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 FIGURE1
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 K a h a r ~ . ~
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-mercapto-
ethanol for 30 min at 37” C. Fosfomycin rendered acid insoluble in this reac-
tion 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
FIGURE 1. Structure of fosfornycin (~-cis-1,2- H,
C--c/
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-hydroxypropylphosphonicacid preparations
were purified from the crude reaction mixtures (see text) by analogous means,
which employed gel chromatography for initial desalting of the chemical prep-
arations and retardation on Dowex-50 (Hf) . Cocrystallization of the “isolated
adduct” after thioglycolic acid reduction and preparative electrophoresis de-
scribed 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.

RESULTSAND 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 Annals New York Academy of Sciences
m
E F:
0 control x
H i.0 turbidity
Q 5pM
viability
2. 0
0 50vM -
-2
._
0. 8
0.6
.A
W
I

E
L

al
0. 4
1. 0 ; 0

-
m
+i0. 2
0.6 gc
0
0” 0. 2
7

/
T 4 mucopeptide
4 0 -
s synthesis x
x
E
g 3 P’ 3 :

g
C

2 2
-
._
s
c

ml a
K c
._
0

2
.- 1

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 con-
tained broth supplemented with 20 mM L-lysine, 2 mM L-leucine, 7 0 pM DL-diamino-
pimelic acid (DAPA) , and the indicated level of fosfomycin. For “mucopeptide syn-
thesis,” sufficient [I-”CIDAPA was added to achieve final specific activity in the broth
of 0.8 pCi/pmole; for “protein synthesis,” ~-[l-“C]leucinewas 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 meth-
ods 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.’*. l R 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

TABLE1
FOSFOMYCIN SUPPRESSION O F ACCUMULATION OF CELL WALL PRECURSORS
INDUCIBLE BY PENICILLIN *

N-Acetylamino Sugar Esters

Concentration Exposure (pmoles/ml packed cells t )
Antibiotic (mM) (min) Total Accumulated
None - - 1.04 (0)
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

+ >
Fosfomycin
Penicillin G
Penicillin G
0.3
0.05
0.05
,,}
final 15
15
§ 3.4

7.25
2.4

6.0

* Staphy~ococcusaureus (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 maxi-
mum 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

TABLE2
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
2.0 1.o 45
0.13 70
0.06 37
15 54
2.0 1 .O 0.13 30 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

nmoles/rnin measured at highest indicated substrate concentration.
.t Specific activity, 0.6 mCi/mmole.
 Kahan et al. : Fosfomycin Action Mechanism 3 69
TABLE3
INACTIVATION
OF PYRUVYL * IN THE ABSENCE
TRANSFERASE
OF PHOSPHOENOLPYRUVATE

Product Formed in
Prior Incubation Complete Reaction
without Phosphoenol- Mixture 'i
pyruvate (nmoles)
Fosfomycin UDP-
Concentration GlcNAc Time Inhibition
(PM) (mM) (min) +Drug -Drug (% 1
125 - 0 0.5 1 0.683 26
6 20 0.025 0.505 95
1
25
0
-
20
0
0.28
0.65
0.35
0.683
21
5
6 20 0.122 0.505 76
1
6 -
0 20
0
0.338
0.67
0.35
0.683
4
2
6 20 0.346 0.505 31
1
Crude extract of S. aurerts, 0.2 mg protein/reaction.
>:
iComplete 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 fosfo-
mycin and phosphoenolpyruvate to crude or only partially purified (5-100-
fold) enzyme preparations.

Inactivation o f Pyruvyl Transferase in Vitro Results

in Covalent Attachment of Fosfomycin to Protein

The finding of irreversible inhibition effected by an epoxide-bearing struc-

ture 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 pro-
tein 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 partici-
pation of UDP-GlcNAc in the reaction of fosfomycin with enzyme, radio-
activity from UDP-[14C]GlcNAcdid 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.
370 Annals New York Academy of Sciences

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 (TABLE4) 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
REACTIONOF PARTIALLY PURIFIED PYRUVYL TRANSFERME
WITH RADIOACTIVEFOSFOMYCIN AND UDP-Glc-NAc *

Analysis of Proteins Separated

Direct Analysis of from Substrates on Gel
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
(70PM)
[W]Fosfomycin
UDP-GlcNAc
(3 mM)
+I
UDP-["C]GkNAc
( 3 mM)
0.6

-
40.0

2.0
0.6

9.6
51

31
46.0

1.8

UDP-["C]GlcNAc
+
Fosfomycin
(70 PM) I - 1.5 0.3 32 2.0

* Reaction mixtures contained in 0.75 ml KPM, 15 mg enzyme protein plus the in-
dicated 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 radioac-
tivity 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.
 Kahan et al. : Fosfomycin Action Mechanism 371
TABLE
5
KINETICSOF INACTIVATION
AND LABELINGOF CRUDEPYRUVYL
TRANSFERASE
BY [8H]FOSFOMYCIN *
Acid-Precipitable Transferase Activity
Time Radioactivity (nmoles/min/rng) K,,t
(min) (pmoles/mg) Remaining Inactivated (rnin-I)
0 0.21 1.86 “0” -
1 4.45 1.22 0.64 144
5 8.8 0.17 1.69 192
15 10.7 0.06 1.80 168
UDP-GlcNAc 2.04 - -
l 5 omitted 0.15

* 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 centri-
fugation 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 di-
alyzed 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 radio-
activity 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 fosfo-
mycin, 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 (TABLE6, sample 2). However, only in cells exposed to the inhibi-
tory 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 pro-
vided 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 trans-
ferase 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.

TABLE6
INACTIVATION OF PYRUVYL TRANSFERASE AND INCORPORATION OF RADIOACTIVITY
1NTO PROTEIN OF s. Iyphimifritfnl EXPOSEDin ViVo TO [sH]FOSFOMYCIN <‘

Analysis After Additional

Analysis of Cells Exposed in Vitro Exposure
in Vivo to Labeled Drug
Fosfomycin Fosfomycin Fosfo- Enzyme
Concentration Bound (pmoles/ Enzyme rnycin Activ-
(PM mg protein) Activity KCRt Bound ity Keat
Sam- Me- in Whole Ex- (mu/ (prnolesl (mu/
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 - - 0.03 - 0.1 0.007 -
* 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’ ” fosfomy-
cin concentration. The “in vitro exposure” to 70 p M [3H]fosfomycin, 11 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.

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 enzymo-
logical 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 relation-
ship 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 fosfo-
mycin 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 -hydroxy-
propylphosphonate, 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 METHODSthe isolation from 621 mg of
M . lysodeikticus protein, purified only eightfold with respect to pyruvyl trans-
ferase 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 electro-
phoresis 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-
374 Annals New York Academy of Sciences

ylated cysteine peptides) , we passed the material through the sequence of

reduction and oxidations indicated in FIGURE3. 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- performic
I oxidation
-1
1-1 cm+ dans-OH threo-sulfone

r
t - I
n
- 1
isolated
addud
th re o-thio e the; erythro-thioether

I
thioglycolic
reduction

3I t h re 0- sul foxides
H 0 lHAc
22
reoxidation

FIGURE3. 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 sepa-
ration 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 con-
verted 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 o f 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
 Kahan et al. : Fosfomycin Action Mechanism 3 75

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 (FIGURE4).*
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]phos-
phate incorporated by phosphorolysis of UDP-GlcNAc-enol pyruvyl ether
(the product of pyruvyl transferase). 2. UDP-GlcNAc is an obligatory co-
factor for complex formation with phosphoenolpyruvate but is not found in
the stable intermediate. 3. Phosphoenolpyruvate could be bound in the inter-
mediate 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 ex-
posure 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 re-
action 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, fosfo-
mycin inactivates with a first-order reaction rate that is 14% of the K,,t (15 min-')
of the pyruvyl transferase reaction.
376 Annals New York Academy of Sciences

0 , O NEM. 0.3 mM

\ A foslomycin. 30 mM
70

30

20

10

?
&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 pro-
tein (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 dithio-
threitol 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 remain-
ing enzyme activity were performed by combining a 50-pl aliquot with 50 pl of assay
mixture. The control reaction (“100%”) represents 1200 cpm.
 Kahan et al.: Fosfomycin Action Mechanism 311

substrate within a protein “cage.” Addition of enzyme4 to C-2 of phospho-

enolpyruvate, 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 configu-
ration 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 ex-
amined, namely enolase, pyruvate kinase, phosphoenolpyruvate carboxykinase,
and phosphoeno1pyruvate:shikimate-5-phosphateenolpyruvyl 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 phos-
phate (2,3-epoxypropyl phosphate) inactivates triose phosphate isomerase and eno-
lase.“ Although in these cases, saturating kinetics are found, with KlnaCt values of 1
and 5 m M ,respectively, the maximum inactivation rates are only lo-“ and 2>< of
the respective Kentvalues.
378 Annals New York Academy of Sciences

U-OH
'0 c--- C - C H ~ D +&+ E-PEP
2 4 I1

1
111
p04

U-oH
u-0,
Inactivated Enzyme CrCH D
'02C'

UDP - GlcNAc - 3 - enolpyruvyl ether

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 phospho-
enolpyruvate. The reversible loss of U-OH at step I1 stabilizes the phosphoenolpyru-
vate adduct generated at step I. Note that all reactions with the enolpyruvate sub-
strates are reversibIe but that after the enzyme-fosfomycin adduct is formed, it
cannot be dissociated by further exposure to UDP-GlcNAc.
 Kahan et al. : Fosfomycin Action Mechanism 379

in the analogous position. Because, in addition, the lack of close steric simi-
larity 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 dis-
cussed 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 trans-
ferase 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 b y 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 con-
cluded 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
380 Annals New York Academy of Sciences
to its inhibitory level of 0.7 p M , 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 con-
cluded 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 excep-
tion, 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 sensi-
tivity 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 concentra-
tion 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
 Kahan et al.: Fosfomycin Action Mechanism 381

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 desig-
nation uhp 2 4 ) . This system differs fundamentally from glpT in that orga-
239

nisms 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 fosfo-
mycin, 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 o L cells/ml induced maximum potentiation at 10 pM within
3 0 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
GIu-6-P
...
.'...
......
m_

.'.'
...

~~*
fosfomycin 15 pM
v...

.....-
.-.-.-
.:.:.:
1U mM
Glycerol
..-.-.
.'...*.
....'
v...

inducer ..-...
.:.:.:
...
.:.:.:
...
...
... 0
...
... ...
...'
... .. ext racell ular
... space
MIC, pM 70 ,700 20 >700 2 7

FIGURE 6. Uptake of [3H]fosfomycin by E. coli strains after prior exposure to in-

A..--..m , u a i r a p u i ~J..n
<. ui t-.n..n-.,.-t +,..l T _--..
1- , r n -1, ---...- A - n = --.a ---L:--A
&-
UULCIJ ~ J L C I I I ~ iiiucuid
. (uu i i i i j w c i c g i u w i i LO / ~ ( u o = u . Jiiiiu 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 concen-
tration (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
 Kahan et al.: Fosfomycin Action Mechanism 383
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 fosfo-
mycin entry? As mentioned, only in Klebsiella are we unable to ascribe sensi-
tivity 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.

THETHERAPEUTIC
POTENTIAL
OF INDUCED
ANTIBIOTIC SYSTEMS
TRANSPORT

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 estab-
lished 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 o r 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.
384 Annals New York Academy of Sciences

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;

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

EDmFosfomycin
Glucose-6-phosphate (mg kg-' 1
Immediate Delayed
Infection (mg kg-'1 Route Therapy 4 hr
E. coli 2017 0 - 25.0 1070
glpT+, iihp' i 50 subcutaneous 1.3 3 10
11 LDw J 50 intravenous
intravenous
0.8
0.8
62
-
5
E. coli 2017 0 - 12.0 -
9 LDm 50 subcutaneous 0.8 -
E. coli 2017-A 0 - 8.3 -
glp T', iihp- 50 subcutaneous 14.2 -
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 mul-
tiple 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, re-
spectively, 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.
 Kahan et al. : Fosfomycin Action Mechanism 385
Our basic finding of the inhibitory effect of fosfomycin on pyruvyl trans-
ferase 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 cova-
lently 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 fosfo-
mycin with enzyme is catalyzed by the enzyme in exactly the same manner as
is the formation of an unexpectedly stable enzyme-phosphoenolpyruvate reac-
tion 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.

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