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Journal of Bacteriology, October 2001, p. 5756-5761, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5756-5761.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Analysis of a Pyocin-Resistant
Lipooligosaccharide (LOS) Mutant of Haemophilus ducreyi:
Restoration of Full-Length LOS Restores Pyocin
Sensitivity
Melanie J.
Filiatrault,1,2
Robert S.
Munson Jr.,3,4 and
Anthony A.
Campagnari1,3,5,*
Department of
Microbiology,1 Department of Medicine,
Division of Infectious Diseases,5 and
The Witebsky Center for Immunology and Microbial
Pathogenesis,2 State University of New York at
Buffalo, Buffalo, New York 14214, and Children's Research
Institute3 and Department of Molecular
Virology, Immunology, and Medical Genetics,4
Ohio State University, Columbus, Ohio 43205-2696
Received 29 March 2001/Accepted 29 June 2001
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ABSTRACT |
DNA sequence and Southern blot analyses were used to determine the
genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R
chromosome containing the suspected mutation was amplified, and
sequence analysis detected a 3,189-bp deletion. This deletion resulted
in the loss of the entire waaQ gene, another open
reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an
argininosuccinate synthase homolog, and a change in the 3' sequence of
the lgtF gene. Southern blot analysis confirmed that no
genomic rearrangements had occurred. Isogenic LOS mutants and the
respective complemented mutants were evaluated for susceptibility to
pyocin C. The mutants expressing truncated LOS were resistant to lysis
by pyocin C, and complementation restored sensitivity to the pyocin. We
conclude that HD35000R is defective in both glycosyltransferase genes
and that pyocin resistance is due to truncation of the full-length LOS molecule.
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TEXT |
Haemophilus ducreyi is a
gram-negative organism which causes chancroid. This genital ulcerative
disease is endemic in many developing countries, with infection rates
being highest in Africa and Asia, where prostitution is an important
risk factor (40). Occasionally, sporadic epidemics occur
in the United States; however, most of these outbreaks are associated
with drug use and the sale of sex for drugs or money (8,
9). Although it is difficult to obtain reliable data, the World
Health Organization estimated that approximately 7 million new cases of
chancroid infection occurred in 1995 worldwide (43). In
addition, chancroid is one of a number of genital ulcer diseases that
serve as cofactors for human immunodeficiency virus transmission
(7). Because the actual skin lesions are the likely site
of human immunodeficiency virus entry, identification and
characterization of virulence factors that contribute to ulcer
formation have become an important goal of current H. ducreyi research efforts.
One potential virulence factor of H. ducreyi is the
lipooligosaccharides (LOS). The LOS of H. ducreyi
structurally resembles LOS from other gram-negative mucosal pathogens,
such as Haemophilus influenzae, Neisseria
meningitidis, and Neisseria gonorrhoeae (5,
22-24). The LOS molecules from these three human pathogens are
important virulence factors involved in adherence to host epithelial
cells, serum resistance, and evasion of the host immune system
(16, 25, 31, 32, 39, 42). While these data suggest similar
functions for H. ducreyi LOS, the actual role of LOS in
chancroid is currently undefined. The inability to identify or
construct LOS mutants in H. ducreyi was initially a major
obstacle; however, this was overcome using pyocin lysis (4,
13) and transposon-based mutagenesis (15, 38) to
identify mutants defective in expression of LOS biosynthesis from
H. ducreyi strain 35000.
Pyocins, bacteriocins produced by Pseudomonas aeruginosa,
have structures similar to contractile bacteriophage tails and are thought to use the lipopolysaccharide or LOS molecule as a receptor (18). The mechanism of action of these particles is
through formation of a pore in the membrane and subsequent disruption of membrane potential resulting in cell death (41).
Pyocins also arrest protein and nucleic acid synthesis of the bacteria (17, 19, 27, 29). Although the mechanism(s) allowing
bacteria to survive pyocin lysis is unknown, pyocin-resistant strains
of N. gonorrhoeae and H. ducreyi have truncated
LOS molecules (4, 11, 13, 26). These pyocin mutants have
been used to identify genes involved in LOS biosynthesis (13, 21,
34), and several pyocin-resistant gonococci have been
characterized at the DNA level (35).
More recently, we described an H. ducreyi pyocin C survivor,
designated HD35000R (13). This pyocin mutant, derived from H. ducreyi strain 35000, was used to clone genes involved in
LOS biosynthesis using complementation. These data suggested that HD35000R had disruptions in both the waaQ and
lgtF glycosyltransferase genes. The aim of this study was to
investigate the genetic defect of HD35000R using Southern blot and DNA
sequence analyses and to begin to define the interaction of pyocin C
with LOS.
Bacterial strains and culture conditions.
H.
ducreyi strain 35000 is a wild-type strain isolated in Winnipeg,
Canada. HD35000R was derived from 35000 as a pyocin-resistant mutant
(13). The isogenic LOS mutants, 35000glu-, 35000hep-, and
35000hepglu- were constructed in our laboratory and have been described previously (13). pLS88 is an H. ducreyi shuttle vector (10). 35000glu-(pGLU),
35000hep-(pHEP), and 35000hepglu-(pLS88HG.13) are complemented strains
derived from the electroporation of plasmids containing the respective
wild-type gene(s) into the respective mutant (13).
35000hepglu-(pGLU) was derived from the electroporation of the plasmid
containing the lgtF gene. H. ducreyi 35000HP is a
human passaged variant of 35000 and has been described previously (1). All H. ducreyi strains were grown at
35°C in 5% CO2 on chocolate agar plates
(6). Escherichia coli strains were grown at
37°C on Luria-Bertani agar plates or in Luria-Bertani broth containing
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
(X-Gal) (40 µg/ml) and/or
isopropyl--D-thiogalactopyranoside (IPTG) (25 µg/ml) and kanamycin (50 µg/ml) as needed. P. aeruginosa
strain C was grown in Pseudomonas broth (11).
Chemicals, reagents, and enzymes.
Plasmid DNA was isolated
using Qiagen purification kits. Restriction enzymes were purchased from
New England Biolabs, Inc. Standard methods were used for restriction
endonuclease analysis, ligations, and transformation of plasmid DNA
(33).
Identification of the genetic defect in HD35000R.
Previously,
we reported the identification and characterization of a
pyocin-resistant LOS mutant designated HD35000R (13). Matrix-assisted laser desorption ionization mass spectrometry analysis
of O-deacylated LOS from HD35000R indicated that the LOS terminated
with a core consisting of only two of the three heptose residues
with no additional branch structures (Fig.
1B), compared to the wild-type strain
35000 (Fig. 1A). This mutant was complemented with a plasmid containing
the waaQ and lgtF genes (13).
Further, an isogenic mutant disrupted in both genes produced the same
LOS phenotype as HD35000R (Fig. 1B); therefore, we hypothesized that
HD35000R had mutations in both glycosyltransferase genes. Repeated
attempts to amplify these genes using primers developed to portions of
the waaQ and lgtF genes were unsuccessful for
reasons which will be explained below.
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FIG. 1.
Biochemical structures of H. ducreyi
35000 LOS and LOS structures expressed by genetically defined 35000 mutants, as determined by Filiatrault et al. (13). (A) The
major LOS structure from H. ducreyi 35000 consists of
lipid A, keto-deoxyoctulosonic acid, a triheptose core, and one main
oligosaccharide branch. This structure can also be substituted for by
sialic acid. (B) HD35000R synthesizes a LOS molecule which lacks the
main oligosaccharide branch and the third heptose residue of the core.
LOS synthesized by HD35000R and 35000hepglu- were identical. (C) The
LOS produced by the lgtF mutant, 35000glu-, consists of
lipid A, keto-deoxyoctulosonic, and the triheptose core. (D) The
waaQ mutant, 35000hep-, produces an LOS molecule similar
to the wild-type strain 35000; however, the structure lacks the third
heptose of the core and additional lactosamine repeats are added to the
terminal portion of the molecule. In addition, no sialic acid is
present in the waaQ LOS structure. The locations of
where the gene products of the 1,4 glucosyltransferase (LgtF) and
the heptosyltransferase III (WaaQ) act are indicated by dashed lines.
All core heptoses are L-glycero-D-manno-heptose
with the exception of the branch heptose (asterisk), which is
D-glycero-D-manno-heptose. Abbreviations are as
follows: LacNAc, N-acetyllactosamine; NeuAC, sialic
acid; Gal, galactose; Glu, glucose; GlcNAc,
N-acetylglucosamine; Hep, heptose; KDO,
keto-deoxyoctulosonic acid; PEA, phosphoethanolamine.
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The wild-type plasmid library constructed in pLS99 was used a second
time to complement HD35000R, and additional sequence
5' to the
waaQ gene was obtained (
13). This complementing
clone
was designated pMF1 (Fig.
2A).
Using this sequence, primers were
designed for the argininosuccinate
synthase homolog (P1), which
is 5' to
waaQ, and the
pgpB homolog (P2), which is upstream of
lgtF
(Fig.
2A). PCR was performed with
H. ducreyi 35000 and
HD35000R
genomic DNA. The expected 2.8-kb product was amplified from
the
wild-type strain. A slightly larger amplicon was amplified from
HD35000R chromosomal DNA, using the same primer pair. These amplicons
were purified using the GeneClean II kit (Bio 101), cloned into
the
pCR2.1 vector (Invitrogen), and the nucleotide sequences of
both
strands were determined. The nucleotide sequence of the plasmid
clone
containing the PCR product from the wild type was identical
with the
nucleotide sequence of
H. ducreyi 35000HP
(
http://www.ncbi.nlm.nih.gov /Microb_blast/unfinishedgenome.html;
data not shown). Sequence
analysis of the DNA fragment from HD35000R,
designated pCR35R3
(Fig.
2B), identified an open reading frame (ORF)
which encodes
a polypeptide that shares 59% identity and 72%
similarity to the
colicin V production proteins of
H. influenzae (
14) and
E. coli (
3) and therefore has been designated
cvpA.
Colicin V is a
proteinaceous bacterial toxin produced by many
strains of
E. coli and other members of the
Enterobacteriaceae and is associated
with the pathogenicity
of these organisms (
2). Interestingly,
the genetic
organization of the S-type pyocins of
P. aeruginosa is
similar to that of colicins (
12,
36). While
production
of a colicin may allow survival in a stressful environment
and
provide
H. ducreyi with an advantage over other
organisms, more
studies are needed to determine if this organism
possesses a similar
bacteriocin system and if production of this toxic
factor contributes
to the pathogenesis of
H. ducreyi.
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FIG. 2.
Partial ORF map of the H. ducreyi genome
and the plasmids characterized in this study. The ORFs are indicated by
open arrows which designate the direction of transcription. The solid
arrows represent the oligonucleotide primers used in PCR. The hatched
bars indicate the probes used for Southern blot analysis. The point
where the deletion occurred in HD35000R is indicated ( ). (A) Plasmid
pMF1 is a plasmid which complements HD35000R. (B) Plasmid pCR35R3
contains a 3.1-kb PCR product from HD35000R chromosomal DNA using
primers P1 and P2. (C) Plasmid p35RPFU#2 is pCRBlunt vector with a
2.16-kb PCR product from HD35000R chromosomal DNA using primers P3 and
P4. (D) Partial ORF map of the HD35000R genome. The deleted region is
designated by the hatched bar.
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The derived amino acid sequence of the next ORF has significant
homology with numerous RNases, including the RNase H from
H. influenzae (76% identity and 85% similarity) (
14).
The next
ORFs were the
lgtF, HI1333, and a portion of
pgpB (Fig.
2B). An
unexpected observation was the lack of
the ORF which has a derived
amino acid sequence similar to the
argininosuccinate synthase
of
H. influenzae (ArgG)
(
14), the region where primer P1 was
developed. Using dot
plot matrix analysis, we found that this
particular segment of DNA in
the
H. ducreyi genome contained a
sequence at the 5' end
which was the reverse complement of the
sequence at the 3' end. PCR
using a single primer, P2, confirmed
that this DNA fragment could be
amplified (data not
shown).
In order to analyze the DNA region surrounding the LOS genes and
identify the position of the colicin V gene in relation to
the
LOS biosynthesis genes, the
H. ducreyi 35000HP genomic
sequence
was obtained from the NCBI unfinished microbial genome BLAST
database
(
http://www.ncbi.nlm.nih.gov/icrob_blast /unfinishedgenome.html)
and
the High Throughput Sequencing Center at the University of
Washington,
Seattle. A homology search of the putative colicin
V gene with
the
H. ducreyi 35000HP genome revealed that this gene
is
~3 kb upstream of the glycosyltransferase genes (Fig.
2 [top]).
In
this gene cluster
cvpA is preceded by the gene encoding the
RNase H-like protein and an ORF which has a derived amino acid
sequence
with 42% identity and 66% similarity to a conserved hypothetical
membrane protein of
H. influenzae (HI0461) (
14)
and
N. meningitidis (
30) (Fig.
2 [top]).
These results suggest that the chromosomal
DNA of HD35000R contains a
large
deletion.
Based on the unexpected results obtained initially with primers P1 and
P2 (above), a second PCR of this region was performed
using another
primer set to further verify the suspected deletion
in strain HD35000R.
The entire region spanning the colicin V gene
to the
lgtF
from
H. ducreyi 35000 and HD35000R chromosomal DNA
was
amplified with oligonucleotide primers P3 and P4 (Fig.
2 [top]).
A
5.35-kb fragment was amplified from wild-type 35000 and a 2.16-kb
fragment was obtained from HD35000R as predicted. The smaller
size of
the PCR fragment obtained from HD35000R was consistent
with a 3.18-kb
deletion. This 2.16-kb product was cloned into
the pCRBlunt vector
(Invitrogen) to form p35RPFU2 (Fig.
2C), and
the sequence was
determined and analyzed. DNA sequences of p35RPFU2
were aligned to
35000HP. Sequence from p35RPFU2 was identical
to 35000HP except for a
3,189-bp deletion. In Fig.
2D the location
of the deletion is depicted
by a cross-hatched bar. Sequence analysis
did not reveal any unique
characteristics of the region where
the deletion occurred. These
results are consistent with the structural
and complementation data of
HD35000R LOS (
13) (Fig.
1).
In analyzing the DNA sequence obtained from HD35000R, we observed that
a portion of the 5' end of the HI0461 ORF had been
fused to the 3' end
of the
lgtF gene in frame, resulting in the
replacement of
the last 37 bp of
lgtF. The derived amino acid
sequence of
the HD35000R protein differs from the sequence of
the 35000HP protein
by 10 amino acids (data not shown). This suggests
that a fusion protein
would be generated between LgtF and a portion
of the HI0461 ORF in
HD35000R, although the LOS analysis suggests
that this is a
nonfunctional protein. LgtF is assigned to family
2 of the
glycosyltransferase enzymes
(
http://afmb.cnrs-mrs.fr/~pedro /CAZY/db.html). These enzymes
are inverting glycosyltransferases
that possess a nucleotide binding
domain (domain A) within the
N-terminal region (
37).
However, glycosyltransferases from the
same family demonstrate little
to no sequence conservation throughout
their C-terminal region
(
20). Therefore, residues which may
be involved in
substrate recognition and catalytic activity do
not appear to be
conserved. By creation of an in-frame fusion
between the two gene
portions of
lgtF and HI0461, critical residues
in the
C-terminal region of the LgtF may have been eliminated,
resulting in
loss of substrate binding. The LOS structure of HD35000R
suggests that
the mutation, which occurred in HD35000R, disrupts
properties of this
enzyme, and that one or more of the C-terminal
13 amino acids of LgtF
are required for proper function. It is,
however, possible that the
fusion protein may not be produced
or is unstable. Further studies are
required to determine the
specific residues that are important in LgtF
transferase
activity.
Southern blot analysis.
To determine whether a chromosomal
rearrangement or deletion of this region had occurred, Southern blot
analysis was performed as previously described (13), using
the NEBlot Phototope labeling kit and Phototope-Star detection kit (New
England Biolabs), with the exception that hybridizations were performed
at 60°C in 100 ng of denatured biotinylated probe per ml of
hybridization fluid. Chromosomal DNA was isolated from H. ducreyi strains, as previously described (13), and
digested to completion with BglII, electrophoresed on a
0.7% agarose gel, and transferred to Immobilon-Ny+ membrane (Millipore) by capillary blotting overnight. Probes to the
cvpA, rnh, and waaQ genes were
generated by PCR, utilizing the primers listed in Table
1 and the plasmids pCR35R3 and pMF1 as
templates, and were used to probe BglII-digested chromosomal
DNA from H. ducreyi strain 35000 and HD35000R. The
cvpA probe hybridized to a single fragment of ~2.2 kb from
H. ducreyi strain 35000 and HD35000R (data not shown). When
chromosomal DNA from H. ducreyi strain 35000 was probed with
a portion of the waaQ gene, a band of ~4.6 kb was observed
(data not shown). This probe did not hybridize with chromosomal DNA
from HD35000R, demonstrating that HD35000R does not contain this
portion of the waaQ gene (data not shown). The
rnh probe hybridized to a fragment of ~4.6 kb from
H. ducreyi strain 35000 and an ~1.4-kb fragment from
HD35000R chromosomal DNA (data not shown). These results are consistent
with a 3.18-kb deletion in HD35000R, demonstrating that no chromosomal
DNA rearrangements of these genes occurred.
Pyocin sensitivity of the LOS mutants and complemented
mutants.
HD35000R was initially selected based on resistance to
pyocin C. In order to confirm that pyocin resistance was a result of the truncation or loss of LOS genes, LOS mutants were assayed for
sensitivity to pyocin C. Isogenic LOS mutants of H. ducreyi strain 35000 lacking expression of the heptosyltransferase III (WaaQ),
the 1,4 glucosyltransferase (LgtF), and both glycosyltransferases were constructed and characterized previously (13). The
heptosyltransferase mutant produces an LOS molecule which is similar to
the wild type but lacks the third heptose of the triheptose core and
contains additional lactosamine repeats (Fig. 1D) (13).
The glucosyltransferase mutant produces a truncated LOS that terminates
after the triheptose core and lacks the main oligosaccharide branch
(Fig. 1C) (13). The LOS glycoform expressed by the double
mutant contains two of the heptose sugars of the core (Fig. 1B)
(13). Pyocin was isolated from cultures of P. aeruginosa strain C by the method described by Morse et al.
(28), and the pyocin lysis assay was performed as
described previously (4, 11).
Both the glucosyltransferase mutant (35000glu-) and the double mutant
(35000hepglu-) were resistant to pyocin lysis (Fig.
3B and D). However, the
heptosyltransferase mutant (35000hep-)
was sensitive to lysis by
pyocin C, suggesting that pyocin interacts
with the
full-length chain (Fig.
3C). To test whether sensitivity
could be
restored to 35000glu- and 35000hepglu-, the complemented
mutants were
analyzed. Complementing the glucosyltransferase with
the wild-type
lgtF and the double mutant with
lgtF and
waaQ restored
sensitivity to pyocin C (Fig.
3E and F). The
complemented
waaQ mutant remained sensitive, as expected
(data not shown). In addition,
the double mutant was complemented with
only the
lgtF gene. This
strain, which displays the same LOS
phenotype as the
waaQ mutant,
was also sensitive to the
pyocin (data not shown). These results
suggest that the main
oligosaccharide branch is involved in susceptibility
to this pyocin,
since the heptosyltransferase mutant and the wild
type, which possess a
common oligosaccharide chain, are sensitive.
These studies demonstrate
that the core is not involved in pyocin
attachment, since the LOS
synthesized by the heptosyltransferase
mutant lacks the third heptose
of the core and since this mutant
remains sensitive.
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FIG. 3.
Pyocin C sensitivity of H. ducreyi 35000, isogenic LOS mutants, and complemented LOS mutants. Wild-type
strain 35000 and isogenic LOS mutants were electroporated with the
pLS88 shuttle vector or pLS88 containing the respective wild-type
gene(s) as previously described (13). All strains were
then tested for pyocin sensitivity and observed for a zone of
lysis, as described in Materials and Methods. (A) 35000(pLS88);
(B) 35000hep-(pLS88); (C) 35000glu-(pLS88); (D) 35000glu-(pGLU);
(E) 35000hepglu-(pLS88); (F) 35000hepglu-(pLS88HG.13).
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Conclusions.
Genetic evaluation of several pyocin-resistant
gonococci revealed a 12-bp deletion and a point mutation in the
phosphoglucomutase gene and a nonsense mutation in the rfaF
gene (35). The authors hypothesized that because there
were no large deletions, it was unlikely that there was an interaction
of pyocin DNA with the bacterial DNA. However, these studies did not
investigate whether there were other deletions or rearrangements in the
remainder of the chromosome. Therefore, the possibility that there were large chromosomal deletions in these mutants cannot be excluded.
Because all pyocin mutants described to date synthesize truncated LOS
molecules compared to the parent strain, the lipopolysaccharide
and LOS molecules have been implicated as the receptor for
R-type
pyocins in
P. aeruginosa (
28) and
N. gonorrhoeae (
11,
18,
44), respectively.
Although many
N. gonorrhoeae prototype and
pyocin mutant
strains have been tested for pyocin sensitivity
(
11),
several drawbacks of this method include the instability
of pyocin
mutants, the possibility of other mutations in pyocin-resistant
strains, and the ability of
N. gonorrhoeae to phase vary its
LOS
molecule. The novelty of this study is the evaluation of stable
isogenic
H. ducreyi LOS mutants and their respective
complemented
mutants for pyocin sensitivity, demonstrating that
sensitivity
or resistance to pyocin is due solely to the LOS
molecule.
In this study, we evaluated isogenic mutants previously constructed in
our laboratory. As expected, the mutants, which produced
truncated LOS
molecules (35000glu- and 35000hepglu-), were resistant
to pyocin lysis
by pyocin C. Complementation of the
H. ducreyi lgtF and
waaQ lgtF double mutant with the wild-type genes in
trans restored sensitivity to pyocin C. In addition,
complementation
of the
H. ducreyi waaQ lgtF mutant with the
lgtF gene alone restored
sensitivity to pyocin. This finding
suggests that the main oligosaccharide
branch of the LOS molecule is
involved in susceptibility to this
pyocin and that the third heptose of
the core is not required.
In addition, novel genes present in the
H. ducreyi genome were
identified, suggesting that this
organism may contain a bacteriocin-like
system similar to those
described for other
pathogens.
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ACKNOWLEDGMENTS |
This work was supported by the National Institutes of Health grant
R01 AI30006 (to A.A.C.). M.J.F. was partially supported by training
grant AI07614-01 from the National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, State University of New York at Buffalo, Biomedical
Research Bldg., Rm. 143, 3435 Main St., Buffalo, NY 14214. Phone: (716) 829-2673. Fax: (716) 829-3889. E-mail:
AAC{at}acsu.buffalo.edu.
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Journal of Bacteriology, October 2001, p. 5756-5761, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5756-5761.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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