In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once Keywords lipooligosaccharide; moiety; Moraxella catarrhalis; outer membrane; ro
Trang 1molecule in biological activities of the Moraxella
catarrhalis outer membrane
Daxin Peng1,*, Wei-Gang Hu1,†, Biswa P Choudhury2, Artur Muszyn´ski2, Russell W Carlson2 and Xin-Xing Gu1
1 Vaccine Research Section, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville,
MD, USA
2 Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
Moraxella catarrhalis, once considered a
nonpatho-genic bacteria to humans, is now one of the leading
causes of bacterial otitis media in children, after
Strep-tococcus pneumoniae and Haemophilus influenzae [1–3]
In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once
Keywords
lipooligosaccharide; moiety; Moraxella
catarrhalis; outer membrane; role
Correspondence
Xin-Xing Gu, 5 Research Court,
Room 2A31, Rockville,
MD 20850, USA
Fax: +1 301 435 4040
Tel: +1 301 402 2456
E-mail: guxx@nidcd.nih.gov
Present addresses
*School of Veterinary Medicine,
Yangzhou University, Yangzhou,
Jiangsu, China
†Defence Research and Development
Canada-Suffield, Medicine Hat, Alberta,
Canada
Database
The nucleotide sequence of lgt3 gene in
Moraxella catarrhalis strain O35E has been
submitted to the GenBank database under
the accession number DQ208195
(Received 19 June 2007, revised 30 July
2007, accepted 21 August 2007)
doi:10.1111/j.1742-4658.2007.06060.x
Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS) The core OS can be dissected into a linker and three
OS chains To gain an insight into the biological activities of the LOS molecules of M catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a sero-type A O35Elgt3 LOS mutant MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted
of a Glc-Kdo2 (linker)-lipid A structure Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents However, the mutant was sensi-tive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells These data, combined with our previ-ous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and sta-bility to preserve normal cell growth Both the lipid A and linker moieties contribute to the LOS toxicity
Abbreviations
BHI, brain-heart infusion; CFU, colony forming units; COPD, chronic obstructive pulmonary disease; DPBSG, Dulbecco’s phosphate-buffered saline containing 0.05% gelatin; EU, endotoxin units; Kdo, 3-deoxy- D -manno-2-octulosonic acid; kds, 3-deoxy- D -manno-2-octulosonic acid transferase; LOS, lipooligosaccharide; lpxA, UDP-N-acetylglucosamine acyltransferase; OMPs, outer membrane proteins; OMVs, outer membrane vesicles; OS, oligosaccharide.
Trang 2with otitis media, and M catarrhalis is responsible for
15–25% of those cases [4,5] M catarrhalis is also a
common cause of lower respiratory tract infections in
adults with chronic obstructive pulmonary disease
(COPD), the fourth leading cause of death in the
Uni-ted States [6,7] Lower respiratory tract infections have
been shown to contribute to the progression of the
COPD Approximately 20 million cases of such
exacer-bations are reported each year in the United States, up
to 35% of them resulting from M catarrhalis
infec-tions
The pathogenesis of M catarrhalis infection is not
well understood As a gram-negative bacterium,
M catarrhalis without capsular polysaccharides is
surrounded by an outer membrane, consisting of
lipo-oligosaccharide (LOS), outer membrane proteins
(OMPs) and pili outside phospholipids [2] The outer
membrane of gram-negative bacteria was initially
con-sidered to protect the bacteria from several antibiotics,
dyes, and detergents (hydrophobic compounds) which
would normally damage the cell wall However, more
outer membrane biological activities have become
apparent over years of extensive research and there is
indication today that the outer membrane of
gram-negative bacteria has some other biological activities,
such as adherence and invasion in vitro, colonization
in vivo, maintenance of cell surface charge and serum
bactericidal resistance [8–13]
Which component on the outer membrane of
M catarrhaliscontributes to the above-mentioned
bio-logical activities? Several OMPs of M catarrhalis were
identified, but their functions were mainly reported as
potential vaccine antigens, although some of them
were indicated to be adhesins [5,14–17] Pili were
con-sidered to be associated with bacterial attachment, but
both piliated and nonpiliated M catarrhalis strains
could adhere to human mucosal cells in vitro [18] LOS
has mostly been investigated for these biological
activi-ties of the M catarrhalis outer membrane [19–24]
M catarrhalis LOS is a major component of the bacterial outer membrane with three main serotypes A,
B and C, of which serotype A is the major type [25,26] Quite a few studies have demonstrated that such an LOS molecule is an important virulence factor for some other respiratory tract pathogens, such as Neisseria meningitidis, Neisseria gonorrhoeae, and
H influenza [27–29] Studies have also implicated the
M catarrhalis LOS as important in the pathogenesis
of the M catarrhalis infection [13,21,23,24] We recently reported that a mutant which lacked LOS showed decreased toxicity, reduced resistance to nor-mal human serum killing, and reduced adherence to human epithelial cells in vitro and in vivo, while show-ing hypersensitivity to hydrophobic compounds, indi-cating that LOS contributes to most biological activities of the M catarrhalis outer membrane [24] The molecular structure of the M catarrhalis LOS is smaller than its lipopolysaccharide counterparts in gram-negative pathogens like Escherichia coli and Sal-monella typhimurium and does not have the O-anti-genic side chain of the repeating units characteristic of classical lipopolysaccharide As showed in Fig 1, the LOS consists of lipid A and a core oligosaccharide (OS) The latter can be further dissected into three OS chains and a linker composed of a central Glc and two 3-deoxy-d-manno-2-octulosonic acids (Kdo) How do the three moieties play their roles in biological activi-ties of the outer membrane? Biosynthesis of LOSs in
M catarrhalis is complex and strictly sequential, requiring many enzymes such as UDP-glucose-4-epi-merase, Kdo-8-phosphate synthase, Kdo transferase (kdt), glycosyltransferase enzymes, and UDP-N-acetyl-glucosamine acyltransferase (lpxA) Recently, several genes encoding these enzymes have been reported [20,22–24,26,30–32] A series of stepwise-truncated LOS mutants can therefore be established by knocking out some of the genes and then the biological activities
of the different truncated LOS mutants can be
Fig 1 Schematic structure of M catarrhalis
LOS [8] Lipooligosaccharide (LOS) consists
of two major moieties: lipid A and a core
oligosaccharide (OS) The core OS can
further be dissected into three OS chains
and a linker (boxed) composed of a Glc and
two Kdo p, pyranose.
Trang 3compared to decipher potential roles of each moiety of
the LOS Previously, we identified an lpxA gene
encod-ing UDP-N-acetylglucosamine acyltransferase and a
kdtAgene encoding Kdo transferase [23,24] The lpxA
or the kdtA mutant contained no LOS or lipid A-only
structure Both mutants showed a variety of changes
of biological activities in vitro as well as in vivo In this
study, we further found an lgt3 gene by a random
transposon mutagenesis approach followed with a
screen by an LOS-specific mAb A subsequently
iso-genic lgt3 mutant contained a truncated core OS and
consisted of a linker (Glc-Kdo2)-lipid A structure The
phenotype of this mutant was examined to investigate
the roles of different moieties of the LOS molecule in
biological activities of the M catarrhalis outer
mem-brane
Results
Construction of M catarrhalis O35Elgt3 mutant
M catarrhalis O35E mutants were constructed by
in vitrotransposon mutagenesis The resulting
kanamy-cin-resistant colonies were screened for loss of
reactiv-ity to a specific anti-LOS mAb 8E7 Two clones were
selected and the EZ:TN transposon was found to be
inserted at position 901 and 1203 of an open reading
frame (ORF) of 1605 bp, respectively (Fig 2) BLAST
searches at GenBank with the deduced polypeptide
sequence of the ORF revealed 99.6% and 100%
iden-tity with a recently identified lgt3 gene encoding b(1–4)
glucosyltransferase of M catarrhalis serotype B strain
7169 [30] and a putative lgt3 gene from a serotype A
strain 25238 [26], respectively
An lgt3 knockout mutant of M catarrhalis O35E
was constructed by allelic exchange with an insertion
of a kanamycin-resistant cassette and deletion of an
822 bp fragment between two HindIII sites of the lgt3
(Fig 2)
Silver staining revealed that the mutant LOS mole-cule migrated more rapidly and showed less density than those of the wild-type, indicating a truncated LOS molecule resulting from the mutant (Fig 3A) Western blot analysis demonstrated that the LOS from O35Elgt3 lost the binding activity to the mAb 8E7 when compared with the wild-type strain (Fig 3B) Purified LOSs from both strains also showed similar results to those described above (data not shown) To further compare LOS expression levels between the wild-type and mutant strains, the amounts of Kdo from both strains were determined as an indicator They were 73 ng and 70 ng per 80 lg of outer mem-brane vesicles (OMVs) for the wide type and mutant strains, respectively, indicating the LOS expressions in both strains were similar
Composition analysis by GC-MS showed that the carbohydrates derived from the wild-type O35E strain consisted of Glc, Gal, GlcNAc and Kdo, whereas the carbohydrates derived from the mutant O35Elgt3 con-sisted exclusively of Glc and Kdo The glycosyl linkage analysis by GC-MS showed that the OSs from the wild-type strain consisted of terminal-, 2-, 4-, and 3,4,6-linked glucosyl residues, terminal- and 4-linked galactosyl residues, and a terminal-linked N-acetylglu-cosaminosyl residue These linkage results were consis-tent with the structure of the LOS reported for serotype A strain 25238 [33] The only hexosyl residue
in the mutant LOS structure was terminally linked Glc
Fig 2 Strategy for construction of the lgt3 mutant in M catarrhalis
O35E Large arrows represent the direction of transcription; the
site of the transposon insertion identified in the O35E is denoted
as EN:TN, and the location of the deletion replaced by the
kanamy-cin-resistant gene (kanR) is between two HindIII cleavage sites.
The sites of primers used are indicated as small arrows (25, 26,
and 40 of Table 1).
Fig 3 LOS patterns of SDS ⁄ PAGE followed by silver staining (A)
or western blotting (B) of M catarrhalis wild-type strain O35E (lane 1) and mutant O35Elgt3 (lane 2) Each lane represents extracts from proteinase K-treated whole cell lysates from 1.9 lg
of each bacterial suspension An anti-LOS MAb 8E7 was used at
1 : 100 dilution (B) Molecular markers (Mark 12; Invitrogen) are indicated on the left.
Trang 4The de-O-acylated LOSs from the wild-type O35E
and the mutant O35Elgt3 were further analyzed by
MALDI-TOF MS (Fig 4) The mass spectral data of
de-O-acylated LOS from O35E was consistent with the
reported structure of serogroup A LOS Among
the major ions m⁄ z 2672.8(M-H)– corresponds to the
molecular mass of the de-O-Ac LOS containing five
Glc, two Gal, one GlcNAc, two Kdo and
O-acyl-ated lipid-A bearing two phosphate moieties The
de-O-acylated lipid-A unit consists of two GlcNAc and
two 3-OH C12:0 groups Among the other ions m⁄ z of
2654.8, 2694.8 and 2717.4 indicates the presence of the
anhydro form and mono- and di-sodiated form of the
molecular ion 2672.8 (Fig 4A) Among other
charac-teristic ions, 1558.2 and 1778.4 represent the mass of
intact OS with one and two Kdo residues, respectively
The low molecular mass with m⁄ z of 895.4 is from the Y-type fragment ion from the de-O-acylated lipid-A portion (as observed in the negative mode) The ions with m⁄ z of 917.4 and 877.4 (unmarked) are mono-sodiated and anhydro forms of 895.4 ion, respectively The ion with m⁄ z of 797.4 was from the anhydro frag-ment ion of de-O-acylated lipid-A bearing only one phosphate group (Fig 4A)
The MALDI-TOF mass spectral data of de-O-acyl-ated LOS for the mutant O35Elgt3 showed the pres-ence of ions with m⁄ z of 1479.6, 1497.6, 1519.6 and 1541.6, respectively The ion at m⁄ z 1497.6 (M-H)– corresponds to the deprotonated molecular mass, con-taining one Glc, two Kdo, de-O-acylated lipid-A and two phosphate moieties The ions with m⁄ z of 1519.6 and 1541.6 are mono- and di-sodiated forms of the de-O-acylated LOS and the ion with m⁄ z of 1479.6 is the anhydrous form of the de-O-acylated LOS The ion 1277.6 is the de-protonated molecular ion from de-O-acylated LOS containing only one Kdo residue (Fig 4B) The low molecular ions with m⁄ z of 917.4 and 877.4 are from the mono-sodiated and anhydro forms of the Y-type fragment ion 895.4 arising from the de-O-acylated lipid-A
Finally, proton NMR spectra of carbohydrates from both wild and mutant strains showed that the spec-trum for the O35E carbohydrates was identical to that published from strain 25238 LOS (Fig 5) [33] The mutant carbohydrate spectrum showed only one ano-meric resonance, a result consistent with a single termi-nal-linked glucosyl residue in the above GC-MS analysis
Taken together, the O35Elgt3 mutant contained only
a linker-lipid A (Glc-Kdo2-lipid A) structure without
OS chains (Fig 1)
Morphology, growth rate, and OMV⁄ OMP profiles of M catarrhalis O35Elgt3 mutant There were no significant differences in morphology, growth rate, or OMV⁄ OMP profiles between the wild-type and the lgt3 mutant (data not shown) The yields
of OMVs from both strains were similar (data not shown)
Susceptibility of M catarrhalis O35Elgt3 mutant
A broad range of hydrophobic agents and a hydrophilic glycopeptide were used to determine the susceptibility of the mutant O35Elgt3 O35Elgt3 showed similar resis-tance or intermediate susceptibility to hydrophobic anti-biotics, reagents [azithromycin (15 lg), deoxycholate (100 mgÆmL)1), fusidic acid (10 mgÆmL)1), novobiocin
A
B
Fig 4 MALDI-TOF MS spectrum (negative mode) of the
de-O-acyl-ated LOS from M catarrhalis wild-type strain O35E (A) and the
mutant O35Elgt3 (B) This analysis was done in the negative mode,
and all ions represented as deprotonated [M-H]–ions The source
of the ions was as indicated in the structure shown in the inset.
The m ⁄ z 917.4 and 797.4 are due to sodiated and dehydrated
forms, respectively, of the m ⁄ z 895.4 fragment ion.
Trang 5(5 lg), polymycin B (300 international units), rifampin
(5 lg), Triton X-100 (5%, w⁄ v), Tween 20 (5%, v ⁄ v)]
and a hydrophilic glycopeptide, vancomycin (5 lg), as
the wild-type strain (data not shown)
Biological activity of M catarrhalis O35Elgt3
mutant
The mutant was tested for LOS-associated biological
activity In a Limulus amebocyte lysate assay, whole
cell suspensions (D620¼ 0.1) gave 3.7 · 103 endotoxin
units (EU) mL)1 for O35E and 2.0· 103 EUÆmL)1 for
the O35Elgt3 mutant In a bactericidal assay with
normal human serum, strain O35E survived at 25% normal human serum However, 75% of the mutant cells died at 25% normal human serum (P < 0.05, Fig 6) indicating a reduced resistance from the mutant
To test the adherence of the O35Elgt3 mutant to human epithelial cells, Chang and HeLa cell lines were used The adherence percentage of O35Elgt3 to Chang and HeLa epithelia were 19.1 ± 4.6 and 27.3 ± 8.4, respectively, whereas those of the wild-type were 41.7 ± 9.1 and 47.0 ± 4.1 (P < 0.01)
To investigate the effect on survival of the O35Elgt3 mutant in a murine model of nasopharyngeal clear-ance, mice were challenged with the wild-type or mutant strains by aerosolization (Fig 7) Mutant
A
B
Fig 5 Proton NMR spectra of LOS derived from M catarrhalis
O35E (A) and the mutant O35Elgt3 (B) For the O35E strain, the
assignments were made based on the identity of this spectrum
with that of the published structure for the LOS from type A
M catarrhalis The spectrum for the mutant lgt3 disaccharide
shows numerous resonances for the H3 protons of Kdo due to the
fact that a 5-linked Kdo residue can, on mild acid hydrolysis, form
4,8-anhydroKdo, 4,7-anhydroKdo, and 2,7-anhydroKdo; all of which
have different H3 resonances.
5.5 6 6.5 7
Normal human Serum (%)
*
Fig 6 Bactericidal activity of normal human serum against
M catarrhalis wild-type strain O35E (black bar) and the mutant O35Elgt3 (gray bar) ‘HI’ represents the group of 25% heat-inactivated normal human serum The data represent the average
of three independent assays An asterisk represents statistical sig-nificance between the wild-type and mutant strains.
0 1 2 3 4 5
0
Nasal wash
*
*
Time post challenge (h)
Bacterial recovery (log CFU/mouse)
Fig 7 Time courses of bacterial recovery in mouse nasal washes after an aerosol challenge with M catarrhalis wild-type strain O35E (r) and the mutant O35Elgt3 (h) Each time point represents a geometric mean of six mice Asterisks represent statistical signifi-cance between the wild-type and mutant strains.
Trang 6O35Elgt3 present in the nasopharynx showed an
accel-erated clearance at 3 h (85.5% versus 60.2%,
P< 0.01) or 6 h (98.0% versus 86.8%, P < 0.01)
compared to the wild-type
Discussion
In this study, an lgt3 gene was found and its products
confirmed from a M catarrhalis serotype A strain
O35E through an isogenic O35Elgt3 mutant Structural
analysis revealed that the O35Elgt3 mutant contained
a truncated core OS and consisted of a linker
(Glc-Kdo2)-lipid A structure, similar to that reported
previ-ously [30,34] The O35Elgt3 mutant demonstrated no
significant difference in its growth rate, LOS
expres-sion level and susceptibility to the hydrophobic
com-pounds compared with the wild-type strain However,
our previous mutants which lacked LOS [24] or
con-tained only lipid-A structure [23] showed reduced
growth rate and hypersusceptibility to the hydrophobic
compounds It implies that the linker moiety of the
LOS might be critical for maintenance of outer
mem-brane integrity, stability or flexibility by resistance to
foreign hydrophobic compounds to preserve normal
cell growth In addition, the O35Elgt3 mutant was
sen-sitive to the bactericidal activity of normal human
serum at 25% as compared with the wild-type strain,
but less sensitive than that of the mutant with lipid
A-only structure [23] It indicates that the linker moiety
(Glc-Kdo2) might also contribute to the serum
bacteri-cidal resistance However, another possibility also
exists As the susceptibility of the lipid A-only mutant
to hydrophobic compounds was higher than that of
the O35Elgt3 mutant, the permeability barrier of the
outer membrane might significantly contribute to the
bactericidal activity of normal human serum, and
might cause a much more sensitive phenotype of the
lipid A-only mutant than that of the O35Elgt3 mutant
Nevertheless, as the O35Elgt3 mutant did not change
its susceptibility to hydrophobic compounds but was
shown to be more sensitive to the bactericidal activity
of the human serum as compared with the wild-type
strain, it suggests that a complete OS chain moiety
contributes to the serum bactericidal resistance
LOS toxicity was assumed to be associated only with
the lipid A moiety By using Limulus amebocyte lysate
assay, we found that the O35Elgt3 mutant did not
show significant changes of the toxicity, whereas the
lipid A-only mutant [23] showed decreased toxicity
(6· 102EU Æ mL)1) six-fold as compared with the
wild-type strain (3.7· 103EU Æ mL)1), suggesting that
the LOS toxicity is attributable not only to the lipid A
moiety but also to the linker moiety (Glc-Kdo2)
Bacterial adherence to the surface of epithelial cells plays a critical role in colonization and is believed to
be the first step in the pathogenesis of microbial infec-tions It has been reported that several LOSs from respiratory tract bacteria are associated with bacterial adherence [29,35,36] Consistent with our previous results from the lipid A-only mutant, the O35Elgt3 mutant showed more than a 50% reduction in attach-ment to Chang or HeLa cells and enhanced clearance from the mouse nasopharynx after an aerosol chal-lenge when compared to the wild-type strain [23] Our data suggest that the M catarrhalis adherence to epi-thelial cells may not be associated with the linker and lipid A moieties, but with the OS chain moiety
In summary, we constructed a serotype A
M catarrhalisO35Elgt3 mutant that produced a linker (Glc-Kdo2)-lipid A structure The mutant showed sig-nificant changes in its biological activities in vitro and
in vivo These data, combined with our previous data from mutants which lacked LOS or contained only lipid A structure, suggest that a complete OS chain moiety of the M catarrhalis LOS is important in bio-logical activities such as serum resistance and adher-ence of epithelial cells, the linker moiety is critical for maintenance of outer membrane integrity and stability
to preserve normal cell growth, and both lipid A and linker moieties contribute to the LOS toxicity
Experimental procedures
Strains, plasmids, and growth conditions Bacterial strains, plasmids and primers are given in Table 1
M catarrhalis strains were cultured on chocolate agar plates (Remel, Lenexa, KS), or brain-heart infusion (BHI) (Difco, Detroit, MI) agar plates at 37C in 5% CO2 Mutant strains were selected on BHI agar supplemented with kanamycin at 20 lgÆmL)1 Growth rates of wild-type and mutant were measured as follows: an overnight culture was inoculated in 10 mL of BHI media (adjusted D600¼ 0.05) and shaken at 37C at 250 r.p.m The bacterial cultures were monitored spectrophotometrically at D600 Escherichia coli was grown on Luria-Bertani agar plates or broth with appropriate antibiotic supplementation The antibiotic concentrations used for E coli were as follows: kanamycin, 30 lgÆmL)1; and ampicillin, 50 lgÆmL)1
General DNA methods DNA restriction endonucleases, T4 DNA ligase, E coli DNA polymerase I Klenow fragment, and Taq DNA poly-merase were purchased from Fermentas (Hanover, MD) Preparation of plasmids, purification of PCR products and
Trang 7DNA fragments were performed using kits manufactured
by Qiagen (Santa Clarita, CA) Bacterial chromosomal
DNA was isolated using a genomic DNA purification kit
(Promega, Madison, WI) DNA nucleotide sequences were
obtained via 3070xl DNA analyzer (Applied Biosystems,
Foster City, CA) and analyzed with dnastar software
(DNASTAR Inc., Madison, WI)
Transposon mutagenesis and identification
of lgt3 gene
In vitro transposon mutagenesis of M catarrhalis was
per-formed as described previously [23] The resulting
kana-mycin-resistant colonies were screened by colony blot
assay and further identified by whole cell enzyme-linked
immunosorbent assay (ELISA) for loss of binding
reactiv-ity to an anti-LOS MAb (8E7) generated by serotype A
strain O35E [21] The transformants without the 8E7
binding activity were subsequently evaluated by
examin-ing the LOS profiles from proteinase-K-treated whole cell
lysates [37] on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS⁄ PAGE) and LOS silver staining [38]
The clones with different LOS bands from the wild-type
LOS were selected for isolation of chromosomal DNA
The DNA fragment with the transposon insertion was
subsequently sequenced and the lgt3 homologue from
strain O35E identified by BLAST searches at GenBank of
the National Center for Biotechnology Information,
Bethesda, MD
Cloning of lgt3 homologue and construction
of the knockout mutants The entire lgt3 was amplified from the chromosomal DNA
of M catarrhalis strain O35E using primers 25 and 26 (Table 1, Fig 2) The PCR product was cloned into pCR2.1 using a TOPO TA cloning kit (Invitrogen, Carls-bad, CA) to obtain pCRlgt3 The insert was released by EcoR I-SalI digestion and then subcloned into an EcoRI-SalI site of pBluescript SK(+) to form pSlgt3 The kana-mycin-resistant gene from pUC4K was digested with EcoRI and subsequently inserted into the lgt3 gene using the HindIII site to form pSlgt3K
After verification by sequence analysis, the mutagenic lgt3 gene with the inserted kanamycin-resistant gene in pSlgt3K was amplified by PCR and purified for electropo-ration to O35E-competent cells After 24 h incubation, the resulting kanamycin-resistant colonies were selected for PCR analysis of chromosomal DNA using primers 40 and
26 (Table 1, Fig 2) An inactivated lgt3 mutant was verified
by sequence analysis and named as O35Elgt3
LOS determination
A crude LOS extraction was performed from both the wild-type strain O35E and the mutant O35Elgt3 using the proteinase-K-treated whole cell lysate method [37] The resulting extracts from each bacterial suspension (1.9 lg of protein amount) were resolved by 15% SDS⁄ PAGE and
Table 1 Strains, plasmids, and primers used in this study.
Plasmids
inserted into blunted HindIII site of pSlgt3 with internal deletion
This study
Primers
lgt3 sense, SalI site underlined
lgt3 antisense, EcoRI site underlined
5¢ flanking DNA of lgt3 sense
Trang 8visualized by silver staining [38] Western blot using MAb
8E7 was performed [21]
To further quantify LOSs expressed on both the
wild-type and mutant strains, a Kdo assay was applied [39] with
OMVs prepared using an EDTA-heat induced method [40]
as whole cells were not applicable for the Kdo assay The
amount of Kdo was determined by using 80 lg of OMVs
as samples and Kdo ammonium salt (Sigma, St Louis,
MO) as a standard
Structural analysis of LOS
For the composition analysis, 30–35 g of wet cells from
O35E and O35Elgt3 were prepared for LOS purification by
phenol–water extraction [41] The purified LOSs were
washed with a 9 : 1 ethanol⁄ water (v ⁄ v) mixture to remove
phospholipids The OSs were prepared by mild acid
hydro-lysis of LOSs in 1% aqueous acetic acid (v⁄ v) for 2.5 h at
100C and gel-filtration chromatography using Bio-Gel P-2
with deionized water as the eluent The glycosyl
composi-tions of OSs in O35E and O35Elgt3 were determined by the
preparation and GC-MS analysis of trimethylsilyl
methyl-glycosides The glycosyl linkages were determined by the
preparation and GC-MS analysis of partially methylated
aldiol acetates Analysis of GC-MS was performed on an
HP)5890 GC interfaced to a mass selective detector
5970 MSD using a Supelco DB1 fused silica capillary
col-umn (30 m· 0.25 mm Internal diameter, J & W Scientific,
Folsom, California) For MS analysis, the LOS was
O-deacylated by treatment with anhydrous hydrazine for
20 min at 37C [42] The de-O-acylated LOS samples were
dissolved in deionized water at 1 lgÆlL)1concentration and
mixed with equal volumes of 0.5 m 2,5-dihydroxy benzoic
acid (matrix) in methanol and spotted with 1 lL on a
100-well stainless steel MALDI plate The mass spectra were
col-lected on a MALDI-TOF instrument (Applied Biosystems)
in the negative reflectron mode NMR was performed on
the OSs by lyophilizing each sample from D2O (99.999 atom
percentage D, Sigma) twice, dissolved in D2O (100 atom
percentage D), and acquiring spectra using a Varian
Ino-va 500 or 600 MHz spectrometer (Varian, Palo Alto, CA)
Limulus amebocyte lysate assay
The chromogenic Limulus amebocyte lysate assay for
endotoxin activity was performed using the QCL-1000 kit
(Bio-Whittaker Inc., Walkersville, MD) Overnight cultures
from chocolate agar plates were suspended in BHI broth to
D620of 0.1 and serial dilutions of these stocks were tested
Susceptibility determination
The sensitivity of strains to a panel of hydrophobic agents
or a hydrophilic glycopeptide was performed using
standard disk-diffusion assays [43] Bacteria were cultured
in BHI to a D600of 0.2 and 100 lL portions of the bacte-rial suspension were spread onto chocolate agar plates Antibiotic disks or sterile blank paper disks (6 mm, Becton Dickinson, Cockeysville, MD) saturated with the various agents were plated on the lawn in triplicate at 37C for
18 h Sensitivity was assessed by measuring the diameter of the zone of growth inhibition in two axes and the mean value was calculated
Bactericidal assay to normal human serum
A complement-sufficient normal human serum was pre-pared and pooled from eight healthy adult donors A bacte-ricidal assay was performed in a 96-well plate [23] Normal human serum was diluted to 0.5, 2.5, 5.0, 12.5, and 25% in
pH 7.4 Dulbecco’s phosphate-buffered saline (NaCl⁄ Pi) containing 0.05% gelatin (DPBSG) Bacteria (10 lL of 106
colony forming units, CFU) were inoculated into 190 lL reaction wells containing the diluted normal human serum, 25% of heat-inactivated normal human serum, or DPBSG alone and incubated at 37C for 30 min Serial dilutions (1 : 10) of each well were plated onto chocolate agar plates The resulting colonies were counted after 24 h of incuba-tion
Adherence assay Chang (conjunctival; CCL20.2) and HeLa (cervix; CCL-2) human epithelial lines were cultured in Eagle’s minimal essential medium (ATCC, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum in an atmo-sphere of 5% CO2at 37C A quantitative adherence assay was performed on a 24-well tissue culture plate (Corning Incorporated, Corning, NY) [34] Adherence was expressed
as the percentage of bacteria attached to the human cells relative to the original bacteria added to the well The data represent the average of three independent assays
Nasopharyngeal clearance pattern in animal model
Female BALB⁄ c mice (6–8 weeks of age) were obtained from Taconic Farms Inc (Germantown, NY) The mice were housed in an animal facility in accordance with National Institutes of Health guidelines under animal study protocol 1158–04 Bacterial aerosol challenges were carried out in mice using the same D540value for wild-type strain O35E (1.24· 109 CFUÆmL)1) or mutant O35Elgt3 (8.2· 108 CFUÆmL)1) in a 10 mL DPBSG [44] The num-bers of bacteria present in nasal washes were measured at various time points postchallenge The minimum detectable number of viable bacteria was 4 CFU per nasal washing Clearance of M catarrhalis was expressed as the percentage
Trang 9of bacterial CFU at each time point compared with the
number deposited at time zero
Statistical analysis
The adherence and clearance percentages were analyzed by
a Chi-square test
Acknowledgements
We thank Eric J Hansen for providing strain O35E,
Wenzhou Hong for assisting in the animal challenge,
and Robert Morell and Yandan Yang for helping in
DNA sequencing This research was supported by the
Intramural Research Program of the NIH, NIDCD
The analytical work was supported in part by the
Department of Energy-funded (DE-FG09–93ER20097)
Center for Plant and Microbial Complex
Carbohy-drates
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