J. Mol. Microbiol. Biotechnol. (2001) 3(2): 179-184.
Enterococcus faecalis MDR Transporters 179
JMMB Symposium Enterococcus faecalis Multi-Drug Resistance Transporters: Application for Antibiotic Discovery Deborah vR. Davis1, James B. McAlpine1*, Christopher
class, the first members of which were discovered in 1953
J. Pazoles1 , M. Kelly Talbot1, Elisabeth A. Alder1, Abbie
(Charney et al., 1953). Virginiamycin, a naturally occurring
C. White1, Brandie M. Jonas2, Barbara E. Murray2,
streptogramin A and B combination, was discovered in 1955
George M. Weinstock2, and Bruce L. Rogers1,3
(Somer et al., 1957) and has been used as a growthpromoter in animal feed until recent years. It has been
1Phytera, Inc., 377 Plantation St., Worcester, MA 01605,
proposed that this use has selected for virginiamycin-
resistant strains of E. faecium, which are cross-resistant
2University of Texas Medical School, 6431 Fannin St.,
to Synercid® (Werner et al., 1998). Although Synercid® is
active against most MLS (macrolide-lincosamide-
3 Present address: Genetics Institute, 35 Cambridge
streptogramin) resistant strains, the existence of a known
mechanism directed at this class is cause for concern. Moreover, E. faecalis appears to be intrinsically resistantto Synercid® and virginiamycin (Rende-Fournier et al.,
1993; Maddock et al., 1999; Lewis and Jones, 2000). Inaddition, E. faecalis resistance and/or superinfection was
Using bioinformatics approaches, 34 potential multi-
seen during clinical trials with Synercid® (Blumberg et al.,
drug resistance (MDR) transporter sequences representing 4 different transporter families were
Linezolid® is the first approved member of a class
identified in the unannotated Enterococcus faecalis
(oxazolidinones) for which the pharmacophore was
database (TIGR). A functional genomics campaign
reported in 1987 (Slee et al., 1987). Despite the fact that
generating single-gene insertional disruptions
this is an exceedingly simple chemical class (Figure 1) and
revealed several genes whose absence confers
very amenable to modern approaches for combinatorial
significant hypersensitivities to known antimicrobials.
analog synthesis, it has taken 13 years to bring this drug
We constructed specific strains, disrupted in a variety
to market. The rate and extent of the development of
of previously unpublished, putative MDR transporter
resistance to oxazolidinones remains to be seen. However,
genes, as tools to improve the success of whole-cell
with few new antibacterials directed towards resistant Gram
antimicrobial screening and discovery. Each of the
positive bacteria in the clinic, the identification of new
potential transporters was inactivated at the gene level
pharmacophores continues to be of urgent importance. and then phenotypically characterized, both with single
While many mechanisms have been identified as the
disruption mutants and with 2-gene mutants built upon
cause of emergent drug resistance, the expression, or
a ∆ norA deleted strain background.
overexpression, of multidrug MDR efflux pumps have beendemonstrated to contribute significantly. Antibiotic
resistance has been directly attributed to this mechanismin a number of model organisms, as well as important Gram
Emerging antibiotic resistance, particularly in nosocomial
positive pathogens, including S. aureus (Yoshida et al.,
situations, has received wide exposure even in the popular
1990; Hsieh et al., 1998), E. faecalis (Lynch et al., 1997)
press (Gorman, 1997; Preston, 1999). Awareness has been
and S. pneumoniae (Gill et al., 1999). In Gram positive
heightened by the incidence of vancomycin-resistant
pathogens in particular, pmrA has been identified as an
Enteroccocus faecalis (French, 1998) and the recent
efflux pump associated with fluoroquinolone resistance in
emergence of a similar resistance in Staphylococcus
Streptococcus pneumoniae (Gill et al., 1999) and mreA
aureus (MMWR, 1997). This observation is particularly
from Streptococcus agalactiae has been characterized as
significant since Vancomycin was considered to be the last
a novel macrolide efflux gene (Dib-Hajj et al., 1997). These
line of defense against Gram positive pathogens. Withinthe last 6 months, two new Gram positive antibacterials,Synercid and Linezolid, have been approved by the FDA. These two agents belong to classes of antibiotics not inprior human therapeutic use and as such could give riseto the hope of a battle won. However, severalconsiderations warn against complacency. Synercid®(quinupristin-dalfopristin) is a combination of twosemisynthetic compounds, belonging to the streptogramin
*For correspondence. Email [email protected]
Figure 1. The chemical structure of Linezolid®
Figure 2. Dose/response curves demonstrate observed hypersensitivities in disruption mutants. Each IC50 was calculated using GraphPad Prism software.
export systems can extrude a wide range of structurally
A general problem facing natural product extract-based
dissimilar compounds. (Paulsen et al., 1996; Lewis et al.,
antimicrobial screening is, that because such extracts are
1997). Significantly, MDR efflux pumps have been
complex mixtures of chemicals, the concentration of any
described in phylogenetically diverse organisms including
one particular component is undefined, and can be very
bacteria (Nikaido, 1996), yeast (Kolaczkowski et al., 1998)
low. In addition, the effectiveness of screening using whole
and mammals (Chen et al., 1986), suggesting their
cells is further diminished by the presence of multiple
fundamental importance. Many MDR efflux pumps are members of multi-gene families with the best described being the ATP Binding Cassette (ABC) family of membrane proteins, the Multiple Facilitator Superfamily (MFS) of proteins, Multidrug And Toxic compound Extrusion family (MATE), and the Small Multidrug Resistance (SMR) family, (Michaelis and Berkower, 1995; Paulsen et al., 1996, Brown et al., 1999).
A traditional approach to screening for new
antimicrobials typically involves the inhibition of growth ofa screening strain, preferably of the target pathogen. Suchwhole-cell antimicrobial assays have the inherentadvantage in that advanced knowledge of the mode ofaction of an antibacterial agent is not required. Hence, suchassays are capable of revealing agents with completelynovel molecular targets and/or modes of action.
Figure 3. The chemical structure of Daunorubicin and Doxorubicin.
Enterococcus faecalis MDR Transporters 181
Table 1. E. faecalis putative transporter sequences and demonstrated
Table 2. Table of observed MIC’s in insertional knockout strains of E. faecalis.
homologies to known multidrug transporters from bacteria, yeasts, and
The total panel of drugs tested consisted of enoxacin, ofloxacin, norfloxacin,
mammals. Shaded boxes represent E value calculations generated by
ciprofloxacin, tylosin, erythromycin, spiramycin, clindamycin, lincomycin,
BLAST searching each candidate sequence against GenBank. Unshaded
virginiamycin, Synercid, chloramphenicol, tetracycline, ampicillin, bacitracin,
E values represent hypothetical MDR transporters or transporter genes
novobiocin, puromycin, gentamicin, rifamycin, pentamidine, crystal violet,
known to efflux metabolites, metals, or other non-antibacterial compounds.
CCCP, acriflavin, daunorubicin, doxorubicin, ethidium bromide, rhodamine123, and mithramycin. E.faecalis E.faecalis E.faecalis Sequence sequence sequence MIC µg/ml identifier identifier identifier Wild-type -gene WT/-gene knockout
a A total of 28 known, chemically diverse antimicrobial agents were tested.
None of the 24 other single gene disrupted strains created consistently showed
more than 2 fold increased sensitivity (compared to the parent) towards any of the
b Compounds indicated were not tested against all of the single-gene disruptants.
They were tested only against the specific gene disruption strain that initially was
susceptible to a related chemical compound.
Putative MDR Transporter Identification
a The E value relates to the smallest sum probability of the number of hits one can expect to see bychance when searching a database of a certain size. In practice, an E-value threshold of 0.001-0.1 isoften considered significant.
Active efflux has previously been demonstrated in E. faecalis by studying the effect of energy (glucose fed versusglucose starved) on the accumulation of labeled
multidrug pumps in many bacteria and yeast. In particular,
norfloxacin, chloramphenicol, tetracycline and benzyl
extrusion by MDR pumps of antibiotics present in screening
penicillin by wild-type strains. (Lynch et al., 1997). Individual
samples at low concentrations significantly decreases the
pumps were not characterized, nor was any genetic
chances of antimicrobial discovery. Thus, there is a strong
analysis part of this work. In our study, we used the amino
rationale for increasing the sensitivity of whole-cell screens
acid sequence and membrane topology of several known
for new antibiotics. For intracellular target assays, the
MDR transporters to probe, using a series of BLAST
effective concentration of a test agent is a function of the
searches for homologs, in the TIGR unannotated E. faecalis
rates of influx and efflux and the ability of the agent to bind
database (www.TIGR.org) (Altschul et al., 1990; Worely et
to its target within the cell. Each of these offers opportunity
al.,1995, 1998). From this, an MDR pump gene candidate
for increasing screen sensitivity. For example, one
list of 23 ABC superfamily homologs, 9 MF family homologs,
approach has been to compromise the cell wall and/or cell
1 MATE homolog, and 1 SMR homolog was generated
membrane and hence increase the rate of influx. This is,
(Table 1). All candidate sequences were retrieved from the
however, accompanied by an increase in the rate of passive
unannotated database and examined for open reading
efflux. The prevalence of active efflux via transmembrane
frames. The 23 ABC transporter candidates were also
pumps provides a more targeted approach to constructing
analyzed for predicted transmembrane regions using
supersensitive whole-cell screening strains. In unpublished
SOSUI (Hirokawa et al., 1998) through the BCM search
work, we have improved the success of whole-cell
launcher (Smith et al., 1996) and predicted ABC family
antifungal screening by using specific strains deficient in a
signature sequences and ATP-binding motifs using Motif
variety of novel MDR-transporter genes. These strains are
finder (Hofmann et al., 1999; www.motif.genome.ad.jp).
significantly more sensitive to known antifungal agents and
Candidates containing 4-6 predicted transmembrane
have enabled the discovery of new agents, from both
regions, ABC signature sequences and ATP-binding motifs
natural product and synthetic chemical libraries, where
were placed at a high priority. MF candidates were also
these agents would not have been discovered using wild
examined for predicted transmembrane regions and were
excluded if they did not meet the arbitrary criteria of havinggreater than nine of these regions.
We initiated a comprehensive functional genomics
study aimed at generating site-directed insertionalmutagenesis to inactivate each individual gene listed in
mutant has shown a significant hypersensitivity todaunorubicin, doxorubicin, and ofloxacin. Ethidium bromideand chloramphenicol showed a 2-fold increase insusceptibility, which given the inherent variability of an MIC-based assay can only be regarded as significant in that itwas a consistent result from several independentmeasurements. Figure 2B illustrates an approximately 60-fold increase in hypersensitivity for doxorubicin against theabc7 - strain compared to the wild-type OG1RF (Table 2). Although conventional wisdom dictates that these MDRpumps are generally promiscuous and accept a widevariety of substrates, these data suggest that this may bea misconception arising from the large number of suchpumps present in each organism. For example, the ABC7transporter exhibits a high degree of specificity within astructural type. The increased sensitivity of strains with
Figure 4. The chemical structures of Lincomycin and Clindamycin.
disabled ABC7 to the two anthracyclines, daunorubicin anddoxorubicin is significantly different (8 and 60-fold,respectively) despite the fact that they differ in structure
Table 1. For these studies, a 500-700 base pair internal
only by a single hydroxyl group. (Figure 3)
fragment from the N-terminal end of each target gene was
Similarly, the abc23- disruption strain has shown
recovered from genomic DNA (E. faecalis OG1RF), cloned
significant hypersensitivity to clindamycin, lincomycin,
into vector pTEX4577 and the plasmids were
virginiamycin and Synercid® (Table 2; Figure 2D).
electroporated (Qin et al., 1999) into OG1RF and selected
Coincidentally, the lincosamides and the streptogramins
for transformants on BHI agar supplemented with
(virginiamycin and Synercid® exert their antibacterial
kanamycin. Transformants were individually confirmed by
activity by binding to the bacterial 50S ribosomal sub-unit
PCR to have the targeted insertion of each plasmid. Once
and inhibiting protein synthesis even though they have
confirmed, each single-gene disruption mutant strain was
unrelated chemical structures (Figures 4 and 5). The two
tested against a panel of 28 compounds using a microbroth
components of the streptogramins are thought to act
dilution assay (NCCLS document M7-A5) and its
synergistically by binding to two separate sites of the
susceptibility compared to that of the wild-type.
bacterial 50S ribosomal subunit. Synercid® is a sterile
The TIGR sequence database was generated from
lyophilized formulation of two semi-synthetic pristinamycin
vancomycin-resistant E. faecalis strain, V583. Although we
derivatives, quinupristin (derived from pristinamycin I) and
amplified candidate sequences abc9, abc12, abc20, and
dalfopristin (derived from pristinamycin IIA) in the ratio of
smr1 from this strain, we were unable to amplify them after
30:70 (w/w) (http://www.aventispharma-us.com).
several attempts from working strain OG1RF. The apparent
Lincomycin and clindamycin also inhibit protein synthesis
absence of these genes from OG1RF may be explained
by blocking peptidyl transferase activity of the 50S
ribosomal subunit (Gale et al., 1972).
Resistance to streptogramins can develop by multiple
Novel Transporters and Observed Substrates
mechanisms including; 1) modification of the drug target,2) inactivation of the drug, and 3) impaired permeability
The observed phenotypes of these strains functionally
from active efflux or production of altered permeases
implicated several genes as responsible for drug efflux in
(Quinupristin/Dalfopristin Drug Monograph, 1998). The
E. faecalis. In particular, the abc7 - single-gene disruption
most commonly observed of these is the modification of
Figure 5. Synercid® consists of two semisynthetic pristinamycin derivatives (quinupristin and dalfopristin) in a 30:70 (w/w) ratio.
Enterococcus faecalis MDR Transporters 183
the target, which is mediated by the erm gene. This gene
encodes an RNA methylating enzyme that results inreduced binding of macrolides, lincosamides, and
A total of 34 potential genes coding for efflux pumps were
streptogramin B antibiotics (MLSB) (Quinupristin/
identified in Enterococcus faecalis by mining the TIGR
Dalfopristin Drug Monograph, 1998). Group A
database for motifs from published MDR transporter amino
streptogramin antibiotics such as dalfopristin are not
acid sequences and transmembrane domains. For 30 of
affected by this type of resistance. In E. faecium, resistant
these, we were able to detect and inactivate by insertional
isolates have been associated with the presence of vat(E)
mutagenesis the corresponding sequence in E. faecalis
(satG) or vat(D) (satA) genes responsible for hydrolysis or
OG1RF. The phenotypic susceptibilities of the disruption
acetylation of quinupristin and dalfopristin, and studies
mutants to a battery of 28 structurally diverse antimicrobial
suggest that this is not the only mechanism (Soltani, et al.,
agents were characterized and strains abc7-, abc11-,
2000). It is likely that the abc23 gene identified in E. faecalis
abc16-, and abc23- were significantly more sensitive to at
is responsible for transporter-mediated resistance in this
least one of the agents, demonstrating that these gene
organism and could explain why E. faecalis, in general, is
products are indeed MDR transporters. Significantly, these
intrinsically resistant to streptogramins.
four ABC insertional mutants revealed large fold-differencesthat have not been observed in similar studies with other
Multiple Transporter Knockouts
Gram positive organisms. For example, with L. lactis LmrA,only minor phenotypic differences in susceptibility were
S. aureus mediates resistance to norfloxacin by expression
observed when knockouts were analyzed (Ian Paulsen,
of the norA gene which codes for an MDR transporter. The
personal communication). For the remaining gene insertion
OG1RF strain of E. faecalis was constructed in which a
mutants, no significant drug susceptibility was detected
homologous gene had been removed and examined for
against the battery of compounds tested. More MDR
its susceptibility to a panel of antimicrobial agents. In E.
transporters might be identified if the panel of compounds
faecalis, the ∆ norA strain was only 2 to 3-fold more
were expanded. It is interesting to note that our work in E.
susceptible to norfloxacin (Figure 2A) and it showed little
faecalis indicates that the dominant efflux pumps present
increased sensitivity to ciprofloxacin. Similarly, in S. aureus,
in this organism appear to be ABC transporters, whereas
a norA- strain was only 4-fold more sensitive than the wild-
ad hoc studies in other bacteria have mainly implicated
type to norfloxacin (Hsieh et al., 1998). Nonetheless, we
proton motive force dependent transporters (i.e. QacA,
reasoned that NorA may still recognize and transport
Bmr, MexAB, and AcrAB) (Paulsen et al., 1996).
agents, but if these were substrates for other transporters,
Transporters were identified that play a major role in
the effect of deleting norA on the intracellular concentration
determining the susceptibility of the organism to
could be negligible. If a drug is the substrate for multiple
streptogramins and lincosamides. NorA, although present
transporters, maximal intracellular concentration can only
in E. faecalis, seems to have only a modest effect, even
be achieved by inactivating all pumps recognizing the drug
on the susceptibility to norfloxacin. It is possible that other
and, a priori, one would expect that sequential inactivation
transporters may also pump norfloxacin and may
of the transporters would be synergistic, with the greatest
compensate for the deletion of norA. MDR disruption
phenotypic effect observed when the last transporter is
mutants hold considerable promise as supersensitive
removed. Starting with the norA deletion strain, we created
screening strains for the discovery of novel antibacterial
a series of 2-gene mutants employing the methodology
pharmacophores. However the large number of MDR
used above. The sensitivity of these to a panel of
transporters present in E. faecalis and our results with 2-
antimicrobials exactly matched the susceptibility profile of
gene disruptants suggest that several of these transporters
each of the single gene disruptants described in Table 2.
will have to be inactivated to achieve a strain that is
This result suggests that disruption of 2 transporters may
hypersensitive to a broad structural variety of antibacterial
be insufficient to produce a significant change in drug
sensitivity. Lee and co-workers have studied the effects ofdifferential multiple transporter expression in Gram
negatives, (E. coli and P. aeruginosa). (Lee et al., 2000).
This work was supported by SBIR Phase I Grant# 1R43 AI44543-01. We
Here the situation is more complex in that there are two
would like to thank Aventis for a sample of Synercid®. Preliminary sequence
types of transporters; single component transporters which
data was obtained from The Institute for Genomic Research website athttp://www.tigr.org. Sequencing of E. faecalis was accomplished with support
extrude substrates from the cytoplasm to the periplasm
from the National Institute of Allergy and Infectious Diseases. In addition,
and multi-component pumps which take substrates all the
we wish to thank John Cryan for technical assistance.
way from the cytoplasm to outside the cell. They concludedthat transporters of different structural types were
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