPORATION OF RADIOACTIVITY

1NTO PROTEIN OF s. Iyphimifritfnl EXPOSED in ViVo TO [sH]FOSFOMYCIN <‘

Analysis After Additional in Vitro Exposure

in Vivo to Labeled Drug Analysis of Cells Exposed

Fosfomycin Fosfomycin Fosfo- Enzyme Concentration Bound (pmoles/ Enzyme rnycin Activ-

Sam- Me- in Whole Ex- (mu/ (prnolesl ( m u / (PM mg protein) Activity KCRt Bound ity Keat

ple dium Cells Cells tract mg) (min-l) mg) mg) (rnin-l)

1 0 - - - 2.61 - 11.3 0.02 230 2 0.36 4.9 8.15 9.8 0.83 183 13.1 0.005 200 3 1 . 1 27.0 10.8 14.0 0.14 176 14.1 0.014 185 4 3.6 55.0 10.8 14.2 0.11 177 13.5 0.005 193 5 3.6 unlabeled - -

* Culture was grown to Awu=0.74, harvested, and resuspended in one-half volume of fresh broth. Each experiment represents 40 ml of this suspension incubated at 37” C for 30 min with the indicated level of [*H]fosfomycin (11 pCi/&mole). After centrifugation, the cell pellet (170-200 mg wet weight) was resuspended in 1 ml of cold KPM. A sample was removed and precipitated directly to yield the “whole cells’ ” radioactivity. Crude extracts were prepared as described under TABLE 5 . A sample of the initial sonic extract, counted directly, provided the “in cells’ ” fosfomycin concentration. The “in vitro exposure” to 70 p M [3H]fosfomycin, 1 1 pCi/pmole, and 3 mM UDP-GlcNAc, lasted 15 min at 37” C. The unit of pyruvyl transferase activity, 1 mU/mg, is equivalent to 1 nmole/min/mg.

0.03 - 0.1 0.007 -

It was thus established that inactivation and labeling occurred during growth of the cells rather than in the course of work-up in some “trivial” in vitro fashion.

Finally, we calculated that intracellular levels of free fosfomycin in the harvested cell pellet exceeded broth levels by 10- to 2O-fold, which accounts well for the unusual sensitivity of this strain to the antibiotic. The resultant intracellular concentrations are within the range used in the preceding enzymological studies. A more precise comparison of the kinetics of in vitro and in vivo inactivation would require estimates of the steady-state levels of phosphoenolpyruvate, UDP-GlcNAc, and the rate of synthesis of pyruvyl transferase. For the latter, we are at least able to exclude the possibility that

Kahan et al.: Fosfomycin Action Mechanism 313

derepression of enzyme biosynthesis occurred in response to declining rates of cell wall synthesis, because the total amount of transferase, which is reflected in fosfomycin binding capacity per milligram of protein, remained constant over the range of antibiotic concentrations used.

Structure of the Fosfornycin Adduct with the Catalytic Center of Pyruvyl Transferme

In this and the following section, evidence is adduced for a steric relationship between fosfomycin and phosphoenolpyruvate that applies uniquely to the reaction mechanism and the catalytic site of the pyruvyl transferase. Because the chemical aspect of fosfomycin viewed from the enzyme's reactive site is frozen into the resulting adduct, we elected to isolate and identify the product. Having earlier eliminated the possibility that UDP-GIcNAc was the acceptor, we correctly guessed that a cysteine residue was the most likely candidate in view of the enzyme's activation by free thiols and its rapid and complete inactivation by sulfhydryl reagents.13 The synthesis of two stereoisomers of 2-S-~-cysteinyl-l-hydroxypropylphosphonate needed for identification of the isolated adduct illustrate two mechanisms by which such a fosfomycin adduct could, in principle, arise. A primary attack of the enzyme nucleophile directed, in accord with the existing chemical precedent,'.& at the C-2 of fosfomycin results in a threo configuration that would be retained in the isolated product. The corresponding threo thioether was synthesized by Drs. D. F. Veber and S. L. Varga of these laboratories by reaction of cysteine with disodium fosfomycin in liquid hydrogen fluoride at -40" C. If, however, fosfomycin were first attacked by an amino acid residue other than cysteine to form a reactive adduct and the hydroxypropylphosphonate residue were subsequently transferred to cysteine (perhaps adventitiously during degradation of the native protein), a second inversion at C-2 would occur, and the final product would possess the erythro configuration. The erythro thio ether was prepared by aqueous alkylation of L-cysteine with threo-2-chloro-1 -hydroxypropylphosphonate, which is itself the result of the addition of hydrogen chloride to fosfomycin in dry ether." The above chemical routes were adopted only after all attempts made to react fosfomycin with free amino acids in aqueous solution had resulted either in no product or in hydrolysis of the antibiotic to its diol. The failure of fosfomycin to label cellular proteins, except in the presence of UDP-GlcNAc, similarly reflects the chemical inertness of the epoxide, other than toward its intended enzymatic target.

We detail under MATERIALS AND METHODS the isolation from 621 mg of M . lysodeikticus protein, purified only eightfold with respect to pyruvyl transferase activity, of a tritiated fosfomycin-amino acid adduct equivalent to only 10 pg. The sequence of proteolytic digestion and column chromatography had as its primary objective the recovery of a product of minimum size that was radiochemically homogeneous on electrophoresis. In fact, the unusually acidic nature of the adduct also permitted its separation from the bulk of unlabeled material by simple techniques and in a high overall yield of 40%. To our initial chagrin, the final isolate thus obtained failed to comigrate on electrophoresis with either of the authentic 2-thio ethers! Advised that this might reflect oxidation of the expected adduct to the sulfoxide during purification ( a common experience in the isolation without precaution of S-carboxymeth-


ylated cysteine peptides) , we passed the material through the sequence of reduction and oxidations indicated in FIGURE 3. It was then found, indeed, to comigrate only with the authentic fhreo compound in its respective three states of sulfur oxidation. Completing the proof, and excluding attack on C-1 of fosfomycin for which marker compounds were unavailable, the fully reduced natural product was cocrystallized with the authentic thio ether and yielded a constant ratio of radioactivity to ninhydrin-assayable material in the super- natants and crystals on two successive cycles. We conclude that inactivation of the pyruvyl transferase results from direct nucleophilic attack by a cysteine residue on C-2 of fosfomycin.

er thro 100 cpm origin suYfone-

I - -1 -

1-1 cm+ dans-OH threo-sulfone

r n t I - -

th re o-thio e the; erythro-thioether

3 t h re 0- sul foxides

performic oxidation

1 I I

isolated addud

thioglycolic reduction

H 0 l H A c reoxidation 2 2

FIGURE 3. Coelectrophoresis of [aHIfosfomycin adduct with authentic markers. Distribution of radioactivity in 0.5 cm strips about the origin is indicated by vertical bars; position of markers, located by ninhydrin, by the dark stripes. Electrophoresis, which was conducted in 8% formic acid (pH 1.9, 15 V/cm for 3 hr), causes a separation of 16 cm between neutral dimethylaminonaphthalenesulfonic acid marker (duns-OH) and its singly charged arnide (cathode at l e f t ) . Markers derived from erythro- or threo-2-S-~-cysteinyI-l-hydroxypropylphosphonic acid are designated by the configuration about C-2 and the oxidation state of S. Reduction conditions were 15% aqueous thioglycolic acid for 15 hr at 50" C under N2. Thio ethers were converted to diastereomeric sulfoxides (plus some sulfone) by 10% HIOa in acetic acid for 2 hr at 25" C . Sulfones were generated with 10% H20, in formic acid for 2 hr at 0" C , followed by hydrogen bromide?%

Properties of the Phosphoeno1pyruvate:Pyruvyl Transferase Intermediate

Our present conception of the steric relation between phosphoenolpyruvate and fosfornycin is derived both from the structure of the cysteine adduct just described and from a detailed study of the stable complex that can be generated between enzyme and its physiological substrate.6 That such a complex might


exist as more than a transient stage in enzyme catalysis was first suggested by observations made during a comparison of enzyme inactivation by fosfomycin and the sulfhydryl reagent, N-ethylmaleimide (NEM) . The initial rate at which NEM inactivates exceeded that of fosfomycin at a 100-fold greater concentration and showed no requirement for UDP-GlcNAc (FIGURE 4) .* A significant ( 10-20% ) residue of activity persisted, however, after extended NEM exposure, despite prior treatment with dithiols to ensure the maximally reduced status of input enzyme. This residue became inactivated, as shown, upon subsequent addition of UDP-GlcNAc. Alternatively, enzyme incubated with LJDP-GlcNAc and then exhaustively dialyzed was also totally susceptible to NEM. We postulated that the NEM-resistant residue represented a stable enzyme-"pyruvate" complex (at that time assumed to be a 2-S-acrylate) ,I5 formed in vivo or during extraction, that was capable of surviving the rigors of purification, storage, and dialysis. We again invoked a "gate-keeper'' role for UDP-GlcNAc in the formation of the natural complex with the additional proviso that should UDP-GlcNAc dissociate from the enzyme prior to con- summation of pyruvyl transfer, the pyruvate residue would be locked in to the catalytic site. Upon reassociation with UDP-GlcNAc, the enzyme could proceed to completion of the cycle.

The foregoing considerations led to the following stratagem for generating the stable complex at will. Enzyme was briefly exposed to complete reaction mixtures and next combined with activated charcoal sufficient to suddenly reduce the UDP-GlcNAc level 100-fold. Maximization of complex formation was promoted by conducting reactions at 0" C, at which the catalytic turnover rate was slow relative to the adsorption of UDP-GlcNAc. Analysis of this system has provided the following results and conclusions: 1. The stable complex is a phosphoenolpyruvate intermediate, because it is labeled equally by [32P]phosphoenolpyruvate, ph~spho[~~C]enolpyruvate, and by [32P]phosphate incorporated by phosphorolysis of UDP-GlcNAc-enol pyruvyl ether (the product of pyruvyl transferase). 2. UDP-GlcNAc is an obligatory cofactor for complex formation with phosphoenolpyruvate but is not found in the stable intermediate. 3. Phosphoenolpyruvate could be bound in the intermediate state to an extent that approaches 80% of the available binding sites measured by reaction with labeled fosfomycin. Enzyme preinactivated by fosfomycin did not form a stable complex. 4. Formation of the complex confers NEM resistance on the enzyme. Readdition of UDP-GlcNAc causes release of complexed phosphoenolpyruvate as a mixture of product and free phosphoenolpyruvate. 5. The complex liberates inorganic phosphate on exposure to dilute acid at more than 50 times the rate of acid hydrolysis of free phosphoenolpyruvate.

The last two findings suggest a covalent attachment of phosphoenolpyruvate to a cysteine residue in the enzyme, as opposed to a trapping of the free

* These inactivation studies were performed at 0" C, with all manipulations in a cold-room to minimize reversible autooxidation of pyruvyl transferase during the reaction phase. (Such oxidized enzyme is protected from NEM inactivation and has its activity restored when excess thiols are added during the assay phase.) The Figure also illustrates the direct proportionality between inactivation rates and fosfomycin concentration up to the highest levels that we have tested. At 30 mM and 0" C, fosfomycin inactivates with a first-order reaction rate that is 14% of the K,,t (15 min-') of the pyruvyl transferase reaction.


70

M

30

20

10

7

5

?

\

0 , O NEM. 0 .3 mM

A foslomycin. 30 mM

&treated enzyme

1 2 3 0 6 9

Exposure to inhibitor - min.

FIGURE 4. Kinetics of inactivation of purified pyruvyl transferase by fosfomycin and NEM at 0” C. Fosfomycin is added to reactions that contain 2 mg enzyme protein (19 nmoles/min at 37” C) and 0.3 pmole of UDP-GlcNAc in 0.1 ml of KPM. At intervals noted, I pl is removed and diluted into 0.1 ml of assay mixture, which is supplemented with 3.3 mM phospho[”C]enolpyruvate, so that inactivation during the assay phase at 37“ C remains below 12% in 10 min, even at the highest fosfomycin level tested. The control reaction (“100%”) represents 950 cpm. The “pretreated enzyme” had been incubated at 24 mg/ml in 20 mM KPO, (pH 7.5) that contained 2.5 mM dithiothreitol and 3 mM UDP-GlcNAc for 20 min at 25” C and was then dialyzed against KPM to decrease the residual UDP-GlcNAc below 15 pM. The “pretreated enzyme” and the untreated control were incubated with 2.5 mM dithiothreitol immediately prior to the NEM inactivation procedure. NEM was added to 25 pg “pretreated” or untreated enzyme in 20 mM KPOl (pH 7.5) to give a final volume of 0.1 ml. At the indicated exposure time, reactions were terminated by the addition of mercaptoethanol to 10 mM. To one series, UDP-GlcNAc was added to 3 mM, 3 min after the NEM, and incubation was continued. Assays of the remaining enzyme activity were performed by combining a 50-pl aliquot with 50 pl of assay mixture. The control reaction (“100%”) represents 1200 cpm.


substrate within a protein “cage.” Addition of enzyme4 to C-2 of phosphoenolpyruvate, with proton addition to (2-3, was also implied by the acid lability of the phosphate in the complex, as well as by the incorporation of a proton from water into the enol carbon of the product enolpyruvate (established by mass spectrometry).

Fosfomycin as a Lethal Phosphoenolpyruvate Analog only to the Pyruvyl Transferase

How can we account for fosfomycin’s specificity of action in bacteria, and nontoxicity in animals, if it is a reactive analog of an ubiquitous metabolite, phosphoenolpyruvate? By combining our deductions about the enzyme’s method of attack upon fosfomycin and phosphoenolpyruvate, we have arrived at our current conception of the steric relationship between them at the active site of pyruvyl transferase, which is illustrated in FIGURE 5 .

In the first step pictured in FIGURE 5 , the UDP-GlcNAc serves in some capacity as a cofactor but does not itself react covalently with enzyme or the other substrate. Fosfomycin has been oriented such that a proton donor site, H-B+, which accounts for protonation of C-3 of phosphoenolpyruvate, here serves to activate the epoxide. The attack of the cysteinyl sulfur at C-2 of fosfomycin can thus be viewed as an addition of sulfhydryl across the C-2-0 bond, in analogy with our proposed addition of sulfhydryl across the C=C bond of phosphoenolpyruvate in the physiological reaction.

In this first step, the lack of exact isosterism of the two compounds as written is also apparent in the three-dimensional models. Most notably, phosphoenolpyruvate contains a -COOH group, which may be expected to facilitate tight binding by electrostatic interactions. The lack of such a group in fosfomycin may account for its failure at even high concentrations to saturate the enzyme’s binding site (cf. FIGURE 4) . t This, then, provides one reason for the observed specificity of fosfomycin toward pyruvyl transferase: its greatest similarity to phosphoenolpyruvate resides not in its steric configuration but in the susceptibility of the C-2-0 bond to an enzymatic addition reaction.

Thus, we find that fosfomycin has either no detectable activity or a weak competitive inhibition on the other phosphoenolpyruvate-utilizing enzymes examined, namely enolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, and phosphoeno1pyruvate:shikimate-5-phosphate enolpyruvyl transferase.

In the absence of a close stereochemical similarity between fosfomycin and phosphoenolpyruvate, its failure to significantly inhibit any of the class of enzymes that catalyze nucleophilic attack at the phosphorus atom (pyruvate kinase, phosphoenolpyruvate carboxykinase, and so on) is readily understand- able. The reactions catalyzed by these enzymes require P-0 bond cleavage; this type of reaction is ruled out in fosfomycin by the presence of a P-C bond

f In contrast to the rapidity of inactivation afforded by high, though not saturating, fosfomycin levels are the substantially lower relative rates at which glycidol phosphate (2,3-epoxypropyl phosphate) inactivates triose phosphate isomerase and enolase.“ Although in these cases, saturating kinetics are found, with KlnaCt values of 1 and 5 mM, respectively, the maximum inactivation rates are only lo-“ and 2>< of the respective Kent values.

378 Annals New

1 U-oH

Inac t i va ted Enzyme

York Academy of Sciences

'0 c--- C - C H ~ D 2 4

p04 111

u-0, CrCH D

'02C'

U-OH

I1 +&+ E-PEP

UDP - GlcNAc - 3 - eno lpyruvy l e the r

FIGURE 5 . Schematic diagram that suggests the probable relationship between the reactions of fosfomycin and of phosphoenolpyruvate at the active site of pyruvyl transferase. The reaction with phosphoenolpyruvate is represented in deuterated water. U-OH is UDP-GlcNAc, with the 3'-OH depicted. and E-PEP is the stable enzyme-phosphoenolpyruvate complex. Although U-OH is required for activity at step I in both cases, it reacts covalently only at step I11 of the reaction with phosphoenolpyruvate. The reversible loss of U-OH at step I1 stabilizes the phosphoenolpyruvate adduct generated at step I. Note that all reactions with the enolpyruvate substrates are reversibIe but that after the enzyme-fosfomycin adduct is formed, it cannot be dissociated by further exposure to UDP-GlcNAc.


in the analogous position. Because, in addition, the lack of close steric similarity precludes effective competitive inhibition, fosfomycin cannot inhibit these enzymes either competitively or, as with pyruvyl transferase, by catalyzed reacton with the enzyme.

In the special case of enolase, it is possible to draw a diagram similar to that of FIGURE 5, which depicts fosfomycin at the active site, with its epoxide oxygen in place of the C-2 of phosphoenolpyruvate. In this case, however, even if the enzyme did catalyze a slow reaction with fosfomycin, the net result, following from a currently proposed mechanism for enolase,ls would merely be hydrolysis of fosfomycin, rather than inactivation of the enzyme.

The phosphoenolpyruvate : shikimate-5-phosphate-3-enolpyruvyl transferase is also refractory to inactivation by fosfomycin but is, unlike the enzymes discussed above, similar to the UDP-GlcNAc pyruvyl transferase in catalyzing net displacement of phosphate at C-2 of phosphoenolpyruvate by an alcohol, with overall retention of the double bond in phosphoenolpyruvate.19 It is the only other example of this class of enzyme currently known and is confined to plants and bacteria. The shikimate pyruvyl transferase has, like the UDP- GlcNAc pyruvyl transferase, been found to incorporate deuterium from water into its enolpyruvate product.20 The mechanism suggested for this enzyme differs, however, from our proposed mechanism for the UDP-GlcNAc pyruvyl transferase, in that the 3-hydroxyl of shikimate-5-phosphate, rather than enzymic sulfhydryl, is added across the phosphoenolpyruvate double bond. If this mechanism is correct, the product of such addition across the fosfomycin C-2-0 bond would be a fosfomycin adduct to shikimate-5-phosphate, rather than an inactivated enzyme. Thus, fosfomycin would not be observed to inactivate this enzyme. The data for the shikimate-5-phosphate pyruvyl transferase are also consistent, however, with a sulfhydryl addition mechanism, and it is possible that the mechanism of the two pyruvyl transferases are identical. In this case, we would expect fosfomycin to also inactivate the shikimate- 5-phosphate pyruvyl transferase, and our failure to observe such an inactivation must then be ascribed either to an even lower affinity of fosfomycin for this enzyme than is the case for UDP-GlcNAc pyruvyl transferase or to a slightly different configuration at the active site or, perhaps, to some other, as yet obscure, factor.

How Fosfomycin Enters and Is Accumulated by Bacteria

There is from one bacterial species to the next a wide variation in intrinsic sensitivity to fosfomycin. No major differences were found by us, though, either in the content of pyruvyl transferase or in that enzyme’s sensitivity to the antibiotic in extracts of five species that cover a range of in vivo suscepti- bilities from 1 to 200 pM. Differences were also not found between extracts of a sensitive strain and of a resistant isolate derived from it. Because no evidence could be found for metabolic inactivation of fosfomycin, we concluded that sensitivity is determined primarily by permeability of the bacterium to the antibiotic. Evidence was also obtained for an active participation of the host in antibiotic entry in those cases where high sensitivity was observed. We have already drawn attention to the accumulation within cells of a highly sensitive strain of Salmonella of free fosfomycin to a level much higher than that in the medium (TABLE 6 ) . Similarly, in a Pvoteus mirabilis strain exposed


to its inhibitory level of 0.7 pM, an intracellular level of 30 pM was found. Significantly, a resistant isolate of that straia, on exposure to 1 mM fosfomycin (a noninhibitory level), could not be demonstrated to contain measurable antibiotic activity. This raised the question of how entry and accumulation was mediated by sensitive cells.

We wondered whether any of the known bacterial transport systems for highly ionized nutrients might also be responsible for accumulating fosfomycin. First investigated was the role of the L-a-glycerophosphate transport system (genetic designation, glpT), which had been extensively studied in E. coli by Lin and colleagues z1 and by Hayashi and associates.?' This choice was naturally influenced by the casual similarity in structure between fosfomycin and L-a-glycerophosphate. On the basis of the following evidence, we concluded that the antibiotic was indeed transported by this system, not only in E. coli but in almost every other strain we have examined. 1. Without exception, all strains that exhibit any sensitivity to fosfomycin possess an ability to metabolize L-a-glycerophosphate by the criteria of enhanced growth, acid pro- duction, and release of orthophosphate. This generalization is based on an examination of 24 strains, which include species of Proteus, Pseudoinonas aeruginosa, Salmonella, E. coli, Klebsiellal Aerobacter, Staphylococcus aureus, and Streptococcus faecalis. The more sensitive strains displayed the higher rates of metabolism. 2. With the sole exception of the KlebsielldAerobacter tribe, all of these strains of bacteria, upon acquiring resistance to fosfomycin by mutation, show virtually no metabolism of L-a-glycerophosphate. 3. The uptake of labeled fosfomycin into sensitive strains of E . coli, S. typhiniuriurn, and Proteus vulgaris can be blocked totally by inclusion into the medium of 1 mM L-a-glycerophosphate, presumably as a consequence of competition by the normal substrate for the attention of the common transport mechanism. Phosphate ion, which inhibits L-a-glycerophosphate transport by the gZpT system, also antagonizes fosfomycin action on the bacterium. 4. Particularly convincing was that glpT- mutants of E . coli (provided by Dr. Lin), were also resistant to fosfomycin at levels at least 30-fold higher than their isogenic parent strain. Conversely, isogenic mutants constitutive for the glycerol regu- lon, and therefore for glpT function, were at least threefold more sensitive than the parent. Thus, the significant basal level expression of the g/pT gene, wherever it is found, provides the exclusive pathway of entry of fosfomycin into bacteria in all but specially supplemented environments, which will now be discussed.

The existence of an alternate transport pathway for fosfomycin was dis- covered in the wurse of investigation of the phenomenon first demonstrated by Zimmerman and colleagues a that many strains display an enhanced sensitivity to fosfomycin when grown on media to which small proportions of blood had been added. This potentiating effect was observed on both the majority sensitive population as well as the residue of persistent resisters, shown by us to be glpT-. We succeeded in isolating the potentiator from media incubated with lysed red blood cells and determined it to be a mixture of glucose-6- phosphate, fructose-6-phosphate, and glucose-1-phosphate, at a total concentration of 15-30 p M . We were also able to show that these substances, though present at low levels within fresh red blood cells, originated chiefly from the action of erythrocyte enzymes liberated by hemolysis that acted on broth con- stituents. Specifically, the initial reaction is the phosphorolysis of inosine and guanosine (present in the meat extract portion of the media), and the resultant


ribose-1 -phosphate is converted to hexose phosphates by the seven enzymes of the pentose phosphate pathway. It was a source of some surprise that these coupled pathways could operate with an efficiency of conversion that ap- proached 50% of the theoretically attainable yield, even though the erythrocyte is diluted 40-fold into a complex bacteriological medium.

Just as the divergent sensitivities of various strains could be attributed to the level of activity of the established L-a-glycerophosphate transport system, it was also possible to associate this potentiating effect with another known bacterial transport system, the hexose phosphate uptake system (genetic designation uhp 2 3 9 2 4 ) . This system differs fundamentally from glpT in that organisms fail to express significant activity in the absence of a competent inducer, so that its capacity for transporting fosfomycin became evident only when an inducer was fortuitously generated on an assay plate. An additional difference between the uhp and glpT systems is the more restricted distribution of the former. As defined by the ability of glucose-6-phosphate to potentiate fosfomycin, uhp is confined to the Enterobacteriaceae (excluding Proteus species) and to Staphylococcus. Our evidence that the potentiation effected by glucose- 6-phosphate is mediated by uhp is largely confined to E. coli, because the use of glucose-6-phosphate dissimilation as a criterion of uhp function in other species is unpredictably affected by the occurrence of uncontrolled phos- phatases.

Under the conditions of routine susceptibility testing, the potentiating effect of fructose-6-phosphate approaches the magnitude of that obtained with glucose- 6-phosphate, whereas mannose-6-phosphate and glucose-1-phosphate exhibit lower potencies. Although the fructose and mannose esters are considered substrates of uhp, they are capable of inducing uhp only under conditions that permit isomerization to glu~ose-6-phosphate.~~ Isomerization of glucose-1- phosphate is likewise required for activity. A critical verification of this finding was the failure of fructose-6-phosphate to potentiate when very dilute cell suspensions were exposed to its action. In contrast, glucose-6-phosphate in suspensions of l oL cells/ml induced maximum potentiation at 10 pM within 30 min.

Other evidence for the identity of uhp as the glucose-6-phosphate inducible fosfomycin transport system follow the outline of our proof for the involvement of glpT in fosfomycin transport. Resisters isolated from media that contained both glucose-6-phosphate and fosfomycin (at a concentration below the level inhibitory when glpT alone provides transport) were invariably found to be uhp- by the criterion of diminished growth on media supplemented with glucose- 6-phosphate as the primary carbon source. These retained the glpT+ character. Finally, high levels (>300 pM) of glucose-6-phosphate antagonize the action of fosfomycin when induced cells are tested for sensitivity, presumably due to competition for the uhp binding sites.

Kadner and Winkler have recently provided genetic evidence that establishes that uhp, defined by either the dissimilation of glucose-6-phosphate or direct measurement of its uptake, occupies the same genetic locus as the fosfomycin resistance mutation, which originates from a glpT- parent, in the presence of glu~ose-6-phosphate.~

In FIGURE 6, we show how the expression of the glpT and uhp systems is correlated both with the susceptibility of strains to fosfomycin and with their ability to allow entry and effect accumulation of the antibiotic. In the absence of exogenous inducers, the wild-type glpT+, uhp+ merely equilibrates with the

3 82 Annals New York Academy of Sciences

external levels of radioactive fosfomycin, whereas the glpT-, uhp+ mutant forbids entry; the radioactivity associated with the cell pellet is equivalent to only 0.4 mllml, namely, the extracellular (interstitial) volume. After growth in glycerol, which induces full expression of the glpT system, the glpT+ strain detectably accumulated fosfomycin and became more sensitive, whereas the corresponding parameters of the glpT- strain were uninfluenced. When the strains were cultured on glucose-6-phosphate and resuspended in fresh, unsup-

L - 1 : gIpT+, uhp' 0 10 mM

fosfomycin 15 pM

1U mM Glycerol

inducer

... ... MIC, pM 70 ,700 20 >700

GIu-6-P m_ ... ... ... ... .'.'.' v...

.....- ...

.-.-.- v... .:.:.: ~~* ....'. ..-.-. .'...*

..-... .:.:.:

.:.:.: ... ... ... ... ... ...'.. ... ... ... 2 7

0 ext racell u lar

space

FIGURE 6. Uptake of [3H]fosfomycin by E. coli strains after prior exposure to in- A..--..m ,.< t-.n..n-.,.-t ....n+,....l T _--.. 1- ,rn -1, ---...- &- A -n = --.a ---L:--A UULCIJ ui uairapui~ J ~ J L C I I I ~ . iiiucuid (uu i i i i j w c i c g i u w i i LO /~ (uo=u.J iiiiu C U I I I U ~ I I ~ U

with indicated inducer for an additional 90 min. Cultures were chilled and centri- fuged, and then resuspended at a sixfold higher cell density in broth without inducer, which contained [W]fosfomycin at 6 pCi/pmole. The incubation was conducted at 37" C for 20 min, followed by rapid cooling and centrifugation. Cell pellets, which ranged in weight from 80 to 95 mg, were resuspended and counted directly. Dilute samples that represented 10' cells of the induced inocula were also plated on Nu- trient-Agar medicated with fosfomycin to determine the minimum inhibitory concentration (MIC). The extracellular space was determined in separate experiments that employed Blue Dextran 2000 as an indicator of the extracellular volume of the cell pellet, which was found to be 0.4 ml/g. The true intracellular volume is assumed to be 0.42 ml/g

plemented medium (to minimize competition by unutilized glucose-6-phosphate for the transport system it induces), both showed a fivefold accumulation within cells over external concentrations. The resistant strain now dernon- strated a high susceptibility that approximates that of the parent strain, which is itself greatly sensitized by this procedure. These data illustrate both the primary role of the glpT system in permitting fosfomycin entry into the wild-type


bacterium and the ability of the uhp system to assist or supplant the glpT in this role when the uhp system is induced.

What is the prospect that other transport systems can also mediate fosfomycin entry? As mentioned, only in Klebsiella are we unable to ascribe sensitivity of the wild type exclusively to the glpT system. We still do not know the method of fosfomycin transport in this genus. In no case can we exclude the possibility that yet other transport systems may provide entry when they are evoked by induction or derepression. With regard to the latter possibility, we have been able to confirm an unusual observation made by M. J. Schlesinger that certain strains constitutive for alkaline phosphatase are highly sensitive to fosfomycin; we find that a majority of constitutive mutants isolated from E. coli displayed this property. This observation, when viewed in light of the recent proposal by Willsky and coworkersz6 that the gene products phoS and phoT are involved in Pi transport, would imply that fosfomycin can also avail itself of this system to penetrate the cell.

THE THERAPEUTIC POTENTIAL OF INDUCED ANTIBIOTIC TRANSPORT SYSTEMS

We have answered the question “How does this antibiotic work?” There remained, however, the challenge “How can one utilize the information?”

Enhancement of fosfomycin activity by hexose phosphates provides an opportunity for exploring a novel form of combination therapy against those strains that have the inducible uhp system. In fundamental contrast with established examples of combined antibiotic action, we now used a second agent, glucose-6-phosphate, which is itself devoid of antibiotic activity. In addition, because induction of transport reflects the synthesis of new proteins, we expected the resultant potentiation to persist after the likely removal of the inducer by vigorous host metabolism. We show in TABLE 7 the effect of this strategy when applied to mice experimentally infected with E. coli. The impressive potentiation of fosfomycin’s effectiveness when glucose-6-phosphate was coadministered is comparable to that observed in vitro. The indifference of a uhp- variant to the combination is an important reassurance that the interaction occurs at the bacterial level and is not, instead, a host-mediated response. Finally, the pre- dicted persistence of the induced state, a “memory effect,” was verified by delaying the administration of fosfomycin until 4 hr after the glucose-6- phosphate, which had been administered at the time of infectious challenge. Potentiation of the antibiotic effectiveness persisted relative to the appropriate control, even though the severity of infection in both cases was amplified by delay and obliged greatly increased doses to establish protection.

We believe this to be the first demonstration that antibiotic efficacy can be improved by the deliberate evocation of the bacterium’s genetic potential. The more general applicability of this stratagem depends upon establishing for a given antibiotic whether a permeability barrier limits the rate of entry. Whether or not that antibiotic makes use of preexistent transport systems, the above experience would encourage a search for agents that will induce latent nutrient transport systems whose affinity might extend to the antibiotic. These can augment existing rates of entry or provide alternative paths in the event that mutants deficient in the basal pathway emerge under drug pressure to com- promise antibiotic effectiveness.


CONCLUDING REMARKS

In this report, we have endeavored to describe how fosfomycin kills bacteria. We first demonstrated that the elimination of cell wall synthesis is the major biosynthetic consequence of exposure to fosfomycin. We have identified the specific enzyme inhibited by fosfomycin as the phosphoeno1pyruvate:UDP- GlcNAc-3-enolpyruvyl transferase, which is responsible for the first step in the synthesis of bacterial cell walls. That the inhibition of the pyruvyl transferase is indeed the method by which fosfomycin kills bacteria was established by showing: first, that all bacteria tested possess an inhibitable pyruvyl transferase;

TABLE I

OF FOSFOMYCIN IN THE MOUSE * GLUCOSE-6-PHOSPHATE POTENTIATES THE THERAPEUTIC EFFICACY

EDm Fosfomycin

Immediate Delayed Glucose-6-phosphate (mg kg-' 1

Infection (mg kg-'1 Route Therapy 4 hr

0 - 25.0 1070 i 50 subcutaneous 1.3 3 10 50 intravenous 0.8 62

5 intravenous 0.8 -

E. coli 2017

J glpT+, iihp' 11 LDw

- 12.0 - 50 subcutaneous 0.8 - 0 E. coli 2017

9 LDm

- 8.3 - 50 subcutaneous 14.2 - 0 E. coli 2017-A

glp T', ii hp- 7 LDm

* Mice were infected intraperitoneally with 2.5 x lo' cells of the indicated strains. The resultant severity of infection in each of the three trials is indicated as the multiple of that dilution (1 LDm) that kills 50% of untreated animals. Both strain 2017 and its uhp- derivative, 2017-A, have minimum inhibitory concentrations of 12 pg ml-l in nutrient broth; minimum inhibitory concentrations are 0.4 and 12 pg/ml, respectively, when glucose-6-phosphate (25 pg/ml) is present. Disodium fosfomycin was administered subcutaneously in a single dose either at the time of infection or 4 hr later; disodium glucose-6-phosphate was injected either subcutaneously or intra- venously (tail vein) at the time of infection in each case. The basis for calculating the median protective dose (EDw) and further details are in Reference 27.

second, that the rate of inactivation of the transferase both in vitro and in vivo parallels the rate of binding of fosfomycin to protein, as measured by the formation of acid-insoluble complexes with radioactive fosfomycin; and, lastly, that the levels of fosfomycin required to achieve substantial inhibition of enzyme activity both in vitro and in vivo correlate well with the levels required to kill bacteria and that the degree of enzyme inhibition is sufficient to account for the failure of the bacteria to maintain an adequate rate of wall synthesis. We therefore conclude that the site at which fosfomycin exerts its lethal effect has been established.


Our basic finding of the inhibitory effect of fosfomycin on pyruvyl transferase has recently been reproduced by Venkateswaran and Wu.' These authors have also isolated a fosfomycin-resistant mutant that is distinct from the vast preponderance of transportless mutants commonly observed and whose pyruvyl transferase exhibits properties that suggest that resistance may be a consequence of an alteration in this enzyme.

After establishing the site of action of the antibiotic, we have explored the extent of homology between fosfomycin and the natural substrate it mimics, namely, phosphoenolpyruvate. We demonstrated that fosfomycin reacts covalently with a cysteinyl residue of the enzyme by isolating the threo-2-S-cysteinyl- 1 -hydroxypropylphosphonate that results from proteolytic digestion of the enzyme inactivated by [3H]fosfomycin. We propose that this reaction of fosfomycin with enzyme is catalyzed by the enzyme in exactly the same manner as is the formation of an unexpectedly stable enzyme-phosphoenolpyruvate reaction intermediate, whose properties we have described. The common depend- ence of both of these enzymatic reactions upon the presence of UDP-GlcNAc has greatly simplified these studies with the pyruvyl transferase.

In its mode of action, fosfomycin shares one similarity with the proposed mechanism for penicillin, that is, the endo-alkylation of a thiol group at the active site of the target enzyme.2R These two antibiotics also share an amusing inversion of order: the first cell wall antibiotic inhibits the last step in cell wall synthesis, whereas this latest antibiotic inhibits the first step.

Of the investigations described here, our studies of the method of transport of fosfomycin across the bacterial permeability barrier have the most practical relevance. We have shown that fosfomycin utilizes the L-a-glycerophosphate transport system as the sole method of entry in almost all sensitive bacteria and that the loss of this system is the primary cause for emergence of resistant mutants. We also demonstrated the existence of a second means of fosfomycin transport, namely, the hexose phosphate uptake ( u h p ) system. This system is present only when induced by the presence of glucose-6-phosphate. Under such conditions, the antibiotic is no longer dependent exclusively upon the L-a- glycerophosphate system for its influx into cells. This phenomenon has provided an unusual opportunity for increasing the efficacy of the antibiotic in treating disease by evoking the latent potential of the bacterium to synthesize an even more powerful uptake pathway.

ACKNOWLEDGMENT

We appreciate the continuous encouragement provided us by Dr. H. Boyd Woodruff from the initiation of this research to its present conclusion.

REFERENCES

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ARISON, R. E. ORMOND, F. A . KUEHL, G. ALBERS-SCHONBERG & 0. JARDETZKY. 1969. Science 166 123.

RUFF, J. M. MATA, S. HERNANDEZ & S. MOCHALES. 1969. Science 166: 122. CHRISTENSEN, B. G., W. 3. LEANZh, T. R. BEATTIE, A. A. PATCHET~, B. H. 2.


3.

4. 5 . 6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20.

21.

22. 23. 24. 25. 26. 27.

28.

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the mechanism of action of fosfomycin (phosphonomycin)

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