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Journal of Bacteriology, July 1999, p. 4137-4141, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Cloning and Characterization of a Locus Responsible for
O Acetylation of the O Polysaccharide of Legionella
pneumophila Serogroup 1 Lipopolysaccharide
Chang Hua
Zou,1
Yuriy A.
Knirel,2,3
Jürgen H.
Helbig,4
Ulrich
Zähringer,2 and
Clifford S.
Mintz1,*
Department of Microbiology and Immunology, University of
Miami School of Medicine, Miami, Florida,
331011; N. D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russian Federation3; and
Forschungszentrum Borstel, Zentrum für Experimentelle
Biologie und Biowissenschaften, D-23845
Borstel,2 and Institut für
Medizinische Mikrobiologie und Hygiene, Universitätsklinikum
der Technische Universität Dresden, D-01307
Dresden,4 Germany
Received 1 February 1999/Accepted 26 April 1999
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ABSTRACT |
Complementation experiments, Tn5 mutagenesis, and DNA
sequencing were used to identify a locus (lag-1) that
participates in acetylation of Legionella pneumophila
serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of
lipopolysaccharides from mutant and complemented strains suggest that
lag-1 is responsible for O acetylation of serogroup 1 O polysaccharide.
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TEXT |
Legionella pneumophila,
the causative agent of Legionnaires' disease, is a gram-negative
bacterium that can enter and grow within a variety of eukaryotic cells,
including human monocytes and alveolar macrophages (7) and
free-living amoebae (4). Barker et al. (1) and
Lüneberg et al. (12) showed that structural and
serological changes can occur in L. pneumophila
lipopolysaccharide (LPS) during intracellular growth in both amoebae
and monocyte-like human cell lines. Further, recent evidence suggests
that serological changes in serogroup 1 LPS can result in a net
reduction in L. pneumophila virulence (12).
To assess the contribution of LPS to the interaction between L. pneumophila and eukaryotic cells, we isolated a mutant of strain
Philadelphia 1, designated CS332, that produced LPS which failed to
bind serogroup 1 LPS monoclonal antibodies (MAbs) MAB2 and 33G2
(15, 17). Also, the mutant LPS, in contrast with wild-type
LPS, bound the serogroup 1 LPS MAb 144C2 (17). Nuclear magnetic resonance (NMR) spectroscopic analysis performed in the present study revealed that CS332 LPS was missing the O-acetyl group at
position 8 of its O-repeat unit (Fig.
1A), which was determined to be
5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-D-galacto-nonulosonic acid, an N- and O-acylated derivative of legionaminic acid (Fig. 1B)
(8, 9, 11, 18, 24).
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FIG. 1.
Structure of L. pneumophila serogroup 1 LPS
adopted from Knirel et al. (8-11), Zähringer et al.
(24), and Moll et al. (18). (A) Schematic
representation of the whole LPS structure. (B) Structure of OPS. Sugar
abbreviations: GlcN3N, 2,3-diamino-2,3-dideoxy-D-glucose;
Kdo, 3-deoxy-D-manno-octulosonic acid; Leg and
iso-Leg, derivatives of legionaminic acid and its C4 epimer,
respectively; Rha, L-rhamnose; QuiNAc,
2-acetamido-2,6-dideoxy-D-glucose
(N-acetylquinovosamine); R = Ac in strains Philadelphia
1 and CS338 or R = H in strain CS332.
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In the current study, we used strain CS332 in complementation
experiments to identify and characterize a locus, lag-1,
that is involved with acetylation of the O polysaccharide (OPS) of L. pneumophila serogroup 1 LPS.
Complementation of strain CS332.
A cosmid library containing
genomic DNA from L. pneumophila Philadelphia 1 was
constructed in pAM2 (Table 1) as
described by Marra et al. (13). The library was mobilized en
masse from Escherichia coli DH5
into L. pneumophila CS332 via triparental matings (14), and
nine transconjugants were identified that bound serogroup 1 LPS MAb,
MAB2, by a colony immunoblot assay (16). Plasmid DNA
isolated from the MAB2-positive transconjugants exhibited identical
restriction fragment patterns following digestion with several
restriction enzymes. A 4.5-kb SphI fragment (pLPS16, Table
1) from one of the recombinant cosmids restored binding of MAB2 and
33G2 and eliminated binding of 144C2 following electroporation (13) into strain CS332.
There were no obvious differences in the electrophoretic profiles of
LPSs isolated from either the mutant or complemented
strains (Fig.
2A). However, LPS from strain CS332 that
harbored
pLPS16 was able to bind MAB2 (Fig.
2B) and 33G2 in Western
blotting
experiments (
25).
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FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (A) and Western blot (B) analyses of LPSs from
Philadelphia 1 (wild-type) and lag-1 mutant strains. (A) LPS
was isolated as described previously (19), resolved on a
sodium dodecyl sulfate-14% polyacrylamide gel, and visualized by
silver staining. Equal amounts of LPS (~1.0 µg) were added to each
lane. Lanes: 1, S. enterica serovar Typhimurium; 2, Philadelphia 1; 3, CS332 (lag-1 negative); 4, CS334
(CS332/pLPS16 [lag-1 positive]). (B) Equal amounts of LPS
from strains Philadelphia 1, CS332, and CS338 (CS332/pLPS17
[lag-1 positive]) were resolved on sodium dodecyl
sulfate-14% polyacrylamide gels, transferred to nitrocellulose paper,
and probed with MAB2 as described by Mintz and Zou (15).
Lanes: 1, Philadelphia 1; 2, CS332; 3, CS338.
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Localization and nucleotide sequence of lag-1.
Tn5 mutagenesis (2) of pLPS16.1 (Table 1) showed
that the gene(s) responsible for complementation of CS332 was localized to an ~1.0-kb region of a 2.3-kb EcoRI fragment contained
within the cloned 4.5-kb SphI fragment (Fig.
3). This finding was consistent with
results of subcloning experiments which showed that the same 2.3-kb
EcoRI fragment (contained in pLPS17, Table 1) complemented CS332 to wild-type LPS MAb binding pattern.
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FIG. 3.
Localization of lag-1 by Tn5
mutagenesis. Tn5 insertions were introduced into pLPS16.1
and mapped according to the methods of de Bruijn and Lupinski
(2). Each triangle represents the position of a
Tn5 insertion in pLPS16.1. Plasmids containing each of the
Tn5 insertions were electroporated into strain CS332, and
transformants were tested for the ability to bind MAB2, 33G2, and
144C2. ( ), insertions eliminating binding of MAB2 and 33G2; (+),
insertions having no effect on the binding of MAB2 and 33G2. Wild-type
LPS has the following MAb binding pattern: MAB2 (+), 33G2 (+), 144C2
(). Restriction sites: C, ClaI; E, EcoRI; H,
HindIII; S, SphI; X, XhoI. The
black box defines the physical location of the lag-1 locus
as determined by DNA sequencing experiments.
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|
As shown in Fig.
3, Tn
5 insertion no. 40.8 in pLPS16.1
disrupted the gene(s) responsible for complementation of strain CS332.
To identify the gene(s) contained within this region, we cloned
the two
BamHI-
EcoRI fragments created by insertion no.
40.8 (Tn
5 contains a single
BamHI site) into
pGEM-7Zf(
) (Promega Corp.,
Madison, Wis.) and determined the
nucleotide sequence of both
strands of the 2.3-kb
EcoRI
fragment from pLPS16.1. The cloned
fragment contained a single open
reading frame (ORF) spanning
1,074 nucleotides. The ORF, designated
lag-1 (lipopolysaccharide-associated
gene), encoded a
357-amino-acid protein with a pI of 10.5 and
a predicted molecular mass
of 40,956 Da. Of interest, we recently
determined that strain CS332
contains a deletion mutation in the
lag-1 gene
(
23).
Distribution of the lag-1 locus.
Southern blot
experiments (14) with a lag-1 probe and genomic
DNA from L. pneumophila serogroups 1 to 5 were performed to determine the distribution of lag-1 in L. pneumophila. Results of these experiments showed that
lag-1 could be detected as a single DNA fragment of various
sizes in four of eight serogroup 1 LPS isolates (25). LPS
from the four serogroup 1 strains containing lag-1 DNA
sequences was able to bind MAB2, whereas three of four of these LPSs
bound 33G2. In contrast, LPS from the four lag-1-negative serogroup 1 isolates failed to bind MAB2 and 33G2. lag-1 DNA
sequences were not detected in chromosomal DNA from LPS serogroups 2 to 5.
lag-1 encodes a polypeptide involved with O acetylation
of serogroup 1 OPS.
The predicted amino acid sequence of
lag-1 showed strong similarity (54% identity) with a
protein called Oac, an O-acetyltransferase encoded by the
Shigella flexneri bacteriophage SF6 (22). The Lag-1 polypeptide was slightly larger than the Oac protein (357- versus
333-amino-acid residues). Nevertheless, similarity with Oac extended
throughout the entire Lag-1 polypeptide.
The lack of the 8-O-acetyl group from legionaminic acid in LPS produced
by the
lag-1 mutant CS332 was consistent with the
idea that
lag-1 encoded an
O-acetyltransferase. To test
this,
we used
1H- and
13C-NMR to characterize
the structures of the OPS of LPSs produced
by strains CS333 (wild
type), CS338 (CS332 plus pLPS17 [
lag-1 positive]), and
CS339 (strain CS332 plus pLPS20 [
lag-1 negative]).
OPSs were isolated from the above-mentioned LPSs and prepared for NMR
analysis as described previously (
11).
1H- and
13C-NMR spectra of OPSs were recorded at 90.6 MHz with a
Bruker
AM-360 spectrometer at pH 4 and 45°C (internal standard
acetone,
delta H 2.225, delta C 31.45) by using standard Bruker
software
(XWINNMR 1.3).
NMR spectroscopy clearly indicated that 8-O-acetylated legionaminic
acid was present in the OPSs from
lag-1-positive strains
CS333 and CS338. In contrast, OPS from the
lag-1-negative
strain
CS339 lacked the O-acetyl group at position 8 of legionaminic
acid. Accordingly, signals for an O-acetyl group at
22.0 (Me)
and
174.4 (CO) were present in the
13C-NMR spectra of OPSs from
wild type (CS333) and the complemented
strain (CS338) but absent from
the mutant (CS339 [Fig.
4]).
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FIG. 4.
13C-NMR spectra and corresponding structures
of OPSs from LPSs produced by wild-type, mutant, and complemented
strains. (A) CS338 (CS332/pLPS17). (B) CS339 (CS332/pLPS20). (C) CS333
(AM511/pLAW300). Signals for the 8-O-acetyl group (Me at 22.0 and
CO at 174.4) are marked by stars. Numbers refer to carbons C1 to C9
of legionaminic acid.
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Concluding remarks.
Helbig et al. (5, 6) showed
that chemical removal of O-acetyl groups from Philadelphia 1 LPS
resulted in loss of reactivity with MAB2 and permitted binding of
several MAbs that failed to bind to unmodified LPS. Consistent with the
findings of Helbig et al., LPS from strain CS332, which contained
8-O-deacetylated legionaminic acid, failed to bind MAB2 but was able to
bind MAb 144C2 (which cannot bind wild-type LPS). Complementation of
the lag-1 mutation in strain CS332 resulted in production of
OPS that contained 8-O-acetylated legionaminic acid (Fig. 4) which, in turn, restored binding of MAB2 but eliminated binding of MAb 144C2. Our
results, along with those of Helbig et al., demonstrate that recognition of serogroup 1 LPS by MAB2 requires an O-acetyl group at
position 8 of legionaminic acid and that the 8-O-acetyl group of
legionaminic acid can block access to other antigenic determinants contained in serogroup 1 LPS.
The limited distribution of the
lag-1 locus among serogroup
1 isolates and its absence in non-serogroup 1 strains suggest
that the
lag-1 gene product is not essential for biosynthesis
of
L. pneumophila LPS. Nevertheless, the MAB2 epitope is
thought
to be associated with the virulence of serogroup 1 LPS strains
that produce it (
3,
21). However, the contribution of the
MAB2 epitope to virulence is unclear, because Mintz and Zou
(
15)
showed that there was no difference in the
intracellular growth
of a
lag-1-positive strain and its
isogenic
lag-1 mutant in monocyte-like
U937 cells and
free-living
amoebae.
Slauch et al. (
20) determined that the Lag-1 polypeptide is
a member of a family of proteins that participate in the acylation
of
exported carbohydrate moieties. This family of proteins includes
Oac
from phage SF6 (acetylation of
Shigella LPS O antigen), OfaA
from
Salmonella typhimurium (acetylation of LPS O antigen),
GumF
from
Xanthomonas campestris pv. campestris (acetylation
of LPS
O antigen and xanthan polysaccharide), and several proteins from
Rhizobium spp. and
Streptomyces spp. that are
involved with the
acetylation of exopolysaccharides and the acylation
of macrolide
antibiotics, respectively. Although many regions of these
trans-acylases
are similar, the most striking homologies can
be found in the
regions corresponding to amino acids 45 to 89 and 143 to 161 in
Lag-1 (
20). TMpred analyses of Lag-1 indicated
many prominent
hydrophobic regions and 10 transmembrane helical
domains, suggesting
that the Lag-1 polypeptide, like other members of
this family
of proteins, is an integral membrane
protein.
The ability of complemented
lag-1 strains to produce OPS
containing 8-O-acetylated legionaminic acid, along with the strong
similarity between the predicted amino acid sequences of Lag-1
and the
Shigella O-acetyltransferase Oac,
suggests that
lag-1 encodes an
O-acetyltransferase. Overexpression of enzymatically
active Lag-1 protein and creation of an in vitro assay to measure
acetyltransferase activity will be necessary to confirm or refute
this
hypothesis.
Nucleotide sequence accession number.
The nucleotide sequence
of lag-1 has been deposited in Genbank under accession no.
U32118.
| |
ACKNOWLEDGMENTS |
We thank Barry Fields, Dick Miller, Marcus Horwitz, and Joe
Plouffe for supplying clinical and environmental isolates of
L. pneumophila. We are indebted to Sarah D'Orazio for help
with manipulation of lag-1 DNA sequence data. The computing
skills of Daniel Gonzalez are gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Present address: BioInsights,
Inc., 153 Dorchester Dr., East Windsor, NJ 08520. Phone: (609)
426-1751. Fax: (609) 426-4530. E-mail: cmintz{at}home.com.
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Journal of Bacteriology, July 1999, p. 4137-4141, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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