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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, September 2001, p. 2432-2435, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2432-2435.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Resistance of Streptococcus pneumoniae
to Deformylase Inhibitors Is Due to Mutations in
defB
Peter
Margolis,
Corinne
Hackbarth,
Sara
Lopez,
Mita
Maniar,
Wen
Wang,
Zhengyu
Yuan,
Richard
White, and
Joaquim
Trias*
Versicor, Inc., Fremont, California 94555
Received 8 January 2001/Returned for modification 23 February
2001/Accepted 29 May 2001
 |
ABSTRACT |
Resistance to peptide deformylase inhibitors in Escherichia
coli or Staphylococcus aureus is due to inactivation
of transformylase activity. Knockout experiments in Streptococcus
pneumoniae R6x indicate that the transformylase (fmt)
and deformylase (defB) genes are essential and that a
def paralog (defA) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in
defB but not in fmt. Reintroduction of the
mutated defB gene into wild-type S. pneumoniae
R6x recreates the resistance phenotype. The altered enzyme displays
decreased sensitivity to actinonin.
| |
INTRODUCTION |
The formylation-deformylation cycle
of the translation-initiating methionine is a characteristic feature of
bacterial protein synthesis. Deformylation of the nascent protein is
catalyzed by an iron metalloenzyme, peptide deformylase (PDF)
(10, 21). The deformylase gene is essential in
Escherichia coli and Staphylococcus aureus
(13-15). Novel antibacterials that are PDF inhibitors,
such as actinonin, recently have been discovered by screening compound collections and combinatorial libraries (1, 5, 7, 9, 11).
Resistance to PDF inhibitors has been reported in S. aureus
and E. coli (7, 13). The mechanism of
resistance is based on the loss of transformylase activity, which
renders deformylase nonessential. However, loss of transformylation
comes at a cost to the bacteria: S. aureus fmt mutants are
slow growers, and the virulence of resistant mutants is attenuated
(13). In the present work, resistance to this new class of
antibiotics is examined in Streptococcus pneumoniae.
(Part of this research was presented at the 40th Interscience
Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario,
Canada, 17 to 20 September, 2000.)
| |
MATERIALS AND METHODS |
Strains and plasmids used in this study are listed in Table
1. Spontaneous PDF inhibitor-resistant
mutants were isolated by plating an exponentially growing culture of
S. pneumoniae ATCC 49619 on blood agar containing 100 µg
of actinonin (Sigma, St. Louis, Mo.) per ml. Growth was determined
spectrophotometrically at 600 nm using Mueller-Hinton broth with lysed
horse blood. MICs were determined as described elsewhere
(5).
E. coli def and fmt sequences were used in BLAST
searches at NCBI (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) to
identify S. pneumoniae homologs. The open reading frames
(ORFs) were amplified from S. pneumoniae R6x by PCR and used
in subsequent experiments.
Genes were inactivated in S. pneumoniae by
insertion-duplication mutagenesis (6). Fragments internal
to defA (codons 43 to 126), defB (codons 77 to
172), or fmt (codons 93 to 195) were PCR amplified and
cloned into pR326, and transformants were selected as described
elsewhere (6, 23). The defB allele was replaced in S. pneumoniae R6x by using a truncated defB
fragment (codon 77 through stop) PCR amplified from S. pneumoniae ATCC 49619 or the resistant mutants. All constructs
were confirmed by PCR and sequencing.
The ability of def gene homologs to code for a functional
deformylase was tested by complementation of the arabinose-dependent phenotype of E. coli VECO2068 with pGEX-5X-3 carrying
def homologs or with vector alone (see Table 1), as
described elsewhere (13). The E. coli VECO2068
strain has the chromosomal copy of the essential def gene
under PBAD control and will grow in the absence of inducer only when an active deformylase is expressed in
trans.
The defB gene (optimized for expression in E. coli by 16 silent mutations in the first 48 codons) was cloned
into pET20b(+) so as to encode a His-tagged protein. This construct was
modified via PCR-mediated site-specific mutagenesis at codon 172 (CAG
to aAa) or codon 123 (GCT to GaT). The resulting plasmids were
introduced into E. coli BL21 for protein expression.
Transformants were grown at 37°C in 500 ml of Luria broth
supplemented with 100 µg of ampicillin per ml to an optical density
at 600 nm of 0.5, at which point IPTG
(isopropyl-
-D-thiogalactopyranoside) was added to a
concentration of 1 mM. After 3 h of induction, the cells were
harvested, resuspended in 35 ml of 10 mM NaCl-20 mM Tris-HCl buffer
(pH 8), and then disrupted, in the presence of catalase, by a French
press. His-tagged deformylase was purified from the cell lysates by
passage over a cobalt affinity column according to the manufacturer's
instructions (Clontech, Palo Alto, Calif.).
Deformylase activity was determined by using a deformylase-formate
dehydrogenase (FDH) coupled assay (12). Experiments were carried out at room temperature in a buffer containing 10 mM NaCl, 0.2 mg of bovine serum albumin per ml, and 50 mM HEPES (pH 7.2). The
reaction was initiated by adding a mixture of 0.5 U of FDH per ml, 1 mM
NAD+, and fMAS at 4 mM (5, 12). Deformylation
was followed by monitoring the reduction of NAD due to the oxidation of
formate by FDH. For inhibition studies to determine the 50% inhibitory concentration (IC50) values, enzyme was preincubated at
different concentrations of actinonin for 10 min prior to the addition
of the substrate (5).
| |
RESULTS AND DISCUSSION |
Identification of deformylase in S. pneumoniae.
A
BLAST search using the E. coli def sequence identified two
S. pneumoniae def homologs, defA and
defB (Fig. 1). In contrast to
many other bacteria (13), neither of these homologs were adjacent to an fmt gene. Several pieces of evidence indicate
that defB, and not defA, encodes the S. pneumoniae R6x PDF. The predicted DefA protein contains two
substitutions (G41C and Q48M) at strictly conserved residues of a key
catalytic domain, GXGXAAXQ (Fig. 1). Substitutions at either of the
analogous residues of E. coli PDF dramatically impair enzyme
activity (3, 4, 8, 18, 20). The S. aureus defA
gene, which also contains two substitutions in this motif (Fig. 1),
encodes a protein lacking PDF activity (13).
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FIG. 1.
Alignment of conserved domains of deformylase proteins.
Partial sequences of the predicted products of the defA and
defB homologs of S. aureus and S. pneumoniae are shown aligned with consensus PDF domains
(13). Residues that diverge from the consensus are
highlighted. Modifications in the deformylase enzyme in the resistant
S. pneumoniae mutants VSPN6501, VSPN6503, and VSPN6504
are indicated by arrows. The positions of the motifs in the S. pneumoniae DefB are as follows: box 1, G69 to Q76; box 2, A123 to
S132; and box 3, Q172 to G182. The sequences of the S. pneumoniae
def homologs from strain R6x have been submitted to GenBank
(accession numbers: defA, AY014508; defB,
AY014509). Sequences of the S. pneumoniae def and
fmt homologs from S. pneumoniae ATCC 49619 were
also submitted to GenBank (accession numbers: defA,
AY014510; defB, AY014511; fmt, AY014512).
|
|
Despite several attempts, defB could not be inactivated by
insertion-duplication mutagenesis. However, defA or
rafE, a nonessential gene (23), were readily
disrupted. This result implies that defB is essential,
although the experiment does not exclude the possibility of a polar
effect on a downstream gene. A plasmid encoding a GST-DefB fusion
protein, but not one encoding GST-DefA, was able to complement the
arabinose-dependent phenotype of E. coli VECO2068.
Expression of the gst-defA and gst-defB fusions was confirmed by Western blotting (data not shown). Purified GST-DefB was associated with a strong PDF activity (1,300 µmol
min
1 mg of protein1). Taken together, these
results argue that defB codes for a true essential
deformylase, whereas defA is a paralog of unknown but nonessential function or has marginal deformylase activity unable to
complement the arabinose-dependent mutant. This is similar to S. aureus RN4220, which also harbors two deformylase homologs, only
one of which, defB, encodes a true PDF (13).
Isolation and characterization of actinonin-resistant mutants.
The frequency of resistance in S. pneumoniae ATCC
49619 was 108, 2 orders of magnitude lower than
that obtained with S. aureus ATCC 25923 or S. aureus 1-63 (13). Three S. pneumoniae
mutants, VSPN6501, VSPN6503, and VSPN6504, were chosen for
further studies. The mutants grew at slower rates, with doubling times
approximately 20% longer, when cultured in broth (Table
2). The slow-growth phenotype is less
pronounced than in S. aureus actinonin-resistant strains
derived from S. aureus ATCC 25923, where doubling times increase approximately 80% (13). These mutants of
S. pneumoniae ATCC 49619 showed resistance to PDF
inhibitors (Table 2) but were unchanged in susceptibility to
penicillin, ampicillin, chloramphenicol, erythromycin,
trimethoprim, or tetracycline. These results suggest that
resistance occurred by a specific mechanism distinct from that for
these other antibiotics and further indicates that the slower growth
rate did not change the overall susceptibility of the mutant strains.
No such differences in susceptibility to PDF inhibitors or doubling
time were observed for strain VSPN7011, which carries a disrupted
defA gene (not shown).
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|
TABLE 2.
Genotypes and susceptibilities to PDF inhibitors, and
doubling times of S. pneumoniae actinonin-resistant and
-susceptible strainsa
|
|
Mutation of fmt leads to resistance in S. aureus
or E. coli (7, 13). The fmt gene
from S. pneumoniae ATCC 49619 actinonin-resistant strains
was PCR amplified and sequenced. None of the resistant strains carried
a mutation in fmt or flanking DNA. The inactivation of
fmt was attempted to assess whether the lack of
transformylase activity could provide resistance in S. pneumoniae R6x. Despite multiple attempts, the fmt gene
could not be inactivated. In contrast, the homologous gene can be
readily inactivated in strains of E. coli, Pseudomonas
aeruginosa, or S. aureus (13, 14,
19). The inability to inactivate fmt
suggests that the transformylase gene itself is essential in
S. pneumoniae R6x, although it is possible that strains
disrupted in fmt were not obtained because of a polar effect
on a downstream gene.
The sequences and flanking DNA of both defA and
defB were PCR amplified and sequenced from S. pneumoniae ATCC 49619 and mutant strains. No change was
observed among the sequences of the defA homolog. However,
each of the resistant strains possessed a single missense mutation in
defB (Fig. 1). For S. pneumoniae VSPN6501, a
Q172K substitution occurs at a residue that is strongly conserved among
PDF proteins (Fig. 1). This position lies immediately upstream of
the 173HEXXH177 (S. pneumoniae
numbering) motif shared by all PDF proteins and characteristic of zinc
hydrolases (22). A nonconservative substitution (Q131A) at
the equivalent position in the E. coli enzyme has been shown
to decrease enzyme activity (8). The analogous pair of His
residues in the E. coli PDF has been shown by genetic and
structural studies to bind the metal ion in the catalytic pocket
(2-4, 8, 16-18). S. pneumoniae VSPN6503 and VSPN6504 have a A123D substitution in the predicted protein. This residue is not conserved among PDF proteins. However, the mutation introduces a charged amino acid five residues upstream of the 128EGCLS132 motif, which has been shown to be
involved in binding the metal ion (Fig. 1) (2-4, 7, 8,
16-18). The mutated residue corresponds to a position two
residues upstream of an Ile involved in defining a substrate-binding
pocket of the E. coli enzyme (7). Thus, in all
three cases, a mutation in S. pneumoniae defB is predicted
to cause a substitution close to conserved domains involved in binding
the metal ion or the substrate, essential for PDF activity.
Mutated defB leads to resistance.
Genes encoding
the wild-type and mutated PDFs were introduced into S. pneumoniae R6x (6). Strains expressing either of the
two mutated defB genes displayed reduced susceptibility to actinonin (Table 3). In addition, the
mutated enzymes are indeed less sensitive to inhibition than the
wild-type parent PDF (Table 3). These results confirm that, in
these mutants, resistance is due to modification of the target rather
than the lack of transformylation activity.
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|
TABLE 3.
Susceptibilities to actinonin of S. pneumoniae
R6x strains transformed with wild-type or mutated
defB and inhibition of purified His-tagged PDF enzymes
by actinonin
|
|
Resistance to PDF inhibitors can occur by at least two distinct
mechanisms, with different consequences predicted in each case.
Resistant mutants of S. aureus or E. coli,
obtained in vitro, lack transformylase activity, bypassing the
essentiality of the def gene (7, 13). Mutation
of fmt should lead to cross-resistance to any antibiotic for
which PDF is the major target, because inhibition of deformylase would
have no consequence for protein synthesis if nascent peptides were not
formylated. However, mutation of fmt does have consequences
for cell growth, as seen in S. aureus and E. coli
fmt mutants. Notably, S. aureus fmt mutants have
attenuated virulence in abscess or septicemia models, decreasing the
chance that such mutants would survive during infection (7,
13). In S. pneumoniae R6x, fmt cannot be
disrupted; instead, resistance to PDF inhibitors derives from
modification of the target. In contrast to strains resistant via a lack
of transformylation activity, it is possible that these S. pneumoniae defB mutants will not be cross-resistant to all PDF
inhibitors. More potent inhibitors, or compounds that bind differently
to PDF, should be active against these resistant S. pneumoniae strains. The essentiality of fmt makes PDF
an attractive target for the discovery and development of novel
antibiotics active against S. pneumoniae.
| |
ACKNOWLEDGMENTS |
Sequencing of S. pneumoniae was accomplished with
support from TIGR, the National Institute for Allergy and Infectious
Diseases, and the Merck Genome Research Institute.
We thank Dennis Young and Carsten Rosenow for advice.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Versicor,
Inc., 34790 Ardentech Ct., Fremont, CA 94555. Phone: (510)
739-3025. Fax: (510) 739-3003. E-mail: jtrias{at}versicor.com.
| |
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Antimicrobial Agents and Chemotherapy, September 2001, p. 2432-2435, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2432-2435.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
