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Journal of Bacteriology, April 2000, p. 2292-2298, Vol. 182, No. 8
Children's Research
Institute,1 Department of
Pediatrics,4 and Department of Molecular
Virology, Immunology and Medical Genetics,2
The Ohio State University, Columbus, Ohio 43205-2696, and
Department of Pharmaceutical Chemistry, University of
California, San Francisco, California
94143-04463
Received 29 July 1999/Accepted 27 January 2000
Haemophilus ducreyi is the etiologic agent of
chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is
considered to be a major virulence determinant and has been implicated
in the adherence of H. ducreyi to keratinocytes. Strain
A77, an isolate from the Paris collection, is serum sensitive, poorly
adherent to fibroblasts, and deficient in microcolony formation.
Structural analysis indicates that the LOS of strain A77 lacks the
galactose residue found in the N-acetyllactosamine portion
of the strain 35000HP LOS as well as the sialic acid substitution. From
an H. ducreyi 35000HP genomic DNA library, a clone
complementing the defect in A77 was identified by immunologic screening
with monoclonal antibody (MAb) 3F11, a MAb which recognizes the
N-acetyllactosamine portion of strain 35000HP LOS. The
clone contained a 4-kb insert that was sequenced. One open reading
frame which encodes a protein with a molecular weight of 33,400 was
identified. This protein has homology to glycosyltransferases of
Haemophilus influenzae, Haemophilus somnus,
Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was
insertionally inactivated, and an isogenic mutant of strain 35000HP was
constructed. The most complex LOS glycoform produced by the mutant has
a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to
that of the LOS of strain A77 and lacks the 3F11-binding epitope.
Structural studies confirm that the most complex glycoform of the LOS
isolated from the mutant lacks the galactose residue found in the
N-acetyllactosamine portion of the strain 35000HP LOS.
Although previously published data suggested that the serum-sensitive
phenotype of A77 was due to the LOS mutation, we observed that the
complemented A77 strain retained its serum-sensitive phenotype and that
the galactosyltransferase mutant retained its serum-resistant
phenotype. Thus, the serum sensitivity of strain A77 cannot be
attributed to the galactosyltransferase mutation in strain A77.
Chancroid is a genital ulcerative
disease caused by Haemophilus ducreyi which is prevalent in
several developing countries (30, 46). Although chancroid
has been characterized histologically (16, 17), little is
known about the molecular basis for ulcer formation. A number of
putative virulence determinants of H. ducreyi have been
described, including a hemolytic cytotoxin (3, 35, 36, 45),
cytolethal distending toxin (11), pili (9), a
hemoglobin binding protein (15, 44), and lipooligosaccharide (LOS) (10, 18, 25, 32). The structure of the oligosaccharide portion of the LOS resembles the structure of human paraglobosides. Thus, it has been proposed that the LOS may help the organism evade the
human immune response by molecular mimicry (26, 27). Direct
injection of H. ducreyi LOS causes ulcers in rabbits and mice (10, 25, 47). Injection of H. ducreyi LOS
causes a stronger inflammatory response in rabbits than injection of
rough lipopolysaccharide from Escherichia coli
(10). Other studies have implicated LOS in adherence to
fibroblasts and keratinocytes (1, 18).
Odumeru and coworkers demonstrated that strain A77 was killed by
incubation in normal human serum (32, 33). Further, they observed that the bactericidal activity in normal human serum could be
removed by incubation with heat-killed whole cells or LOS from
serum-sensitive strains but not by incubation with heat-killed serum-resistant strains or LOS from serum-resistant strains. They concluded that LOS composition contributed to the susceptibility of
H. ducreyi to the bactericidal activity of normal human
serum. A serum-resistant derivative of A77 was also isolated, and the LOS composition of this derivative appeared to differ from that of A77.
In order to increase our understanding of the role of LOS in the
pathogenesis of chancroid, the structures of the major glycoforms of
the LOS from H. ducreyi strains have been determined
(8, 28, 29, 41, 42). The major glycoform from strain 35000HP has the following structure:
Gal In this study, we demonstrate that the LOS biosynthetic defect in
strain A77 is in the galactosyltransferase II gene, designated lgtB, and that this defect is not responsible for the
serum-sensitive phenotype.
Bacterial strains and culture conditions.
H. ducreyi
strains (Table 1) were grown at 35°C
with 5% CO2 on chocolate agar (Becton Dickinson).
Chocolate agar plates supplemented with streptomycin at 20 µg/ml,
kanamycin at 20 µg/ml, and/or X-Gal (5-bromo-4-chloro-3-indolyl-
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Characterization of the
Lipooligosaccharide Galactosyltransferase II Gene of
Haemophilus ducreyi
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
1
4GlcNAc1 3Gal14Hep
16Glc1(Hep12Hep1)3,4Hep1 5Kdo.
Approximately one-third of the terminal galactose residues are
substituted with sialic acid (8, 28). The structure of the
LOS of strain A77 was also determined (B. W. Gibson, W. Melaugh, N. J. Phillips, and A. A. Campagnari, Abstr. 99th Gen. Meet.
Am. Soc. Microbiol., abstr. B/D-51, p. 39, 1999). The most complex glycoform of the A77 LOS lacks the galactose found in the
N-acetyllactosamine portion of strain 35000HP LOS. The
monoclonal antibody (MAb) 3F11 binds to the terminal
Gal14GlcNAc13Gal epitope; thus A77 does not bind
MAb 3F11 (40).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-D-galactopyranoside) at 40 µg/ml were prepared as previously described (18, 34).
Brain heart infusion broth supplemented with 5% fetal calf serum,
0.0025% hemin chloride solution (Sigma; predissolved in 20 mM NaOH),
and 1% IsoVitaleX (sBHI) was used for growth of H. ducreyi
in liquid medium. E. coli strains were grown on
Luria-Bertani (LB) plates or in LB broth supplemented with appropriate
antibiotics. Kanamycin was used at 20 µg/ml, chloramphenicol was used
at 30 µg/ml, and ampicillin was used at 50 µg/ml where appropriate.
TABLE 1.
Bacterial strains and plasmids used in this study
Modification of shuttle vector pLS88.
The H. ducreyi-E. coli shuttle vector pLS88 (13) was modified
by addition of the cloning site from pSuperCosI (Stratagene, La Jolla,
Calif.) which contains the following restriction sites in the indicated
sequence:
EcoRI-NotI-BamHI-NotI-EcoRI.
To facilitate construction, the chloramphenicol acetyltransferase gene
(cat) from pUC4DEcat was cloned into the BamHI
site of pSuperCosI. The EcoRI fragment from this construct
was then ligated to EcoRI-digested, dephosphorylated pLS88.
The ligation mixture was transformed into DH5, and clones were
selected on LB plates supplemented with kanamycin and chloramphenicol.
A plasmid with the appropriate restriction map, designated pRSM1937,
was digested with BamHI, religated, and transformed into
DH5, and clones were selected on kanamycin plates. A plasmid lacking
the chloramphenicol resistance gene was saved as pLS99. This plasmid
has a unique BamHI cloning site flanked by NotI sites.
Construction of an H. ducreyi 35000HP genomic DNA
library.
Chromosomal DNA from H. ducreyi 35000HP was
purified with the AGTC bacterial genomic DNA purification kit (Edge
Biosystems, Gaithersburg, Md.) and was partially digested with
Sau3AI. Fragments 3 to 5 kb in size were isolated on a 1%
agarose gel, purified with the Geneclean kit (Bio 101, Inc., La Jolla,
Calif.), and then ligated to BamHI and calf intestine
alkaline phosphatase-treated pLS99. The ligation reaction mixture was
electroporated into DH5 as previously described (34),
yielding approximately 1.2 × 104 recombinant clones.
These transformants were harvested from plates and suspended in 100 ml
of LB broth containing kanamycin. Some cells were kept in 10% glycerol
and stored at 
70°C for future usage. Plasmid DNA was purified from
the remainder of the preparation using the Wizard DNA purification
system (Promega Corp., Madison, Wis.).
Immunologic screening of library.
The plasmid library was
electroporated into H. ducreyi strain A77 as previously
described (34) with a Bio-Rad Gene Pulser II at 200 
, 2.5 kV, and 25 µF. Transformants were transferred to nitrocellulose
filters (Schleicher & Schuell, Keene, N.H.) and screened
immunologically by colony blot assay as described previously using MAb
3F11 (18). An immunologically reactive clone was identified.
The plasmid from this clone was designated pRSM1955.
Nucleotide sequence analysis. The DNA sequence was determined in both directions using an ABI 377 automated DNA sequencer. Contig assembly and sequence analysis were performed with DNASTAR (Madison, Wis.) software. Homology was determined through the National Center for Biotechnology Information BLAST network server, the Megalign program (DNASTAR), and the GAP program from the Genetics Computer Group, Madison, Wis.
Construction of an H. ducreyi 35000HP isogenic mutant
deficient in galactosyltransferase II.
The insert in pRSM1955
contains an internal EcoRI site as well as the two
EcoRI sites at the vector insert junction. The large fragment in the insert was isolated as an EcoRI fragment and
subcloned into pUC19 to form pRSM1956. Plasmid pRSM1956 was partially
digested with MunI. Full-length linear DNA was isolated on
an agarose gel, dephosphorylated, and ligated to the Km-2 element,
which had been isolated as an EcoRI fragment from pJRS102.0
(38). The ligation mixture was transformed into DH5, and
clones were isolated on LB plates supplemented with kanamycin and
ampicillin. EcoRI and BamHI restriction enzyme
analysis was performed to select a plasmid in which the kanamycin
cassette was inserted in the putative galactosyltransferase II gene. A
plasmid with the appropriate restriction map was saved as pRSM1975.
-galactosidase can be used as a
counter-selectable marker. The EcoRI fragment of pRSM1975
was isolated, blunt ended, and cloned into the NotI site of
pRSM1791 to form pRSM1977. pRSM1977 DNA was electroporated into
H. ducreyi 35000HP, and kanamycin-resistant clones were
selected. Kanamycin-resistant clones were then streaked for isolation
on chocolate agar containing both kanamycin and X-Gal. Clones that were
white and that grew normally were further characterized by Southern
blotting to verify the allele exchange and loss of plasmid sequences. A
representative mutant, designated 35000HP-RSM210, was chosen for LOS
biochemical structure analysis.
Southern blot hybridization. Chromosomal DNA was isolated from H. ducreyi strains and digested with EcoRI, subjected to electrophoresis on a 0.7% agarose gel, and transferred to a nylon membrane using the Turbo Blotter kit (Schleicher & Schuell). The plasmid DNA of pRSM1956 was labeled with 32P using the RadPrime DNA labeling system (Gibco/BRL, Gaithersburg, Md.). Hybridization and posthybridization were performed as described previously (34).
Complementation of the glycosyltransferase II mutation. Competent cells of 35000HP-RSM210 were prepared and transformed with pRSM1955 as described above. Clones were selected on chocolate plates supplemented with streptomycin.
Preparation and analysis of LOS.
Crude LOS preparations from
H. ducreyi strains were prepared by a modified microphenol
method and analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on a 14% acrylamide gel as previously
reported (10). To obtain larger amounts of the mutant strain
LOS (1 mg) for subsequent mass spectrometric characterization studies,
this strain was grown in liquid media (1 liter) and LOS was prepared as
described elsewhere (5, 24, 48). Crude LOS preparations
isolated from both the mutant and wild-type strains were O-deacylated
with hydrazine (37°C for 30 min) (21) to increase
solubility and make the LOS more amenable to mass spectrometric
analysis (19). In addition, a portion of the O-deacylated
LOS (150 µg) was further treated with 48% aqueous HF (at 4°C for
16 h) to remove phosphoethanolamine (PEA) and phosphate groups
(29). Finally, an oligosaccharide fraction was prepared by
hydrolysis of the crude LOS (175 µg) in a solution of 1% acetic acid
(350 µl) at 100°C for 2 h (29). The resulting water-soluble oligosaccharides were then separated from the lipid A
fraction by centrifugation (5,000 × g, 4°C) and
purified by size exclusion chromatography using two 300- by 7.8-mm
BioSelect SEC 125-5 columns (Bio-Rad) connected in series. Samples were eluted in 50 mM pyridinium acetate (pH 5.2) at a flow rate of 1 ml
min
1. Fractions were collected in 0.5- to 1-ml volumes.
The O-deacylated LOS, the HF-treated and O-deacylated LOS, and the
acid-released oligosaccharide fractions were all analyzed by mass
spectrometry as described below.
-MSH (CZE mixture; Bio-Rad), and adrenocorticotropin 1-24 (Sigma). All samples were prepared using a 320 mM 2,5-dihydroxybenzoic acid solution in 4:1
(vol/vol) acetone-water containing 175 mM 1-hydroxyisoquinoline (19). In all cases, 1 µl of analyte (0.1 to 1 µg of
material) was mixed with 1 µl of matrix solution, desalted with
cation exchange resin beads (DOWEX 50X, NH4+)
(31), and then air dried at room temperature on a stainless steel target. Typically, 20 to 50 laser shots were used to record each
linear and reflectron spectrum, or spectral segment if PSD conditions
were used.
Bactericidal assay. H. ducreyi cells grown for 48 h on a chocolate agar plate were washed in phosphate-buffered saline, then subcultured into sBHI broth, and grown to early log phase (optical density at 600 nm [OD600] = 0.2). The cells were pelleted and resuspended to the same OD in Hanks' balanced salt solution-0.1% gelatin (HBSSg). The cells were diluted 1:100 in HBSSg, and 30 µl was used in each sample. The cells were assayed in HBSSg with 40 and 60% normal human serum (pooled from four subjects) in a total volume of 300 µl. Two controls were employed for each sample. A control without serum was used to determine the total cell count, and a heat-inactivated serum control was used to verify that killing of the bacteria was due to complement. Serum was inactivated by heating at 56°C for 30 min. Except for the control without serum, samples were incubated at 35°C with shaking (160 rpm). Dilutions of the control without serum were plated immediately. The other samples were plated after 60 and 120 min. All dilutions were plated in triplicate on chocolate agar plates. Bactericidal activity was measured as percent survival compared to that of the time zero control.
Nucleotide sequence accession number. The sequence of the insert of pRSM1955 obtained in this study has been assigned GenBank accession no. AF224466.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Structural characterization of strain A77 LOS.
H.
ducreyi strain A77, a strain from the Paris collection, is serum
sensitive (32, 33), poorly adherent to fibroblasts, and
deficient in microcolony formation (2, 4). The structure of
the LOS of strain A77 was determined by nuclear magnetic resonance, carbohydrate, and mass spectrometric analysis (Gibson et al., Abstr.
99th Gen. Meet. Am. Soc. Microbiol.). This LOS lacks the sialic acid
substitution as well as the galactose residue found in the
N-acetyllactosamine portion of strain 35000HP and therefore is defective in 3F11 binding (Fig. 1).
Since galactose was present in the LOS of strain A77, we hypothesized
that A77 could synthesize UDP-galactose, produced a functional
galactosyltransferase I, and was likely deficient in the
galactosyltransferase II gene.
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Screening of H. ducreyi 35000HP genomic DNA
library.
The A77 strain was used as the recipient for an H. ducreyi 35000HP genomic plasmid library. Clones which reacted with
MAb 3F11 were identified. One plasmid, designated pRSM1955, conferred 3F11 reactivity to strain A77. The SDS-PAGE profile of the LOS isolated
from strain A77/pRSM1955 was similar to the profile of the LOS from
strain 35000HP (Fig. 2).
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Nucleotide sequence analysis.
The sequence of the insert of
pRSM1955 was determined. This insert contains three complete open
reading frames (ORFs) (Fig. 3). The 5'
portion of the sequence contains a partial ORF, designated orf1,
encoding the H. ducreyi homologue of pseudouridylate
synthase (P44445). orf2 begins at nucleotide 905 and is 783 bp in
length. The derived amino acid sequence has homology with those of
hypothetical proteins from Haemophilus influenzae (HI0177)
and other gram-negative organisms. orf3 begins at nucleotide 1772 and
is 1,047 bp in length. The derived amino acid sequence is 87%
identical to that of the product of the sialylglycoprotease
(gcp) gene of P. haemolytica, and therefore orf3
has been designated gcp. orf4 starts at nucleotide 2833 and
is 843 bp in length. The hypothetical protein encoded by orf4 has
homology with the products of numerous glycosyltransferase genes
including genes from H. influenzae (12, 22),
Haemophilus somnus (Genbank accession no. AF096997)
(unpublished data), Neisseria meningitidis (23),
Neisseria gonorrhoeae (20), and P. haemolytica (39). As orf4 encodes a glycosyltransferase
that catalyzes the transfer of galactose to GlcNAc (see below) and has
homology to Neisseria galactosyltransferases with similar specificities (encoded by a gene previously designated
lgtB), we have tentatively designated this gene
lgtB pending a unified nomenclature for the
glycosyltransferase genes of Haemophilus and
Neisseria.
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Insertional mutagenesis of the putative glycosyltransferase gene. Given the observed homology between the H. ducreyi lgtB gene and other glycosyltransferase genes, it was extremely likely that the observed complementation in A77/pRSM1955 was due to the lgtB gene. In order to demonstrate directly that a mutation in lgtB would result in a strain producing a truncated LOS, we insertionally inactivated the lgtB gene in pRSM1956 and constructed an isogenic mutant in the 35000HP background. Presumptive mutants were identified as described in Materials and Methods, and Southern blot hybridization was performed to confirm proper allelic exchange. The plasmid pRSM1956 containing the glycosyltransferase gene was used to probe blots containing EcoRI-digested chromosomal DNA from six putative mutants. A hybridization band of 5.5 kb was observed with strain 35000HP. All mutants had a single hybridizing band of 8.5 kb (data not shown), and one mutant, designated 35000HP-RSM210, was saved for further analysis.
Analysis of the LOS from the isogenic galactosyltransferase mutant of H. ducreyi. LOS isolated from strain 35000HP-RSM210 was separated by SDS-PAGE along with LOS obtained from the parental strain, 35000HP (Fig. 2). The most complex major glycoform produced by strain 35000HP-RSM210 ran with a mobility identical to that of the major glycoform produced by strain A77. The major glycoform produced by strains A77 and 35000HP-RSM210 ran considerably faster than the two major glycoforms present in strain 35000HP LOS. One of these two major glycoforms terminates in N-acetyllactosamine, and the other terminates in sialyl-N-acetyllactosamine (Fig. 1).
The structure of the strain 3500HP-RSM210 LOS glycoforms was determined by mass spectrometric experiments on several chemically degraded forms of the LOS in a fashion similar to that previously reported for structural studies on the parental strain 35000 (8, 18). To obtain the precise molecular masses of the LOS glycoforms, LOSs from both the mutant and parental wild type strains were converted into their O-deacylated forms and analyzed by negative-ion MALDI-TOF mass spectrometry. As shown in Fig. 4A, the wild-type O-deacylated LOS gave a series of singly deprotonated molecular ion peaks, with the four most abundant peaks corresponding to the LOS glycoforms terminated in N-acetyllactosamine (m/z 2709.4) and sialyl-N-acetyllactosamine (m/z 3001.1), both of which were partially replaced with a single PEA group (m/z 2832.6 and 3123.9, respectively). These masses and assignments were consistent with structures previously reported for the wild-type strain passaged in humans (8). In contrast, O-deacylated LOS from the mutant showed three abundant peaks at lower masses, (M H) at m/z 2671.2, 2548.1, and 2468.4 (Fig. 4B). The two higher peaks at m/z
2671.2 and 2548.1 yielded molecular masses consistent with the expected
composition for this O-deacylated LOS, as shown in Fig. 1B, involving
the loss of terminal Gal (162 Da; m/z 27102548 and
28332671) or its sialylated counterpart, NeuAc23Gal (453 Da; m/z 30012548 and 31242671), from the four major
LOS glycoforms present in the parental strain. Indeed, these same
species are also present in the parental wild-type strain but at much
lower abundances. The remaining lower-mass peak at m/z 2468 appears to arise from the additional loss of a HexNAc (203-Da)
moiety, and can be attributed to the loss of terminal GlcNAc from the major LOS glycoform at m/z 2671. In addition to the
molecular ions, several "prompt fragments" (data not shown)
resulting from gas phase fragmentation at the labile Kdo-lipid A were
present. Fragmentation of this bond among the three molecular ions
gives rise to the conserved diphosphoryl-di-N-acyl lipid A
peak at m/z 952 and the three oligosaccharides that differ
in PEA content at m/z 1718.8, 1595.7, and 1515.2. Fragmentation analysis of the major LOS glycoform at m/z
2671.4 by MALDI-PSD, a technique that allows for specific ion selection
followed by detection of metastable decomposition fragments, confirmed
this interpretation. Moreover, the PSD spectrum showed that the
oligosaccharide fragment at m/z 1718.2 undergoes further
decomposition with losses of PEA (m/z 1595.9) and/or
phosphorylphosphoethanolamine (PPEA; m/z 1515.7), consistent
with a structure containing a single PEA linked exclusively to the
phosphate of phospho-Kdo, i.e., Kdo(PPEA), and not the heptose
core. As the pattern of phosphorylation can vary in LOS, particularly
when oligosaccharide truncations are introduced, the corresponding
dephosphorylated O-deacylated LOS from this mutant was examined by
MALDI and MALDI-PSD after HF treatment. The most abundant molecular ion
species was (M + Na)+ at m/z 2331.60 (exact
mass), consistent with the expected loss of two phosphates (160 Da)
from the lipid A moiety and a loss of PPEA (203 Da) from the
oligosaccharide moiety. This phosphate substitution pattern is
therefore similar, if not identical, to that defined in the parental
wild-type strain.
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-elimination under mild acid conditions, yielding
terminal anhydro-Kdo. A MALDI-PSD spectrum of the most abundant
oligosaccharide (Fig. 5) yielded a series
of sequence ions that correspond to cleavage of the carbohydrate chain,
including a complete Y-ion series (m/z 1336.2, 1173.9, 981.7, 819.6, and 243.8) and B-ion series (m/z 388.2, 580.8, 742.9, and 934.9). (Ion nomenclature is according to that proposed by Domon and Costello [14]). Together with previous mass
spectrometry data, these studies support a structure in which the most
abundant LOS glycoforms from 35000HP-RSM210 are identical to those from the parental strain LOS except that they now terminate prior to the
second galactose residue. Furthermore, the LOS of this mutant strain
directly corresponds to the LOS of the "atypical" wild-type strain
A77.
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Sequence of the galactosyltransferase II gene from strain A77. The galactosyltransferase II genes from strains 35000HP and A77 were amplified by PCR. Residual PCR primers were removed by spin column purification, and the PCR products were directly sequenced. The nucleotide sequence of the gene from 35000HP agreed with the sequence determined from the plasmid clone, while the sequence of the gene amplified from strain A77 differed by a single nucleotide. The derived amino acid sequence of the A77 protein differs from the sequence of the strain 35000HP protein by an alanine-to-threonine change at position 207 (data not shown).
Serum sensitivities of strains 35000HP and 35000HP-RSM210. In order to determine whether the serum sensitivity of strain A77 was due to the LOS mutation, as previous data suggested, we determined the serum sensitivities of strains 35000HP, A77, 35000HP-RSM210, and A77/pRSM1955. As reported by other investigators, strain 35000HP is resistant to the bactericidal activity of 40 or 60% normal human serum. In our experiments, the survival of strain 35000HP in 60% normal human serum was 269% ± 26%, while the survival of strain A77 under the same conditions was 1% ± 1%. Surprisingly, complementation of the LOS defect in strain A77 (strain A77/pRSM1955) did not result in a serum-resistant phenotype (<1% survival in 60% serum). Similarly, introduction of a galactosyltransferase II mutation into strain 35000HP did not make the strain serum sensitive (222% ± 53% survival).
LOS (lipopolysaccharide) is a critical virulence factor in many gram-negative pathogens, and mutations in LOS biosynthesis frequently result in strains with a decreased resistance to the bactericidal activity of normal human serum. Characterization of the LOS from strain A77 and identification of the glycosyltransferase mutation in strain A77 were of particular interest as strain A77 was considered to be serum sensitive because of a LOS defect. However, our data fail to support this earlier hypothesis, as repair of the LOS defect in A77 failed to confer a serum-resistant phenotype to the strain and an isogenic mutant of strain 35000HP deficient in the lgtB gene retained its serum-resistant phenotype.| |
ACKNOWLEDGMENTS |
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We thank Huachun Zhong for excellent technical assistance.
This work was supported by National Institutes of Health grants R01 AI38444 (R.S.M.) and R01 AI31254 (B.W.G.) and by PE Biosystems, which kindly provided instrumentation to B.W.G. The DNA sequence was determined by the Core Facility at Children's Research Institute, which was supported in part by National Institutes of Health grant HD34615.
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ADDENDUM IN PROOF |
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Elkins and coworkers recently identified a protein, designated DrsA, that is in part responsible for the serum resistance of Haemophilus ducreyi (C. Elkins, K. J. Morrow, Jr., and B. Olsen. Infect. Immun. 68:1608-1619, 2000).
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FOOTNOTES |
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* Corresponding author. Mailing address: Children's Research Institute, Room W402, 700 Children's Dr., Columbus, OH 43205. Phone: (614) 722-2680. Fax: (614) 722-3273. E-mail: munsonr{at}pediatrics.ohio-state.edu.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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